CN116699135A - Application of cancer immunotherapy target and diagnosis and prognosis prediction biomarker - Google Patents

Abstract

The invention discloses an application of a cancer immunotherapy target and a diagnosis and prognosis prediction biomarker, which comprises the following steps of determining and detecting cancer cells of a patient or blood of the patient: elevated EGFR activation levels, wnt pathway activation levels, activated β -catenin levels, long non-coding RNA LINC00973 expression levels, LNC1574203 expression levels, CD55 protein and mRNA expression levels, CD59 protein and mRNA expression levels, CD73 protein and mRNA expression levels, as compared to reference levels, for use in diagnosis, prognosis prediction and treatment of cancer.

Description

Application of cancer immunotherapy target and diagnosis and prognosis prediction biomarker

Technical Field

The invention relates to the field of oncology, in particular to an application of a cancer immunotherapy target and a diagnosis and prognosis prediction biomarker.

Background

The complement system is an important component of the immune response. However, its role in tumor immune escape induced by oncogenic signaling and its relationship to cytotoxic T cell activation remain largely uncertain.

Disclosure of Invention

The invention aims to provide an application of a cancer immunotherapy target and a diagnosis and prognosis prediction biomarker.

The invention provides application of a marker in preparation of a detection reagent or a kit for cancer diagnosis and prognosis prediction, wherein the marker is one or more of EGFR, wnt, beta-cateni, long non-coding RNA LINC00973, LNC1574203, CD55, CD59, CD73, C4d, C3a, C3b, C5a and C5 b-9.

According to one embodiment of the invention, an elevated EGFR activation level, wnt pathway activation level, activated β -catenin level, long non-coding RNA LINC00973 expression level, LNC1574203 expression level, protein and mRNA expression level of CD55, protein and mRNA expression level of CD59, protein and mRNA expression level of CD73, and various combinations of these elevated levels, as compared to a reference level, predicts that the patient has an invasive cancer, has an invasive cancer in the progressive stage, or has a poor prognosis.

According to another embodiment of the invention, a reduced C4d expression level, C3a expression level, C3b expression level, C5a expression level, C5b-9 expression level, and various combinations of these reduced levels compared to a reference level predicts that the patient has an invasive cancer, has an invasive cancer in the stage of progression, or has a poor prognosis.

According to another embodiment of the invention, the reference level is a level in blood from non-cancerous or early cancer cells or healthy individuals, or early cancer patients.

According to another embodiment of the invention, the cancer is an oral cancer, an oropharyngeal cancer, a nasopharyngeal cancer, a respiratory cancer, a genitourinary system cancer, a gastrointestinal cancer, a central or peripheral nervous system tissue cancer, an endocrine or neuroendocrine system cancer or a hematopoietic system cancer, a glioma, a sarcoma, an epithelial cancer, a lymphoma, a melanoma, a fibroma, a meningioma, a brain cancer, a renal cancer, a biliary system cancer, a pheochromocytoma, a pancreatic islet cell cancer, a lif Mei Niliu, a thyroid cancer, a parathyroid cancer, a pituitary tumor, an adrenal tumor, a osteogenic sarcoma tumor, a neuroendocrine system tumor, a breast cancer, a lung cancer, a head and neck cancer, a prostate cancer, an esophageal cancer, a tracheal cancer, a liver cancer, a bladder cancer, a stomach cancer, a pancreatic cancer, an ovarian cancer, a uterine cancer, a cervical cancer, a testicular cancer, a colon cancer, a rectal cancer or a skin cancer.

The invention also provides the use of an EGFR inhibitor, a Wnt inhibitor, a β -catenin inhibitor, a LINC00973 inhibitor, an LNC1574203 inhibitor, a protein and mRNA inhibitor of CD55, a protein and mRNA inhibitor of CD59, a protein and mRNA inhibitor of CD73 or a pharmaceutical composition comprising the inhibitor for the preparation of a product for the treatment of cancer.

According to one embodiment of the invention, the EGFR inhibitor, wnt inhibitor, β -catenin inhibitor, LINC00973 inhibitor, LNC1574203 inhibitor, protein and mRNA inhibitor of CD55, protein and mRNA inhibitor of CD59, protein and mRNA inhibitor of CD73, including small molecule inhibitors, polypeptides, complementary inhibitory oligonucleotides or neutralizing antibodies to EGFR, wnt, β -catenin, LINC00973, LNC1574203, protein and mRNA of CD55, protein and mRNA of CD59, protein and mRNA of CD 73.

The invention further provides the use of a C4d agonist, a C3a agonist, a C3b agonist, a C5a agonist, a C5b-9 agonist, or a pharmaceutical composition comprising the agonist, in the manufacture of a product for the treatment of cancer.

According to an embodiment of the invention, the C4d agonist, C3a agonist, C3b agonist, C5a agonist, C5b-9 agonist includes small molecule agonists for C4d, C3a, C3b, C5a, C5 b-9.

The present invention illustrates a previously unknown mechanism by which oncogenic EGFR or Wnt signaling inhibits the complement system through LINC 00973-mediated upregulation of CD55 and CD 59. This was the first report that demonstrates that oncogenic signaling inhibits cytotoxic cd8+ T cells in a complement inhibition-dependent manner, revealing a new type of intrinsic cell regulation between complement and cd8+ T. Importantly, preclinical evidence is also provided for the first time, suggesting that combined blockade of mCRP function and PD-1/PD-L1 checkpoints may promote complement and activation of cd+ T cells may be a rational strategy for treatment of human NSCLC. The clinical significance of this modulation was demonstrated by positive correlation of EGFR activation with the expression levels of active β -catenin, LINC00973, CD55 and CD59 in human NSCLC specimens, which is associated with clinical aggressiveness of the tumor. These findings reveal that the unknown mechanism of oncogenic signal-dependent inhibition of the complement system and subsequent cd8+ T cells (fig. 19 l) is activated by tumor cells and underscores the importance of EGFR/Wnt/β -catenin transactivation-mediated upregulation of CD55 and CD59 for tumor immune evasion.

Drawings

Figures 1 and 2 show that EGFR activation increases the expression of CD55 and CD59 and inhibits the activation of the complement system and cd8+ T cells.

Figures 3 and 4 show that EGFR activation upregulates CD55 and CD59 by inhibiting miR-216b and miR-150, respectively.

Figures 5, 6 and 7 show that EGFR activation induces LINC00973 expression to adsorb miR-216b and miR-150 up-regulates CD55 and CD59.

Figures 8 and 9 show that EGFR activation-induced β -catenin transactivation enhances LINC00973 expression and subsequent CD55 and CD59 upregulation.

FIGS. 10 and 11 show that GFR/β -catenin activation inhibits complement activation by upregulating miR-216b and miR-150-mediated CD55 and CD59 adsorbed by LINC 00973.

Figures 12, 13 and 14 show that EGFR/β -catenin activation inhibits immune cell function by upregulating CD55 and CD59.

Figures 15, 16, 17 and 18 show that EGFR/β -catenin transactivation enhanced CD55 and CD59 expression promotes tumor growth by inhibiting complement activation and immune cell activation in mice.

Figure 19 shows that CD55 and CD59 silencing mediated complement activation promotes immune checkpoint blockade therapy.

FIG. 20 shows that EGFR/β -catenin transactivation, LINC00973 expression, and CD55 and CD59 levels are positively correlated with each other in human NSCLC specimens, and with clinical aggressiveness of the disease.

FIG. 21 shows that EGFR signaling might up-regulate Lnc1574203 expression by beta-catenin transcription.

FIG. 22 shows that knock down of β -catenin inhibits the up-regulation of CD73 expression by EGF treatment.

FIG. 23 shows that knockdown Lnc1574203 inhibits the up-regulation of CD73 expression by EGF treatment.

FIG. 24 shows that inhibition of Dicer blocked down-regulation of CD73 expression by Lnc1574203 knockdown.

FIG. 25 shows expression of LINC00973 and C5 in lung adenocarcinoma and normal tissues.

Fig. 26 shows a correlation analysis of CD55 and CD59 expression in lung adenocarcinoma.

FIG. 27 shows survival analysis of LINC00973 expression in lung adenocarcinoma patients.

FIG. 28 shows the expression of C3 and C5 in lung squamous carcinoma and normal tissue.

Fig. 29 shows a correlation analysis of CD55 and CD59 expression in lung squamous carcinoma.

Figure 30 shows survival analysis of CD59 expression in lung adenocarcinoma patients.

Fig. 31 shows the expression of CD55, CD59, C3 and C5 in cholangiocarcinoma and normal tissues.

Fig. 32 shows a correlation analysis of expression of CD55 and CD59 in cholangiocarcinoma.

FIG. 33 shows the expression of CD55, CD59 and C5 in liver cancer and normal tissues.

Fig. 34 shows a correlation analysis of the expression of CD55 and CD59 in liver cancer.

Fig. 35 shows survival analysis of C3 expression in liver cancer patients.

FIG. 36 shows expression of LINC00973, CD55, CD59 and C5 in pancreatic cancer and normal tissues.

Fig. 37 shows a correlation analysis of CD55, CD59 and LINC00973 expression in pancreatic cancer.

Fig. 38 shows survival analysis of LINC00973 and CD59 expression in pancreatic cancer patients.

FIG. 39 shows the expression of CD55 and C5 in gastric adenocarcinoma and normal tissues.

Fig. 40 shows a correlation analysis of CD55 and CD59 expression in gastric adenocarcinoma.

Fig. 41 shows survival analysis of CD59 expression in gastric adenocarcinoma patients.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings, in order to make the objects and technical solutions of the present invention more apparent.

The complement system is a phylogenetic conserved branch of the innate immune response that acts through a series of more than 30 coordinated cascades of proteins and zymogens and recognizes foreign pathogens and self cells expressing abnormal surface molecules, triggering the release of inflammatory mediators, the recruitment of immune cells, phagocytic response and cell lysis. The complement system can be activated by the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway is triggered by direct binding of antibody or complement component C1q to the pathogen surface; the lectin pathway is triggered by binding of mannan-binding lectins to pathogen-associated molecular patterns (PAMPs) or apoptotic host cells; while an alternative pathway, which may account for 80% of total complement activation, is triggered by spontaneous cleavage of C3 in serum to produce C3b, followed by binding of C3b to the surface of pathogens (e.g., tumor cells) and amplification of complement activation. All activation pathways lead to the formation of serine protease C3 convertase complexes that catalyze proteolytic cleavage of C3 plasma into biologically active C3a (a major anaphylatoxin) and C3b (an opsonin) that coat antigen surfaces and label them for phagocytosis by circulating macrophages. The C3b complexes with other cleavage fragments of the circulating complement protein to form C5 convertases that cleave C5 to trigger the terminal pathway, yielding C5a and C5b; the latter then binds to C6, C7, C8 and multiple C9 to form the C5b-9 terminal complement complex, also known as the Membrane Attack Complex (MAC), which is deposited in the lipid bilayer of the cell, ultimately leading to membrane destruction and cell lysis. The complement system is generally considered a protective mechanism against tumor formation, although some reports suggest that the complement system has a pro-tumorigenic potential under certain cancers and certain conditions. The contribution of the complement system to cancer pathophysiology may depend largely on the nature of the tumor microenvironment.

The complement system is tightly down-regulated by membrane-bound complement regulatory proteins (mCRP), such as CD46, CD55, and CD59.CD55 (also known as decay accelerating factor, DAF) accelerates the decay or breakdown of C3 and C5 convertases, resulting in reduced formation of anaphylatoxins (C3 a and C5 a) and opsonin, and prevents MAC formation. CD59 inhibits MAC formation by preventing C9 polymerization and inserting additional C9 molecules into the MAC complex and by interfering with pore formation of C5 b-8. Interestingly, CD55 and CD59 are overexpressed in a variety of human cancer cells, and can be used as biomarkers for tumor development and targets for cancer immunotherapy. Cytokines have been reported to increase or decrease the expression of mCRP on tumor cells; however, it is not known whether oncogenic signals, particularly those triggered by mutations in receptor tyrosine kinases, up-regulate CD55 and CD59, thereby inhibiting complement activation and protecting tumor cells from immune attack. Furthermore, despite the promising response of several types of cancers to immune checkpoint blockade, a large proportion of cancer patients, including non-small cell lung cancer (NSCLC) patients with Epidermal Growth Factor (EGF) receptor (EGFR) mutations, are resistant to anti-PD-1 antibody therapies. It is currently unclear whether tumor cells coordinate the regulation of complement system and cytotoxic T cell activity, and subsequently regulate immune checkpoint blockade responses.

In this patent, we demonstrate that EGFR activation or Wnt signaling in NSCLC cells increases CD55 and CD59 expression, inhibits the complement system, and inhibits subsequent macrophage phagocytosis and cd8+ T cell activation by β -catenin-mediated transcriptional upregulation of long-chain non-coding RNA (lncRNA) LINC00973, adsorption of CD 55-targeting microRNA (miR) -216b and CD 59-targeting miR-150. Inhibition of this modulation restores EGFR-induced complement inhibition and sensitizes EGFR mutated NSCLC patients to immune checkpoint blocking therapies.

The complement system is an important component of the immune response. However, its role in tumor immune escape induced by oncogenic signaling and its relationship to cytotoxic T cell activation remain largely uncertain. Here we demonstrate that EGFR activation or Wnt signaling increases expression of the complement regulatory proteins CD55 and CD59, thereby inhibiting secretion of the complement system and C3 and C5 convertase-dependent cytokines that contribute to cd8+ T cell activation. The enhanced expression of CD55 and CD59 was due to beta-catenin mediated upregulation of lncRNA LINC00973, which adsorbed CD 55-targeting miR-216b and CD 59-targeting miR-150. Knock down of CD55 and CD59, CD55/CD59 neutralizing antibody treatment or knock-in mutation of TCF/LEF binding elements in LINC00973 promoter region, activate complement system and cd8+ T cells and inhibit tumor growth, which is eliminated by depletion of C3 and C5 in the liver of mice. Importantly, the combination therapy of anti-CD 55/CD59 antibodies and anti-PD-1 antibodies produces synergistic tumor suppression. In addition, EGFR phosphorylation, activated β -catenin, LINC00973 expression, and CD55 and CD59 levels are inversely related to infiltration of M1 macrophages and cd8+ T cells in human non-small cell lung cancer (NSCLC) specimens, positively related to clinical aggressiveness of NSCLC. These findings underscores the important role of EGFR/Wnt induction and β -catenin mediated upregulation of CD55 and CD59 in inhibiting complement and cd8+ T cell activation to facilitate tumor immune evasion and immune checkpoint blocking resistance, and reveals the great potential of novel cancer therapies with anti-CD 55/CD59 antibodies in combination with immune checkpoint inhibitors.

The invention provides an application of a cancer immunotherapy target and a diagnosis and prognosis prediction biomarker, which comprises the following steps: (1) Determining the detection of cancer cells in the patient or blood in the patient comprises: elevated EGFR activation levels, wnt pathway activation levels, activated β -catenin levels, long non-coding RNA LINC00973 expression levels, LNC1574203 expression levels, protein and mRNA expression levels of CD55, protein and mRNA expression levels of CD59, protein and mRNA expression levels of CD73, and; (2) Determining the detection of cancer cells in the patient or blood in the patient comprises: reduced C4d expression level, C3a expression level, C3b expression level, C5a expression level, C5b-9 expression level compared to a reference level; and (3) blocking one or more states of EGFR activation, wnt pathway activation, activated β -catenin, long non-coding RNA LINC00973 expression, LNC1574203 expression, CD55 protein and mRNA expression, CD59 protein and mRNA expression, CD73 protein and mRNA expression with an inhibition method; and (4) promoting C4d expression, C3a expression, C3b expression, C5a expression, C5b-9 expression by an activation method; and/or (5) predicting the favorable response of the patient to the method of treatment; the reference level is a level in blood from a non-cancerous or early cancer cell or a healthy individual, or from a patient with early cancer.

In alternative embodiments, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, cancer of the central or peripheral nervous system tissue, cancer of the endocrine or neuroendocrine system or hematopoietic system, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, renal cancer, biliary system cancer, pheochromocytoma, islet cell cancer, li-furo Mei Niliu, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

In alternative embodiments, the method of determination comprises performing ELISA, immunoassay, radioimmunoassay, immunohistochemistry, immunoradiometric assay, fluoroimmunoassay, gel electrophoresis, immunoblot analysis, in situ hybridization, flow cytometry, or microscopic assay using a specific antibody.

In alternative embodiments, the method of inhibition comprises the use of an EGFR inhibitor, wnt inhibitor, β -catenin inhibitor, LINC00973 inhibitor, LNC1574203 inhibitor, protein and mRNA inhibitor of CD55, protein and mRNA inhibitor of CD59, protein and mRNA inhibitor of CD73, or any other method of inhibiting EGFR activation, wnt pathway activation, activated β -catenin, long non-coding RNA LINC00973 expression, LNC1574203 expression, protein and mRNA expression of CD55, protein and mRNA expression of CD59, protein and mRNA expression of CD 73.

In alternative embodiments, the method of activation comprises the use of a C4d agonist, a C3a agonist, a C3b agonist, a C5a agonist, a C5b-9 agonist, or any other method of activating C4d expression, C3a expression, C3b expression, C5a expression, C5b-9 expression.

In alternative embodiments, the EGFR inhibitor, wnt inhibitor, β -catenin inhibitor, LINC00973 inhibitor, LNC1574203 inhibitor, protein and mRNA inhibitor of CD55, protein and mRNA inhibitor of CD59, protein and mRNA inhibitor of CD73, including small molecule inhibitors, polypeptides, complementary inhibitory oligonucleotides or neutralizing antibodies to EGFR, wnt, β -catenin, LINC00973, LNC1574203, protein and mRNA of CD55, protein and mRNA of CD59, protein and mRNA of CD 73.

In alternative embodiments, the C4d agonist, C3a agonist, C3b agonist, C5a agonist, C5b-9 agonist, includes small molecule agonists to C4d, C3a, C3b, C5a, C5 b-9.

In alternative embodiments, the beneficial response includes a decrease in tumor size or burden, a retardation of tumor growth, a decrease in tumor-associated pain, a decrease in cancer-associated pathological conditions, a decrease in cancer-associated symptoms, an increase in progression-free phase of cancer, an increase in disease-free interval, an induced remission, a decrease in metastasis, an increase in patient survival or an increase in sensitivity of the tumor to an anti-cancer treatment, in particular an immunotherapy.

The invention also provides an application of the cancer immunotherapy target and the diagnosis and prognosis prediction biomarker, which comprises the following steps: (1) Determining whether detecting cancer cells in the patient or blood in the patient comprises: elevated EGFR activation levels, wnt pathway activation levels, activated β -catenin levels, long non-coding RNA LINC00973 expression levels, LNC1574203 expression levels, protein and mRNA expression levels of CD55, protein and mRNA expression levels of CD59, protein and mRNA expression levels of CD73, and various combinations of these six elevations as compared to a reference level; (2) Determining whether detecting cancer cells in the patient or blood in the patient comprises: reduced C4d expression levels, C3a expression levels, C3b expression levels, C5a expression levels, C5b-9 expression levels, and various combined levels of these five reductions as compared to a reference level; (3) Predicting that the patient has an aggressive cancer if the cancer cells or the patient's blood contain an elevated level of any of (1); (4) Predicting that an aggressive cancer that the patient has is in a stage of progression if the cancer cells or the patient's blood contain an elevated level of any of (1); (5) Predicting that the patient has a poor prognosis if the cancer cells or the patient's blood contain an elevated level of any of (1); (6) Predicting that the patient has an aggressive cancer if the cancer cells or the patient's blood contain a reduced level of any of (2); (7) Predicting that the patient has invasive cancer in the stage of progression if the cancer cells or the patient's blood contain a reduced level of any of (2); (8) Predicting that the patient has a poor prognosis if the cancer cells or the patient's blood contain a reduced level of any of (2); the reference level is a level in blood from a non-cancerous or early cancer cell or a healthy individual, or from a patient with early cancer.

In alternative embodiments, if the patient is tested for invasive cancer, inhibitor anticancer therapy is performed by blocking one or more of EGFR activation, wnt pathway activation, activated β -catenin, long non-coding RNA LINC00973 expression, LNC1574203 expression, protein and mRNA expression of CD55, protein and mRNA expression of CD59, protein and mRNA expression of CD 73.

In alternative embodiments, if the patient is tested for invasive cancer, the activator anti-cancer treatment is performed by activating one or more of C4d expression, C3a expression, C3b expression, C5a expression, C5b-9 expression.

The invention is explained in detail below in connection with specific experiments. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to product specifications; the reagents and materials, unless otherwise specified, are commercially available.

The materials used in the experiments of the invention are as follows:

anti-CD 55 (E7G 2U) (# 31759), anti-CD 46 (D6N 7H) (# 13241), anti-Dicer (D38E 7) (# 5362), anti- β -catenin (D10 A8) (# 8480), anti-EGFR (L858R mutant specificity) (43B 2) (# 3197) and anti-EGFR (D38B 1) (# 4267) antibodies (for immunoblotting); an anti-non-phospho- β -catenin (Ser 33/37/Thr 41) (D13A 1) (active- β -catenin) (# 8814) antibody (for immunohistochemistry); the aSignalStainDAB substrate kit (# 8059), anti-rabhiIgG (HRP-conjugated) antibody (# 7074), anti-mouseigG (HRP-conjugated) antibody (# 7076), anti-i-c-Jun (60A 8) (# 9165), anti-human Frizzled6 (# 5158) (for immunoblotting), anti-CD 8 antibody (# 85336) (for immunohistochemistry), anti-SNIP/p 140Cap (# 3757) antibody (for immunoblotting), and anti-human EGFR phosphorylating Y1173 antibody (# 7) (for immunohistochemistry) were purchased from cell 4401 technology (Danvers, mass.). Anti-tubulin (DM 1A) (T9026) antibodies (for immunoblotting), liberase (5401054001), lepirudin (recombinant form of hirudin, H0393) and Lipopolysaccharide (LPS) from E.coli O127: B8 (L4516) were purchased from Sigma (St. Louis, mitsu.). BAMBANKER (4987481582811) is available from wako chemicals (Osaka, japan). Human cord blood CD34+ hematopoietic stem cells (CBP 3401C) were obtained from STEMEXPRESS (Folsom, calif.). XAV939 (HY-15147) is purchased from MCE (MonmouthJunction, NJ). Anti-mouse C3b antibody (HM 1065), anti-mouse C4d (HP 8033) (for immunohistochemistry) were purchased from hycult biotech (Wayne, PA). Anti-mouse C5b-9 (bs-2673R) (for immunohistochemistry) was purchased from Bioss (Woburn, mass.). Immobilon protein chemiluminescent HRP substrate (WBKLS 0100), immobilon-PPVDF membrane (IPVH 00010), chromatin immunoprecipitation kit (17-10085), RNA immunoprecipitation kit (17-704), anti-Ago 2 antibody (03-110) (for immunoprecipitation) and anti-TCF-4 antibody (clone 6H 5-3) (for electrophoretic mobility shift assay) were obtained from Millipore (Billerica, mass.). Anti-human C5b-9 antibody (aE 11) (LS-C663067) (for flow cytometry) WAs purchased from Life span Biosciences (Seattle, WA). anti-CD 59 (MEM-43/5) (ab 9183) antibody (for immunoblotting), anti-CD 80 antibody (ab 134120) (for immunohistochemistry), anti-CD 86 antibody (ab 220188) (for immunohistochemistry), anti-CD 64 antibody (ab 140779) (for immunohistochemistry), anti-human C3B antibody (ab 11871), anti-human C4d antibody (ab 183311), anti-human C5B-9 antibody (ab 55811) (for immunohistochemistry and immunofluorescence), human complement factor HELISA kit (ab 252359), human complement B factor ELISA kit (ab 137973), human complement C3ELISA kit (ab 108823), human complement C5 kit (ab 125963), mouse complement C3ELISA kit (ab 263884) and mouse complement C5 kit (ab 264609) were purchased from Abcam (sisal ELISA). PE-Cy7 anti-mouse CD8a antibody (53-6.7) (561097) (for flow cytometry), fixative/permeabilization solution kit (554714) and PE-Cy7 anti-human CD8 antibody (HIT 8 a) (566858) (for flow cytometry) were purchased from BDbiosciences (SanJose, calif.). APC anti-human/mouse granzyme B antibody (QA 16a 02) (372204), PE anti-mouse perforin antibody (S16009A) (154306) (for flow cytometry), PE anti-human perforin antibody (B-D48) (353303) (for flow cytometry), zembiovifloxablialinit (423113), alexaFluor647 anti-human CD11B antibody (M1/70) (101218), trusainfcx anti-mouse CD16/32 monoclonal antibody (93) (101319), brilliant villet 510 anti-mouse CD11B antibody (M1/70) (101245), PE anti-mouse CD45 antibody (30-F11) (103106), PE/Cyanine7 anti-mouse F4/80 antibody (BM 8) (123114) (for flow cytometry), anti-human CD3 (t 3 a) (300302) (for co-stimulation), anti-human CD28 (CD 28.2) (302902) (for co-stimulation) from bio-gene (dio, D). Nano-Glo luciferase report analysis System (N1610) and luciferase report analysis System (E1910) were purchased from Promega (Madison, wis.). ABCA protein assay kit (23225), EGF recombinant human protein solution (PHG 0311L), revertFirststrandcDNA synthesis kit (K1622), powerUpSYBRgreenMastermix (A25742), NE-PER nuclear and cytoplasmic extraction reagent (78833), anti-human/mouse complement C3b monoclonal antibody (6C 9) (MA 1-70053) (for flow cytometry), enzyme-free cell dissociation buffer (13150016), iscove Modified Dulbecco's Medium (IMDM) (12440053), trypLE expression enzyme (12605010), pHrodod, SE (P36600), 4',6-diamidino-2-phenylindole (DAPI) (P36931), ACK lysis buffer (A1049201), ultraCompends (01-2222-41), cytivaFicoll-Paque PLUS medium (45-001-750)), fixagel air dying cell staining kit (L34965) and Dynasty CD14 (Flowersham) available from Flowersham (FleerD 82). The anti-mouse C5b-9 antibody (2A 1) (sc-66190) (for flow cytometry) was obtained from SantaCruzBiotechnology (SantaCruz, calif.). Human M-CSF (300-25), human IFN-gamma (300-02) and human IL-4 (200-04) were purchased from PeproTech (Israel). Recombinant human/mouse Wnt-5a protein (645-WN-010), human IFN-gamma quantikineELISA kit (SIF 50), mouse IFN-gamma quantikineELISA kit (SMIF 00), human IL-1 beta quantikineELISA kit (SLB 50), mouse IL-1 beta quantikineELISA kit (SMLB 00C), human IL-6quantikineELISA kit (S6050), mouse IL-6quantikineELISA kit (SM 6000B), human IL-17quantikineELISA kit (S1700), mouse IL-17quantikineELISA kit (SM 1700), human TNF-alpha quantikineELISA kit (STA 00D), mouse TNF-alpha quantikineELISA kit (SM00B), mouse IL-1 beta quantikineELISA kit (DY 0) and human complement C5aELISA kit (DY 2037) are purchased from R & DSS. Mouse complement C3aELISA kit (NBP 2-70037) and human complement C3aELISA kit (NBP 2-66755) and recombinant human Wnt-7b protein (H00007477-P01) were purchased from Novus biologicals (Centennial, CO). RNeasy Mini kit (74104), QIAquick gel extraction kit (28706), QIAamp DNAMini kit (51306) and Plasmid midi kit (12145) are purchased from QIAGEN (Germanown, MD). Anti-human PD-1 antibody (J116) (BE 0188) (for blocking), anti-mouse PD-1 antibody (29 F.1A12) (BE 0273) (for blocking), anti-human CD8 antibody (OKT-8) (BE 0004-2) (for depletion) and IgG (MOPC-21) (BE 0083) (for isotype control) were obtained from BioXcell (Lebanon, NH).

DNA construction and mutagenesis

Polymerase Chain Reaction (PCR) -amplified human WTCCTNNB 1, EGFR L858R, CD, CD59, TCF-4, MLINC00973 and LINC00973 were cloned into pcDNA3.1/hygro (+) -Flag, pCDH-CMV-MCS-EF1-Puro-SFB, pCDH-CMV-MCS-Flag-EF 1-puromycin, pCDH-CMV-MCS-Flag-EF1-Geneticin (G-418), pCDH-CMV-MCS-Flag-EF 1-blasticidin, pCDH-CMV-MCS-Flag-EF1-Zeocin or pET32a vectors.

EGFRL858R and short hairpin RNA (shRNA) resistant beta-catenin (rbeta-catenin) were constructed. pGIPZshRNA is constructed by ligating an oligonucleotide targeting human beta-catenin, LINC00973, CD55 or CD59 into an XhoI/MluI digested pGIPZ vector. The following pGIPZshRNA target sequences were used: control shRNA oligonucleotides, 5'-GCTTCTAACACCGGGAGGTCTT-3'; human beta-cateninsshRNA oligonucleotides, 5'-GCATAACCTTTCCCATCATCG-3'; mouse β -cateninsshrna oligonucleotide, 5'-CGTGAAATTCTTGGCTATTAC-3'; LINC00973shRNA oligonucleotide, 5'-GGCACGACTTCTGGTCTATT-3'; MLINC00973shRNA oligonucleotide, 5'-GCGTAACTTCTGGTCATTTAG-3'; human CD55shRNA oligonucleotides, 5'-TGGTCCACAGCAGTCGAATTT-3'; mouse CD55shRNA oligonucleotide, 5'-GTTAGTCTAGCTTATGATTAA-3'; human CD59shRNA oligonucleotides, 5'-GATGCGTGTCTCATTACCAAA-3'; mouse CD59shRNA oligonucleotides, 5'-CGGTGGTTTCTTCATGCAATA-3'; mouse C3shRNA oligonucleotides, 5'-CCATCAAGATTCCAGCCAGTA-3'; and mouse C5shRNA oligonucleotides, 5'-GCACGACTCCTGGTCTATTAC-3'.

Cell lines and cell culture conditions

H1395, H322M and 293T cells were obtained from ATCC. LA795 and MC38 cells used in the experiments were identified in the national academy of medical science and the beijing institute of synergetics medical science using a short tandem repeat spectrum. Cells were maintained in complete medium containing Dulbecco's Modified Eagle's Medium (DMEM), 10% Fetal Bovine Serum (FBS), 1,000U/ml penicillin and 100. Mu.g/ml streptomycin. Cells were serum starved for 16 hours prior to EGF treatment. EGF at a final concentration of 100ng/ml was used for cell stimulation. The cell lines used in this study were not found in the common false recognition cell line database maintained by the international cell line authentication committee and ncb ihasample. Cell lines were identified by short tandem repeat analysis and mycoplasma contamination was routinely detected. Cells were incubated at 4X 10 for 18 hours prior to transfection ⁵ Is inoculated in 60mm dishes or 1X 10 per well in 6 well plates ⁵ Is a density inoculation of (3). Transfection procedures were performed as described previously.

Immunoblot analysis

Proteins were extracted from the cultured cells by modified buffers and immunoblotted with the corresponding antibodies as described previously.

5 'and 3' Rapid Amplification of CDNA Ends (RACE)

According to the manufacturer's instructions, we have used the SMARTERRACCDNA amplification kit (Clontech, paloAlto, calif.) to determine the transcription initiation and termination sites of LINC00973 and MLINC00973 using 5' -RACE and 3' -RACE assays. Gene specific primers used for PCR in RACE analysis were as follows: LINC00973, 5'-CCATGGACAAAGCCAAGGATTCAGTAAAG-3' (reverse) (5 '-RACE) and 5'-GAAGGGGAGGAATTACTTATCCTTTGGC-3'(front) (3' -RACE); MLINC00973, 5'-TGCTACTCAAAATCTCTGCTTGGAAAGA-3' (reverse) (5 '-RACE) and 5'-ATTTGTTTGACATTGAATCTGAGCCTTG-3'(front) (3' -RACE).

Fluorescence in situ hybridization in cells

The Stellaris In Situ Hybridization (ISH) probe was designed against human LINC009734.2 using a stellarisrnaishpprobe designer (BiosearchTechnologies, inc., petaluma, CA, USA) at http:// www.biosearchtech.com/stellarisdesigner (StellarisProbeDesignerversion). Human H1395 cells were slightly modified by hybridization with the StellarisRNAISH probe to LINC00973 labeled with Quasar570 according to the manufacturer's instructions provided on line on http:// www.biosearchtech.com. Briefly, cells were incubated with 20nMQuasar 570-labeled probes in hybridization buffer and hybridized overnight at 42 ℃. Cells were washed in hybridization buffer at 42℃and briefly in 0.1 XSSC. Cells hybridized with the Quasar 570-labeled probes were initially incubated in 3% hydrogen peroxide to block potential endogenous peroxidases. Probes were detected using peroxidase conjugated anti-fluorescein-Ab (Roche applied sciences, mannheim, germany) at a dilution of 1:400, followed by Cy 3-labeled TSA substrate for 10 min (Perkinelmer, waltham, mass., USA). Cell samples were mounted using Prolonggold anti-fade mounting agent containing DAPI for nuclear staining (ThermoFisher scientific, waltham, mass., USA).

In situ hybridization

Formalin-fixed (4% paraformaldehyde; sigma-Aldrich, st. Louis, mo.) paraffin-embedded (FFPE) sections of human NSCLC samples were probed with in situ hybridization probes (Exiqon, vedbaek, denmark) using Digoxin (DIG) -labeled Locked Nucleic Acid (LNA) as previously described. The fixed and permeabilized cells were prehybridized in hybridization buffer, then hybridized with 25 nLNA probe for 1 hour at 55℃for LINC00973:5'-AATGCGAAGGAGTAACACAGCT-3' (predicted rnatm=84℃), MLINC00973:5'-AGTGATTTATTTGCATGCTAAT-3' (predicted rnatm=81℃), miR-216b:5'-TCACATTTGCCTGCAGAGATT-3' (predicted rnatm=85℃), miR-150:5'-CACTGGTACAAGGGTTGGGAGA-3' (predicted rnatm=83℃) and using the disorder probe 5'-TGTAACACGTCTATACGCCCA-3' (predicted rnatm=87℃) as a negative control. DIG-labeled probes were detected by peroxidase conjugated anti-DIG-Ab (Rocheapplied sciences, mannheim, germany) at a dilution of 1:400, followed by DAB substrate addition for 10 min (CellSignaling technology, danvers, mass., USA).

RNA immunoprecipitation

pMS2-GFP (Addgene) was introduced into H1395, H322M or LA795 cells with pcDNA3.1-MS12, pcDNA3.1-MS12-LINC00973, pcDNA3.1-MS12-MLINC00973, pcDNA3.1-MS12-LINC00973-mut (miR-216 b), pcDNA3.1-co-transfected MS12-LINC00973-mut (miR-150), pcDNA3.1-MS12-MLINC00973-mut (mmu-miR-216 b) or pcDNA3.1-MS12-MLINC00973-mut (mmu-miR-150). 48 hours after transfection, according to the manufacturer's instructions. RNA fractions isolated by RIP were quantified by a NanoDropND1000 instrument (Thermo-Fisher scientific, waltham, mass.).

For anti-Ago 2RIP, H1395 and H322M cells were transfected with miR-216b, miR-150 or microRNA negative controls. RIP experiments were performed using cells with anti-Ago 2 antibodies (Millipore) 48 hours after transfection.

Subcellular separation

The nuclear extract kit of ActiveMotif (Carlsbad, CA) separates the nucleus from the cytosol.

Chemometric analysis

The copy numbers of LINC00973, miR-216b and miR-150 in H1395 cells treated with EGF for 0, 2, 4, 6 and 8 hours were determined. The increased number of LINCs 00973 and the decreased number of miR-216b and miR-150 were calculated between 0 and 2 hours, 2 and 4 hours, 4 and 6 hours and between 6 and 8 hours. The stoichiometry of binding of LINC00973 to miR-216b and miR-150 was calculated based on the average of the increase or decrease in the number of LINC00973, miR-216b and miR-150 over 8 hours.

Chromatin immunoprecipitation (ChIP) assay

ChIP was performed using upstateBiotechnology kit. Chromatin prepared from cells (in 15cm dishes) was used to determine total DNA input and samples were incubated overnight with specific antibodies or normal rabbit or mouse immunoglobulin G. The human LINC00973 promoter specific primers used for PCR were as follows: TBE1,5'-TATTGAGAATCACAATTATG-3' (forward) and 5'-TGAACCCCAACAGGAAAATA-3' (reverse); TBE2,5'-ATTTCAAATTATTGAGGGACT-3' (forward) and 5'-CTACTTAAGAAGCATACAGAA-3' (reverse); and TBE3,5'-GCAGGGGAAGGGTTATGAACA-3' (forward) and 5'-TTCCTCGGATGGTTTCCCACA-3' (reverse). The mouse MLINC00973 promoter specific primers used in PCR were as follows: TBE4,5'-TAGGTTTGGTCTTCTCATTGT-3' (forward) and 5'-AGAATACAAGAGATGGAAGAG-3' (reverse); and TBE5,5'-GGATTACCTTCTTGTTTTTTC-3' (forward) and 5'-AGAAACCAAAGTATTCCACGA-3' (reverse).

RNA pulldown

LINC00973, LINC00973-mut (miR-216 b), LINC00973-mut (150) or lncRNA-225205 are transcribed in vitro from pSPT19-LINC00973, pSPT19-LINC00973-mut (miR-216 b), pSPT19-LINC00973-mut (150) and pSPT19-225205 vectors, respectively. These lncRNA were biotin-labeled with biotin RNA labeling mix (Roche) and T7RNA polymerase (Roche), treated with no RNaseDNaseI (Roche) and purified using rneasy mini kit (Qiagen, valencia, CA). Whole cell lysates of 1mgH1395 and H322M cells were incubated with 3 μg of purified biotinylated transcript for 1 hour at 25 ℃; the complexes were isolated using streptavidin sepharose beads (Invitrogen). Precipitated RNA was detected by qRT-PCR analysis.

Quantitative real-time PCR

Total RNA was extracted from cell and tissue samples using TRIzol reagent according to the manufacturer's instructions (Invitrogen). An equal amount of RNA sample was used for cDNA synthesis by TaqMan reverse transcription kit (applied biosystems). Quantitative PCR analysis was performed using 7500 real-time PCR systems (applied biosystems) and SYBRPremix ExTaq kit (TakaraBio), with beta-actin or U6RNA (for miRNA) as internal controls.

The following primers were used for quantitative PCR: human CDs 55, 5'-CCAGCACCACCACAAATTGAC-3' (forward) and 5'-TCTCCAATCATGGTGAATCCT-3' (reverse); human CD59,5'-AGGCATGCCAAATGTTCCATA-3' (forward) and 5'-GTTTTCATGCCCTGCTATCTG-3' (reverse); human LINC00973,5'-ATGAAGCCACAGAGATTTGCT-3' (forward) and 5'-AGCCTTCAATTCCAGGGAAAG-3' (reverse); humans CFH,5'-AATTCATCCAGGTCTTCACAA-3' (forward) and 5'-ACTCCATTTTCCCATGTAGC-3' (reverse); humans CFB,5'-CGAGCTTTGAGGCTTCC-3' (forward) and 5'-TGATGTAGACCTCCTTCCG-3' (reverse); humans C3,5'-GCTGAAGGAAAAGGCCAAG-3' (forward) and 5'-CGGTGCTGGTTTTATGGTG-3' (reverse); humans C4,5'-CTCCATCTCAAAGGCAAGC-3' (forward) and 5'-AACACCGAGCAGGTCCA-3' (reverse); humans C5,5'-ATCAGGGCACAAAGTCCTC-3' (forward) and 5'-CTTCTGGCACCACTCGTAA-3' (reverse); human CD55 introns 2, 5'-AGTTCTGGGAATGGAATGTATCTTA-3' (forward) and 5'-AGTGTTAGGAAGAAAAACTCTTAAT-3' (reverse); human CD55 intron 3,5'-TCTGGTGTTTGGGGGAAATAGTATC-3' (front) and 5'-TTAGGTAACCTCAAAACTAATTAAAT-3' (reverse); human CD55 intron 8,5'-AAGGCAATTACTGCCCTGAAACTGA-3' (front) and 5'-ATGTAAGCCACAAAACCAATGCTGA-3' (reverse); human CD59 intron 1,5'-GGTGTCCTAGCCGAACGCTGGCTTC-3' (forward) and 5'-CCGCTAGAGCTTCCCTTGAGACGAA-3' (reverse); human CD59 intron 2,5'-GAAGTCTGACACAGGTCTCACAGGG-3' (forward) and 5'-CTAAGAATGGTCCTCAACTGACACT-3' (reverse); human CD59 intron 5,5'-ACTGTAATCCTCATTAGGCTTGCAT-3' (forward) and 5'-CCTGAGTCGGTTACTTAACCATAAT-3' (reverse); human FZD1,5'-ATCGAAGCCAACTCACAGTATTT-3' (forward) and 5'-CACGTTGTTAAGCCCCACG-3' (reverse); human FZD2,5'-GTGCCATCCTATCTCAGCTACA-3' (forward) and 5'-CTGCATGTCTACCAAGTACGTG-3' (reverse); human FZD3,5'-GTTCATGGGGCATATAGGTGG-3' (forward) and 5'-GCTGCTGTCTGTTGGTCATAA-3' (reverse); human FZD4,5'-CCTCGGCTACAACGTGACC-3' (forward) and 5'-TGCACATTGGCACATAAACAGA-3' (reverse); human FZD5, 5'-CATGCCCAACCAGTTCAACC-3' (forward) and 5'-CGGCGAGCATTGGATCTCC-3' (reverse); human FZD6,5'-ATGGCCTACAACATGACGTTT-3' (forward) and 5'-GTTTACGACAAGGTGGAACCA-3' (reverse); human FZD7,5'-GTGCCAACGGCCTGATGTA-3' (forward) and 5'-AGGTGAGAACGGTAAAGAGCG-3' (reverse); human FZD8,5'-ATCGGCTACAACTACACCTACA-3' (forward) and 5'-GTACATGCTGCACAGGAAGAA-3' (reverse); human FZD9,5'-TGCGAGAACCCCGAGAAGT-3' (forward) and 5'-GGGACCAGAACACCTCGAC-3' (reverse); human FZD10,5'-GCTCATGGTGCGTATCGGG-3' (forward) and 5'-GAGGCGTTCGTAAAAGTAGCA-3' (reverse); human LRP5,5'-TGGCCCGAAACCTCTACTG-3' (forward) and 5'-GCACACTCGATTTTAGGGTTCT-3' (reverse); human LRP6,5'-ACGATTGTAGTTGGAGGCTTG-3' (forward) and 5'-ATGGCTTCTTCGCTGACATCA-3' (reverse); humans ACTB,5'-ATGGATGACGATATCGCTGCGC-3' (forward) and 5'-GCAGCACAGGGTGCTCCTCA-3' (reverse); human MIR216B,5'-CGGGCAAATCTCTGCAGGCA-3' (forward) and 5'-CAGCCACAAAAGAGCACAAT-3' (reverse); human MIR150,5'-CGGGCTCTCCCAACCCTTGT-3' (forward) and 5'-CAGCCACAAAAGAGCACAAT-3' (reverse); human RNU6,5'-CTCGCTTCGGCAGCACA-3' (forward) and 5'-AACGCTTCACGAATTTGCGT-3' (reverse); mouse CD55, 5'-ATTGTCCAGAGCCACCAAAAAA-3' (forward) and 5'-TGTCCTACATCAGACTTGCTC-3' (reverse); mouse CD59, 5'-TTCAGATGCTGCCAGTTTAAC-3' (forward) and 5'-AAATGGCCACCAGAACCGAGG-3' (reverse); mouse MLINC00973,5'-ATGATTGCTCATGGGTCCTGT-3' (forward) and 5'-GAGGCAGTGACACAGCTGGGA-3' (reverse); mice C3,5'-TCCAACAAGAACACCCTCA-3' (forward) and 5'-GGCTGGATAAGTCCCACA-3' (reverse); mice C5,5'-ACAGCCCAATCAAGTTCCT-3' (forward) and 5'-TTCAAGTCGTCACCCAGAG-3' (reverse); and mice ACTB,5'-GCTGTGCTGTCCCTGTATGCC-3' (forward) and 5'-GGAGAGCATAGCCCTCGTAGA-3' (reverse).

Lentiviral formulations

We transfected 293T cells in 150mm dishes with 6. Mu.g of lentiviral expression vector for the specific gene or shRNA, 6. Mu.g of pLP1, 6. Mu.g of pLP2 and 6. Mu.g of pLPVSV-G (Invitrogen), a plasmid vesicular stomatitis virus envelope encoding the G protein. The next day the medium was changed. Lentiviral-containing media were harvested 48 hours and 72 hours post-transfection. The virus particles were concentrated and purified by ultra-high speed centrifugation (25,000 g, 2 hours at 4 ℃). Lentiviruses (1X 10) in the presence of 6. Mu.g/ml polybrene (Sigma) ⁶ ) And infecting the cells.

Purification of recombinant proteins

GST-TCF4 is expressed in bacteria and purified as described previously.

Genome editing

As previously described, genomic mutations are introduced into cells using the CRISPR/Cas9 system (84). Single guide RNA (sgRNA) is intended to target sites adjacent to human or mouse LEF/TCF binding element mutation sites or genomic regions mutated by human miR-216b and miR-150 binding elements (miR-216 b/150-BE) using CRISPR design tools (http:// CRISPR. Mit. Edu /). The annealed guide RNA oligonucleotides were inserted into PX458 vectors (Addgene, cambridge, MA) digested with BbsI restriction enzyme (85). Cells were seeded at 60% confluence and co-transfected with sgRNA (0.5 μg), single stranded donor oligonucleotide (ssODN) used as template for introducing mutations (20 pmol) and wild type hscas 9 labeled with GFP. 24 hours after transfection, cells were digested with trypsin, diluted to obtain single cells and seeded into 96-well plates. Genomic DNA was extracted from GFP positive cells. Genotyping was performed by sequencing the PCR products spanning the mutated region. The sgRNA target sequence of TBE1 is 5'-TTATAGAATATAATCAAAG-3'; the single stranded donor oligonucleotide (ssODN) sequence of TBE1 is 5'-TACTAACAAAGTAAAATTATTTATTGAGAATCACAATTATGTACCAGATATTTAAAATAATATTAGTATGACTAGCCCGATAATAGAATATTAGCCAAAGTTCAGAAAGGCTAAGTAAATTATCCAAGTTCAACCATAAATATTATTTTCCTGTTGGGGTTCAAGCGCAAGTCTACCTGACTCTAAAATGCAAACTT-3'. The sgRNA target sequence of TBE2 is 5'-ATTCTCCATCAAAGTTCCTC-3'; the ssODN sequence of TBE2 is 5'-GAAATAGATAAAACATTTAAAATGTCTAGAAAGATTTCAGTTTATTTCAAATTATTGAGGGACTAATGGGCTATTCTCCGCCAAAGTTCCACACGGACATACTGCTCTAATTATATGTATTGATTATTCTATTTTTCTGTATGCTTCTTAAGTAGTAATGATTTTTTCCAGAATATGCCT-3'. The sgRNA target sequence of TBE3 is 5'-CTGAAGATCAAAGAATGTCA-3'; the ssODN sequence of TBE3 is 5'-GGAAGGGTTATGAACAGTTGTAGTTCTCTTTTGCTCTCAGCAAACAGGGACTTCACACATTTTAGTTAATCTGAAGGCCAAAGAATGTGATCGGAGAAAATATGTTAAAAGCAAAACAATCCTTTTGAAATTGTGGGAAACCATCCGAGGAAAGACAAACAT-3'. The sgRNA target sequence of TBE4 is 5'-TTTTCTTTGATTGTTGTGCC-3'; the ssODN sequence of TBE4 is 5'-ACTCCTATTATCCGTAGGTTTGGTCTTCTCATTGTGTCCTGGATTTCCTGGATATTTTGAGTTAGGATCTTTTTGCATTTTCGATATTCTTTGGCTGTTGTGCCGATGTTCTCTATGGAATCTTCTGCACCTGAGATTCTCTCTTCCATCTCTTGTATTCTGTTGCTGATGCTCAAATCTATGGTTCCAGATTGTTTCC-3'. The sgRNA target sequence of TBE5 is 5'-CCTTTGAAGGGCTGGATTCG-3'; the ssODN sequence of TBE5 was 5'-GTCTTCTTTTAGGTTTGTTGAGGGATTACCTTCTTGTTTTTTCTAGGGCATTGTTCCCGTTCTTGTATTGGTTTTTTTCTGTTATTAACCTTTGGGGGCTGGATTGGTAGAGAGATAATGTGTGAATTTGGTTTTGTCGTGGAATACTTTGGTTTCTCCATCTATGGTAATTGAGAGTTTGGCTGGGTATAGTAGCC-3'. The sgRNA target sequence of miR-216b/150-BE is 5'-GAAAGACACTAGAAGCTCTT-3'; the ssODN sequence of miR-216b/150-BE is 5'-CCAGAAAGAGCTGTGTGTATATTTTAGAAAGACACTAGAAGCTCCCAAAGACATGTGGACAGTTGTGGCTGCTCCTGAGCTGACACTAACTGCTCATGACTCCTCTGCAAAGAGAGTAGGTGGTTTCCTAGAGGAAGAAGTTTGGGTAATGAAGCCACAAGAGCCCGCTGATACATTTGCTAGGCACGACTTCTGGTCAT-3'.

Genotyping was performed by sequencing the amplified PCR products using the following primers spanning the mutated region. For TBE1,5'-GCTTCTTATGTTAAAATTAGTG-3' (forward); 5'-ATAGCTTTCTAAATGACCAGA-3' (reverse); for TBE2,5'-TCCAAATGCCATCCCACCTTT-3' (forward); 5'-TGCGCTTGAACCCCAACAGGA-3' (reverse); for TBE3,5'-GACTGGGATACAAGTTCAAGA-3' (front); 5'-CCTAGGTCAGAGTTGACTGCA-3' (reverse); for TBE4,5'-GTCTCTGGTGAAAAATCTGGT-3' (forward); 5'-GTCTCTGGTGAAAAATCTGGT-3' (reverse); for TBE5,5'-GGCTACTCCAGCTTGTTTCTT-3' (forward); 5'-GACCAGCAAACATCTTCAACA-3' (reverse); for miR-216b/150-BE,5'-CTTGCTCTGAATCCTATCATAGCTT-3' (forward direction); 5'-AGGATATGTAGAGGAATCATGTGGG-3' (reverse).

Luciferase reporter detection

pMIR-REPORT, pMIR-REPORT-LINC00973, pMIR-REPORT-MLINC00973, pMIR-REPORT-LINC00973-mut (miR-216 b), pMIR-REPORT-LINC00973-mut (miR-150), pMIR-REPORT-MLINC00973-mut (mmu-miR-216 b) or pMIR-REPORT-MLINC00973-mut (mmu-miR-150) and miR-216b mimics, miR-150 mimics, mmi-miR-216 b mimics, mmi-miR-150 mimics, miR-216b inhibitors, miR-150 inhibitors, mmi-miR-216 b inhibitors, mmi-miR-150 inhibitors or miR-Control are transferred into H1395, H322M or LA795 cells by lipofectamine. The relative luciferase activity was normalized to Renilla luciferase activity 48 hours after transfection.

The pGL4.10 or pGL4.10-LINC00973 promoter and pcDNA3.1 (+) -beta-catenin, pcDNA3.1 (+) -TCF4, pcDNA3.1 (+) -TCF-4ΔN or pcDNA3.1 (+) enter H1395 or H322M cells by lipofectamine mediated gene transfer. The relative levels of luciferase activity were normalized to the Renilla luciferase activity level and control 48 hours after transfection.

TUNEL detection

Mouse tumor tissue was cut to 5 μm thickness. Apoptotic cells were counted using the deaden colorimetric TUNEL system (Promega) according to the manufacturer's instructions.

Complement deposition assay

Tumor cells were incubated with DMEM medium (10% FBS) with or without supplementation of 25% v/v human serum (human complement system source) and 25 μg/ml pirudin (complete preservation of complement activity in human serum (86)) for 3 hours at 37 ℃. After washing, expression of the tumor cell surfaces C3b and C5b-9 was determined by flow cytometry using the indicated antibodies.

Anaphylatoxin Release test

Tumor cells were incubated with DMEM medium (10% fbs) with or without 25% v/v human serum and 25 μg/ml pirudin for 3 hours at 37 ℃. The release of the anaphylatoxins (C3 a and C5 a) from the supernatant was determined by ELISA according to the manufacturer's instructions.

Co-culture of tumor cells with PBMC

Peripheral Blood Mononuclear Cells (PBMCs) were obtained from healthy donors after informed consent and isolated by Ficoll-paque plus density gradient. In plates coated with anti-CD 3 (2. Mu.g/mL) and anti-CD 28 (4. Mu.g/mL) monoclonal antibodies and supplemented with RPMI1640 medium supplemented with 2mM L-glutamine and 10% (v/v) human serum, PBMC were treated with M-CSF (80 ng/mL) for 4 days, then with LPS (100 ng/mL) and IFN-gamma (20 ng/mL) for 48h, then co-cultured with tumor cells. Tumor cells were incubated at 37℃for 3h in DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25. Mu.g/mL piclidin, co-cultured with human PBMCs (PBMC: tumor cell ratio 3:1) in plates coated with anti-CD 3 (2. Mu.g/mL) and anti-CD 28 (4. Mu.g/mL) monoclonal antibodies for 30 h in 100ng/mL LPS medium (87).

Cytokine expression analysis

Tumor cells were co-cultured with human PBMCs. The supernatant was then probed for cytokine protein levels by ELISA.

Complement activation mediated immune cell attack assay

Complement activation mediated immune cell attack (CARIA) was analyzed as previously described (62). Briefly, we constructed pCDH-CMV-nanoluciferase (NLuc) -EF 1-puromycin lentiviral vector and stably expressed NLuc in tumor cells. The NLuc activity reflecting CARIA (specific cleavage) or no (basal NLuc activity) 0.25% Triton X-100 treatment was determined using a Nano-Glo luciferase reporter assay system (N1130, promega, madison, wis.) under experimental conditions (experimental NLuc activity) or under (maximum NLuc) conditions. Tumor cells were co-cultured with human PBMCs. The percentage of CARIA (specific lysis) was calculated using the following equation: specific lysis (%) =100× (experimental NLuc activity-basal NLuc activity)/(maximum NLuc activity-basal NLuc activity).

Cell viability assay

Cell viability was determined using [3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium inner salt; MTS ] assays were performed according to the manufacturer's instructions (G5421, promega, madison, wis.). Tumor cells were co-cultured with human PBMCs. The percentage of living cells was calculated using the following equation: live tumor cells (%) =100× ((experimental absorbance-absorbance of non-co-cultured PBMCs)/absorbance of non-co-cultured tumor cells).

Preparation of primary human macrophages

Enriched monocytes were obtained from isolated PBMCs by Magnetic Activated Cell Sorting (MACS) and Dynabeads selection coated with anti-CD 14 antibodies. Monocytes differentiate into macrophages (M0) in the presence of 80ng/ml M-CSF for 4 days in RPMI1640 medium supplemented with 2 ml-glutamine, 10% (v/v) human serum; m0 macrophages were polarized with LPS (100 ng/ml) and IFN-. Gamma.20 ng/ml to M1 macrophages for 48 hours, or M2 macrophages were polarized with IL-4 (20 ng/ml) for 48 hours. All in vitro phagocytosis assays were performed using M1 macrophages, unless otherwise indicated. Macrophages were harvested using TrypLEExpress.

Phagocytosis assay based on flow cytometry

All in vitro phagocytosis assays were performed by macrophages at 1:1 in ultra low adhesion 96 well U-shaped bottom plates in serum free IMDM: tumor cell ratio Co-culture tumor cells expressing GFP were incubated with donor-derived macrophages in a humidified incubator at 37℃for 1-2 hours with 5% CO 2. GFP-expressing tumor cells were collected from plates using TrypLEexpress, incubated with DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25 μg/ml pirudin for 3 hours at 37℃and washed prior to co-cultivation. For all assays, M1 macrophages were collected from the plates using tryplecexpress. After co-cultivation, plates were placed on ice to stop phagocytosis assay; the samples were centrifuged at 400g for 5 min at 4 ℃ and human macrophages were recognized by staining with a 647-labeled anti-CD 11 b. Samples were analyzed by flow cytometry on an LRSFortessa analyzer (BDBiosciences) or CytoFLEX system (Beckman), all using high throughput autosampler. Phagocytosis was measured as the number of CD11b+GFP+ macrophages, quantified as a percentage of the total number of CD11b+ macrophages. Each phagocytic response (with independent donor and experimental group) was performed in at least triplicate. To account for the innate variability in the original phagocytic levels of donor-derived macrophages, phagocytosis was normalized to the highest technical replication of each donor. All biological replications correspond to independent human macrophage donors.

Phagocytosis assay based on live cell microscopy

Phagocytosis assays based on live cell microscopy were performed as described previously (88). Briefly, tumor cells were collected using TrypLEexpress, labeled with pHrodored succinimide ester (dilution 1:30,000) in PBS for 1 hour at 37℃according to the manufacturer's instructions, and then washed twice with DMEM containing 10% FBS and 100U/ml penicillin/streptomycin. Tumor cells were then incubated with DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25. Mu.g/ml pirudin for 3 hours at 37 ℃. Donor-derived macrophages were collected using tryplecexpress and added to the clear plates and adhered for 1 hour at 37 ℃. Tumor cells with pHrodored markers of serum-free IMDM (macrophage: tumor cell ratio 1:1) were then added to the plates. Plates were gently centrifuged at 50g for 2 min to force tumor cells close to adherent macrophages. The phagocytosis assay plate was then placed in an incubator at 37 ℃ and imaged using IncuCyte (Essen). These images were obtained using a 20 x objective lens at 800ms exposure per field. Phagocytosis events were calculated as the number of pHrodored+ events per well, and these values were normalized to the maximum number of events measured repeatedly across the technology. The threshold for identifying the pHrodored+ event is set based on an intensity measurement of cells labeled with pHrodored without macrophages.

Development of humanized mice

NOG-EXL (NOD.Cg-PrkdcsccidIl 2rgtm1SugTg (SV 40/HTLV-IL3, CSF 2) 10-7 Jic/JicTac) (13395) mice (6-12 weeks old) were purchased from Tacon biosciences. Humanized mice with human cd34+ Hematopoietic Stem Cell (HSC) transfer were developed according to previous studies. Briefly, cryopreserved human cord blood-derived cd34+ cells (StemExpress, folsom, CA) were thawed in an incubator at 37 ℃ and prepared for transplantation according to the manufacturer's instructions. 3-5X 104HSCs were then intravenously transplanted into NOG-EXL mice (MBR-1520R-4, hitachi PowerSolutesCo., ltd., ibaraki, japan) 1 day prior to transplantation that received 1.5Gy of whole body X-rays. Chimerism of human leukocytes (frequency of human cd45+ cells in whole PBMCs) was determined by flow cytometry 8-10 weeks after HSC transplantation. Mice transplanted with >25% chimeric human cd45+ cells in the total PBMC population were used in animal studies.

Animal study

Female "615" mice were purchased from the Tianjin blood institute. Female 5 to 7 week old C57Bl/6 mice were purchased from Experimental animal technology, inc. of Beijing vitamin Tahe. All mice were housed in cages without specific pathogen, provided with standard food, and allowed free access to hypochlorous acid weakly acidic water in a 12:12 light/dark cycle, and lighted 8:00 am. The temperature was maintained at 22℃and the humidity at 45% (40-60%). Animal research design and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the national cancer center/national cancer clinical research center/cancer hospital, academy of sciences of chinese medicine and beijing covarian.

LA795 cells (1.0X10) ⁶ ) Resuspended in Hanks balanced salt solution (Gibco) and subcutaneously injected to the right of "615" mice to examine tumor size and mouse survival. Tumors were resected 23 days after tumor cell inoculation.

MC38 cells (3.0X10) ⁶ ) Resuspended in Hanks balanced salt solution (Gibco) and subcutaneously injected into the right flank of C57Bl/6 mice to examine tumor size. Tumors were resected 20 days after tumor cell inoculation.

Expression by using AAV8 delivery systemMouse C3shRNA and C5shRNA, a mouse model of C3 and C5 consumption was established in "615" mice (90). Briefly, adeno-associated virus 8 (AAV 8) expressing non-targeted shRNA (AAV 8-shControl) or C3shRNA and C5shRNA (AAV 8-C3/C5 shRNA) (vector builder, guangzhou, china) was produced in HEK293T cells. Virus was introduced into the tail vein at 2X 10 ¹² The copy (cp)/kg amount was injected into mice. 4 weeks after virus injection, mice were sacrificed for analysis or received subcutaneous injections of mouse NSCLLA 795 cells (1X 10) ⁶ ) With or without EGFRL858R expression. Tumors were resected 17 days after injection.

H1395 or H322M cells with or without EGFR 858R expression (4.0X10 ⁶ ) Resuspended in hanks balanced salt solution (Gibco) and injected subcutaneously to the right on day 0. In the indicated case, 1.5mL of pooled human serum (mixed samples from 5 healthy individuals as a source of the human complement system) was intraperitoneally (ip) injected (91) into the humanized mice every three days after tumor cell inoculation; anti-human PD-1 (100 μg per mouse, clone J116, BE0188, bioXCell) or anti-human CD55 (100 μg per mouse, clone BRIC216, 9404, international blood group reference laboratory) and anti-human CD59 (100 μg per mouse, clone BRIC229, 9409, international blood group reference laboratory) antibodies or immunoglobulin G (IgG, 100 μg per mouse, clone MOPC-21, BE0083, bioXCell) were intraperitoneally injected into humanized mice on days 3, 6, 9, 12, 16, 20, 23 after tumor cell inoculation; XAV939 (30 mg/kg) was injected intraperitoneally into humanized mice once daily following tumor cell inoculation; humanized mice were treated weekly (120 mm 3) for 5 days after tumor formation with oral gavage vehicle or EGFR inhibitor gefitinib (80 mg/kg) to examine tumor size or survival of mice.

Starting on day 7 after inoculation, the size of all tumors was measured every 3-4 days. The measurement is performed manually by evaluating the longest dimension (length (L)) and longest vertical dimension (width (W)). Tumor volume was estimated according to the equation: (L X W) ² )/2。CO ₂ Inhalation was used to euthanize mice. Mouse tumors were dissected, fixed in 4% formaldehyde and embedded in paraffin. For survival analysis, death was assigned to primary tumor burden reaching 2.5cm and/or animal statusThe number of days with score values below the values allowed for the animal protocol. No statistical method is used to predetermine the sample size. For all experiments, each group included at least six mice, based on available information about the variability of immune checkpoint blocking experiments. Animals arriving at the facility were randomly placed into cages of five mice each and randomly grouped prior to treatment. During the experiment and evaluation of the results, the investigator was not blind to the allocation.

Preparation of Single cell suspension from mouse tumor sample

Single cell suspensions of mouse solid tumor samples were obtained by mechanical dissociation using a straight razor, followed by enzymatic hydrolysis in 10ml RPMI containing 10. Mu.g/ml DNaseI (Sigma-Aldrich) and 25. Mu.g/ml Liberase (Sigma-Aldrich) for 30-60 min at 37℃with vigorous blows every 10 min to promote dissociation. After a maximum of 60 minutes, the dissociation reaction was quenched with RPMI containing 10% FBS cooled to 4℃and filtered through a 100 μm filter and centrifuged at 400g for 10 minutes at 4 ℃. Erythrocytes in the samples were lysed by resuspension of the tumor pellet in 5ml ack lysis buffer for 5 min at room temperature. The cleavage reaction was quenched by addition of 20ml of RPMI containing 10% FBS and the samples were centrifuged at 400g for 10 min at 4 ℃. Samples were analyzed directly or resuspended in BAMBANKER, aliquoted into freezer tubes and frozen prior to subsequent analysis.

Isolation of tumor infiltrating leukocytes

A single cell suspension of mouse tumor samples was obtained (as described above), frozen samples were thawed at 37 ℃ for 3-5 minutes, washed with DMEM containing 10% fbs, and centrifuged at 400g at 4 ℃ for 5 minutes. Tumor infiltrating leukocytes were isolated by gradient centrifugation over 40-80% percoll (P1644, sigma-Aldrich).

Flow cytometer

Tumor infiltrating leukocytes or human PBMC were stained with Phycoerythrin (PE) -Cy7 labeled anti-mouse CD8a (clone 53-6.7) or PE-Cy7 labeled anti-human CD8 (clone HIT8 a) antibodies. Dead cells were excluded using the FixableAqua dead cell staining kit. For intracellular staining, cells were stained with an antibody against a cell surface marker for 30 min, fixed, permeabilized with a fixation/permeabilization solution kit and antibody with APC anti-human/mouse granzyme B (clone QA16a 02), PE anti-mouse perforin (clone S16009A) or PE anti-human perforin (clone B-D48).

FACS and in vivo phagocytosis analysis of mouse tumor samples

A single cell suspension of mouse tumor samples was obtained (as described above), frozen samples were thawed at 37 ℃ for 3-5 minutes, washed with DMEM containing 10% fbs, and centrifuged at 400g at 4 ℃ for 5 minutes. The samples were then resuspended in FACS buffer at a concentration of 100 tens of thousands of cells per milliliter and blocked with monoclonal CD16/32 antibody for 15 minutes on ice before staining with antibodies recognizing the target protein. The samples were stained on ice for 30 minutes, washed twice with FACS buffer and resuspended in buffer containing 1. Mu.g/ml DAPI prior to analysis. Fluorescence compensation was performed using single-dye UltraCompeBeads. Immunolabeling and DAPI gating were performed using fluorescence minus one control. Flow cytometry was performed on a FACSAriaII cell sorter (BDBiosciences) or LRSFortessa analyzer (BDBiosciences). The FACSDiva software is used for data collection. Flow cytometry data were analyzed using FlowJoV 10. Phagocytosis is measured as the percentage of CD11b+F4/80+ tumor-associated macrophages that also express GFP.

Patient tissue sample

Patient groups were randomly distributed by age and sex. The number of samples is sufficient for statistical analysis. We retrospectively collected surgical resected, formalin fixed and paraffin embedded NSCLC tissue samples from national tumor center/national tumor clinical research center/tumor hospital biological library of academy of chinese medicine and beijing synergetic medical college (beijing, china.) tissue samples from 200 naive patients receiving pathological diagnosis cancer surgery from 2003 to 2014 were selected as independent cohorts, including 200 lung adenocarcinoma (LUAD) cases and 200 adjacent normal specimens. All patients received standard treatment after surgery. From our previous clinical trial study (accession number: chiCTR-OIC-17013726) (73), we recruited 24 NSCLC patients as another independent cohort, who received two cycles of sintillimab (an anti-PD-1 antibody) and then underwent clinical response assessment and then surgery. Positron emission tomography-computed tomography is obtained at baseline and pre-operatively. The response was assessed according to RECIST version 1.1. The use, database and study protocol of the human NSCLC specimen was approved by the national tumor center/national tumor clinical research center/the medical ethics committee of the tumor hospital institute, national academy of medical science and Beijing covariant medical college. All tissue samples were collected according to the informed consent policy. All patients received written informed consent at the time of admission for scientific research using their tissue, blood or other samples, and patient privacy was preserved.

We obtained clinical data by reviewing the patient's medical history. The pathological stage was assessed according to united states joint committee for cancer/international cancer control alliance TNM staging system (93) version 8.

Histological evaluation and immunohistochemical staining

Mouse tumor tissue was fixed and prepared for staining. Specimens were stained with Mayer's hematoxylin followed by eosin (H & E) (BiogenexLaboratories, sanRamon, CA). Slides were mounted using universal mount (research genetics, huntsville, AL).

Tissue sections from paraffin-embedded human NSCLC specimens were stained with antibodies or LNA probes. These proteins were scored quantitatively in tissue sections according to the positive cell percentages and staining intensity defined previously. Similarly, we quantitatively scored LINC00973, MLINC00973, miR-216b and miR-150 in tissue sections by ISH. The following ratio fractions were assigned: 0% if 0% of tumor cells are positively stained, 1 if positive cells are from 0% to 1%, 2 if positive cells are from 2% to 10%, 3% to 30% if positive cells are from 11, 4 if positive cells are from 31% to 70%, and 5 if positive cells are from 71% to 100%. We also rated the staining intensity on a scale of 0 to 3: 0, negative; 1. weak; 2. is moderate; 3. strong. As previously described, the ratio and intensity fractions are combined to obtain a total fraction (ranging from 1 to 8). The score is compared to the total lifetime, which is defined as the time from the date of diagnosis to the date of death or last known follow-up.

Statistics and reproducibility

All statistical data are expressed as mean ± SD. All experiments were independently repeated at least 3 times with similar results. Significant differences in the average values obtained in the control group and the experimental group were analyzed. Pair wise comparisons were made using a two-tailed t-test. P values less than 0.05 are considered significant. Unless otherwise stated, the experiments were not randomized, nor were the study personnel blinded to experimental conditions and outcome assessment.

The results obtained in the above experiments were analyzed as follows.

EGFR activation increases the expression of CD55 and CD59 and inhibits complement system and CD8+ T cell activation

To determine whether EGFR activation regulates the expression of mCRPs, we treated H1395 and H322MNSCLC cells with EGF for 24 hours. Wherein a in FIG. 1 is H1395 cells treated or not treated with EGF (100 ng/ml) for 24 hours. Immunoblot analysis was performed with the indicated antibodies. b-e shRNA against CD55 and CD59 are expressed in H1395 cells. Designated cells were treated with or without EGF (100 ng/ml) for 24 hours and then incubated with DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25. Mu.g/ml pellpirudin for 3 hours. The surface expression of the C3b and C5b-9 tumor cells in b was determined by flow cytometry with the indicated antibodies. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001.C the amount of anaphylatoxins (C3 a and C5 a) in the supernatant was determined by ELISA. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001.d cells were co-cultured with human PBMC. The expression of the cytokines indicated in the medium was examined. Data are expressed as mean ± SD of seven independent experiments using different human donors; ns, not significant; * P <0.001.e cells were co-cultured with human PBMC. The expression of granzyme B and perforin in cd8+ T cells was detected by flow cytometry with the indicated antibodies. Data are expressed as mean ± SD of seven independent experiments using different human donors; ns, not significant; * P <0.001.

In FIG. 2, a is H322M cells treated with or without EGF (100 ng/ml) for 24 hours. Immunoblot analysis was performed with the indicated antibodies. b-e shRNA against CD55 and/or CD59 is expressed in H322M cells. Designated cells were treated with or without EGF (100 ng/ml) for 24 hours. The designated cells were then incubated with DMEM medium (10% fbs) with or without 25% v/v human serum and 25 μg/ml pirudin for 3 hours. Representative results of C3B and C5B-9 expression on the surface of tumor cells as determined by flow cytometry with the indicated antibodies in B. C expression of C3b and C5b-9 on the surface of tumor cells was determined by flow cytometry with the indicated antibodies. Data are expressed as mean ± SD (n=6). HS, human serum; ns, not significant; * P <0.001. In d, the amount of anaphylatoxins (C3 a and C5 a) in the supernatant was determined by ELISA. Data are expressed as mean ± SD (n=6); HS, human serum; ns, not significant; * P <0.001.e, co-culturing the cells with human PBMC. The expression of the indicated cytokines in the medium was examined. Data are expressed as mean ± SD of seven independent experiments using different human donors; HS, human serum; ns, not significant; * P <0.001. H1395 cells in f were treated with or without EGF (100 ng/ml) for 12 hours. The relative expression levels of CFH, CFB, C and C5mRNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); ns is not significant. H1395 cells were treated with or without EGF (100 ng/ml) for 12 hours in g. The expression levels of factor H, factor B, C3 and C5 in the cell culture supernatant were examined. Data are expressed as mean ± SD (n=6); ns is not significant. H H1395 cells were treated with or without EGF (100 ng/ml) for 24 hours, then incubated with DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25. Mu.g/ml pellpirudin for 3 hours. These cells were then co-cultured with human PBMCs. The expression levels of granzyme B and perforin in cd8+ T cells were detected by flow cytometry using the indicated antibodies. Representative results are shown.

The results showed that the expression of CD55 and CD59 was significantly enhanced, not that of CD46 (FIG. 1 a). As expected, EGF treatment inhibited complement activation, which was reflected in inhibition of C3b and C5b-9 (markers of all pathway activation) on the tumor cell surface (fig. 1b; fig. 2b and C), reduced release of anaphylatoxins C3a and C5a (fig. 1C, fig. 2 d) and reduced secretion of IFN- γ, TNF- α, IL-6, IL-1 β and IL-17 in co-culture with human Peripheral Blood Mononuclear Cells (PBMC) (fig. 1d, fig. 2 e). This inhibition was eliminated by the combined consumption of CD55 and CD59 induced by the corresponding shRNA expression (fig. 1b-d, fig. 2C-e), which resulted in more C5b-9 deposition than CD55 or CD59 alone (right panel of fig. 2C). Depletion of CD55 and CD59, and depletion of CD55 alone, also significantly enhanced deposition of C3b (FIG. 2C left panel) and release of C3a and C5a (FIG. 2 d), indicating the role of CD55 in complement activation. Notably, depletion of CD55 and CD59 induced complement activation did not occur in the absence of human serum (fig. 2 c-e). Furthermore, EGF treatment did not alter the expression levels of intracellular mRNA (expansion data fig. 2 f) or extracellular protein (fig. 2 g) of complement inhibitor factor H and factor B or complement proteins C3 and C5. These results indicate that EGFR activation in NSCLC cells inhibits complement activation by up-regulating CD55 and CD 59.

EGFR activation in tumor cells reduced the expression of cytotoxic particles B (GzmB) and Perforin (PFN) in cd8+ T cells and inhibited the activation of cd8+ T cells. Cytokines, such as IFN-gamma, TNF-alpha, IL-6, IL-1 beta and IL-17, maintain CD8+ T cell activity through complement-mediated secretion (44) of antigen presenting cells and CD4+ T cells. Determining whether EGFR activation in tumor cells regulated the activity of cd8+ T cells, we co-cultured EGF-treated or untreated H1395 cells with human PBMCs containing cd8+ T cells and showed that EGFR activation inhibited the activity of isolated cd8+ T cells, reflected in reduced granzyme B and perforin expression (fig. 1e, fig. 2H). This inhibition was eliminated by the depletion of CD55 and CD59, which greatly enhanced the expression of granzyme B and perforin PFN (fig. 1 e). These results strongly suggest that EGFR activation increases the expression of CD55 and CD59, thereby inhibiting the complement system, complement system-dependent cytokine secretion and subsequent cd8+ T cell activation.

EGFR activation upregulates CD55 and CD59 by inhibiting miR-216b and miR-150, respectively

To determine the mechanism by which EGFR activation induces increased expression of CD55 and CD59, we expressed luciferase reporter genes driven by the promoters of CD55 and CD 59. In FIG. 3, a is H1395 cells treated or not with EGF (100 ng/ml) for 12 hours. The relative expression levels of CD55 and CD59mRNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); * P <0.001. b. f, h are immunoblots with the indicated antibodies. B, H1395 cells were transfected with small interfering RNA (siRNA) against Dicer. Cells were then treated with or without EGF (100 ng/ml) for 24 hours. c, H1395 cells expressing luciferase reporter gene are fused or unfused with the corresponding Mutant (MUT) of Wild Type (WT) 3' UTR or CD55 and CD59 genes, simulated with miR-Control, miR-150-mics or miR-216 b. The relative luciferase activity in cells expressing the luciferase reporter gene without fusion of the 3' utr is normalized to that in the miR-control. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001.d, using miR-Control, miR-150-inhibitor or miR-transfection to express a luciferase reporter gene H1395 of a corresponding Mutant (MUT) fused or unfused with Wild Type (WT) 3' UTR or CD55 and CD59 genes. The relative luciferase activity in cells expressing the luciferase reporter gene without fusion of the 3' utr is normalized to that in the miR-control. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001. And e, transfecting the H1395 cells by using miR-Control, miR-150-chemicals or miR-216 b-chemicals. Cells were treated for 12 hours with or without EGF (100 ng/ml). The relative expression levels of CD55 and CD59mRNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001. And f, transfecting the H1395 cells by using miR-Control, miR-150-chemicals or miR-216 b-chemicals. Cells were treated with or without EGF (100 ng/ml) for 24 hours. And in g, transfecting the H1395 cells by using miR-Control, miR-150-inhibitor or miR-216 b-inhibitor. Cells were treated for 12 hours with or without EGF (100 ng/ml). The relative expression levels of CD55 and CD59mRNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001. And H, transfecting the H1395 cells by using miR-Control, miR-150-inhibitor or miR-216 b-inhibitor. Cells were treated with or without EGF (100 ng/ml) for 24 hours.

In fig. 4, d, e, k, m, n, immunoblot analysis was performed with the indicated antibodies. The luciferase reporter gene driven by the promoters of CD55 and CD59 in a was co-transfected into 293T cells with EGFR-expressing vectors. Cells were treated with or without EGF (100 ng/ml) for 8 hours. Luciferase activity was measured. Relative luciferase activity was normalized to the group without EGF treatment. Data are expressed as mean ± SD (n=6); ns is not significant. b H1395 cells were treated with or without EGF (100 ng/ml) for 12 hours. The relative RNA expression levels of the transcribed introns of the CD55 and CD59 genes were measured using quantitative PCR. Data are expressed as mean ± SD (n=4); ns is not significant. c H322M cells were treated with or without EGF (100 ng/ml) for 12 hours. The relative expression levels of CD55 and CD59mRNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); * P <0.001. In d, H1395 and H322M cells were transfected with or without one of two different small interfering RNAs (siRNAs) against Dicer, respectively. e H322M cells were transfected with small interfering RNA (siRNA) against Dicer. These cells were then treated with or without EGF (100 ng/ml) for 24 hours. f, transfecting H1395 and H322M cells with siRNA against Dicer. Cells were then treated for 12 hours with or without EGF (100 ng/ml). The relative expression levels of CD55 and CD59mRNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001. The miRNA in g encodes a predictive algorithm (http:// www.mircode.org/index. Php) -predicting miRNAs and their target sequences on CD55 and CD59 mRNAs. H H322M cells expressing the luciferase reporter gene were fused or unfused with the wild-type (WT) 3' UTR or the corresponding mutants of CD55 and CD59 genes (MUT), and were simulated using miR-Control, miR-150-mics or miR-transfection 216 b. It is shown that in cells expressing the luciferase reporter gene, relative luciferase activity was normalized to that in the miR-control without fusion of the 3' utr. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001.i H322M cells expressing luciferase reporter gene are fused or unfused with Wild Type (WT) 3' UTR or corresponding Mutants (MUTs) of CD55 and CD59 genes, and 216b inhibitor is transfected with miR-Control, miR-150-inhibitor or miR-150-inhibitor. It is shown that in cells expressing the luciferase reporter gene, relative luciferase activity was normalized to that in the miR-control without fusion of the 3' utr. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001. And j, transfecting the H322M cells by miR-Control, miR-150-chemicals or miR-216 b-chemicals. Cells were treated for 12 hours with or without EGF (100 ng/ml). The relative expression levels of mRNA of CD55 and CD59 genes were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001. And k, transfecting the H322M cells by miR-Control, miR-150-chemicals or miR-216 b-chemicals. Cells were treated with or without EGF (100 ng/ml) for 24 hours. And transfecting the H322M cells by miR-Control, miR-150-inhibitor or miR-216 b-inhibitor. Cells were treated for 12 hours with or without EGF (100 ng/ml). The relative expression levels of CD55 and CD59mRNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001. And in M, transfecting the H322M cells by using miR-Control, miR-150-inhibitor or miR-216 b-inhibitor. Cells were treated with or without EGF (100 ng/ml) for 24 hours. H1395 and H322M cells in n, o were treated with or without EGF (100 ng/ml) for 24 hours. Immunoblot analysis was performed with the indicated antibodies (n). The relative protein expression levels of c-Jun and SRCIN1 were measured. Data are expressed as mean ± SD (n=3); ns, not significant (o).

The results showed that EGF treatment in H1395 cells did not alter the activity of the CD55 and CD59 promoters (fig. 4 a) or the expression levels of the CD55 and CD59 introns (fig. 4 b), indicating that EGF-enhanced CD55 and CD59 expression was not regulated at the transcriptional level. Nonetheless, quantitative PCR analysis showed that EGF treatment increased the levels of CD55 and CD59mRNA (fig. 3 a). Given that mRNA expression can be regulated by microRNA (miRNA) -dependent regulation, we depleted Dicer (fig. 4 d), a ribonuclease critical for miRNA biogenesis. We found that Dicer consumption increased the protein (fig. 3 b) and mRNA (fig. 4 f) expression of CD55 and CD59 and eliminated the effect of EGF on CD55 and CD59 expression (fig. 3b, fig. 4 e-f), indicating that EGF-induced upregulation of CD55 and CD59 expression was mediated by inhibition of specific mirnas targeting CD55 and CD59mRNA degradation.

Sequence analysis by the miRNA code prediction algorithm (http:// www.mircode.org/index. Php) revealed that miR-216b-5p and miR-150-5p (subsequently miR-216b and miR-150 were used) had potential binding sites located in the 3' untranslated region (UTR) of mRNA of human CD55 and CD59, respectively (FIG. 4 g). The 3 'UTR-luciferase reporter assay showed that luciferase activity was reduced in cells expressing the luciferase gene (wild type (WT) 3' UTR fusing the CD55 and CD59 genes) due to endogenous expression of miR-216b and miR-150, respectively. However, this reduction was alleviated by expression of mutants comprising replacement of agagau with GAGAGCC in CD55UTR and UGGGAG with CAAAGA in CD59UTR (fig. 3 c). Similar remission was observed with inhibitors expressing miR-216b and miR-150, which were oligonucleotides complementary to miR-216b and miR-150, respectively (FIG. 3 d). In addition, overexpression of miR-216b and miR-150 reduced luciferase activity of the luciferase gene (fusion WT, but not mutant, 3' UTR of CD55 and CD59 genes) (FIG. 3 c). Consistent with these findings, EGF-enhanced expression of mRNA (FIG. 3 e) and protein (FIG. 3 f) of CD55 and CD59 was inhibited by overexpression of miR-216b and miR-150. In contrast, in H1395 (FIG. 3 g-H) and H322M cells, the expression of miR-216b and miR-150 inhibitors increased the expression of CD55 and CD59mRNA (FIG. 3 g) and protein (FIG. 3H). miR-150 and miR-216b have been shown to regulate c-Jun and SRCIN1, respectively. However, EGF treatment did not alter the expression levels of c-Jun or SRCIN1 (extension data FIG. 4 n-o), indicating that miR-216b and miR-150 differentially regulated expression of the respective target proteins in the signaling environment. These results indicate that EGFR activation upregulates the expression of CD55 and CD59 by inhibiting miR-216b and miR-150, respectively.

EGFR activation induces LINC00973 expression to adsorb miR-216b and miR-150, and up-regulates CD55 and CD59

In FIG. 5, a, b is a plot of binding sites of miR-216b and miR-150 on lncRNA LINC 00973. The 5 'and 3' sequences of LINC00973 were obtained using Rapid Amplification of CDNA Ends (RACE). PCR products EnsembGenomeBrowser amplified from known regions of the 5 'and 3' sequences of LINC00973 and LINC00973 sequences were separated on agarose gels. The size of the sequence is expressed according to the sequencing result of the PCR product. d is the PCR product obtained using the primer of LINC00973 was sequenced. The full length sequence of LINC00973 is shown. e is the LINC00973 expression level determined using Gene Expression Profiling Interaction Analysis (GEPIA). LUAD, lung adenocarcinoma; TPM, every million achievement list; t, tumor; n, normal; num, number of samples). f is the preparation of total cell lysates and cytosolic and nuclear fractions from H322M cells. RNA was extracted and purified from cell fractions prepared from the same number of cells. The relative expression levels of ACTB (cytoplasmic control), RNU6-1 (control) and LINC00973 RNAs were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); * P <0.001.g is the time period specified for H322M cells treated with or without EGF (100 ng/ml). The relative expression levels of LINC00973RNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); * P <0.05, < P <0.001.h is an RNA Immunoprecipitation (RIP) map of anti-GFP antibody for detection of mirnas endogenously associated with LINC 00973. I is the co-transfection of MS2-GFP with MS12, MS12-LINC00973 or MS12-lncRNA-225205 into H322M cells. GFP-RIP followed by microRNAqRT-PCR was performed to detect the lncRNA-related microRNAs. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001. Schematic j shows mutations in the binding sites of miR-216b and miR-150 on LINC 00973. k. In l MS2-GFP was co-transfected into H322M cells with MS12, MS12-LINC00973-mut (miR-216 b) (k), MS12-LINC00973-mut (miR-150) (l) or control MS 12-lncRNA-225205. GFP-RIP followed by microRNAqRT-PCR was performed to detect the lncRNA-related microRNAs. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001.M is H322M cells were transfected with biotin conjugated LINC00973 or lncRNA-225205, followed by streptavidin pulldown assay and subsequent microRNAqRT-PCR to detect the relevant endogenous micrornas. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001.n, o H322M cells were transfected with biotin-conjugated LINC00973, LINC00973-mut (miR-216 b) (n), LINC00973-mut (miR-150) (o), or control lncRNA-225205, followed by streptavidin pull-down assays and subsequent microRNAqRT-PCR to detect the relevant endogenous micrornas. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001.

In FIG. 6, a is H1395 cells treated or not with EGF (100 ng/ml) for 7 hours. Fluorescence In Situ Hybridization (FISH) assays were performed using probes for LINC00973 (left panel). The distribution ratio of LINC00973 in the nucleus and cytosol was calculated. * P <0.001 (right panel). B is the preparation of total cell lysates and cytosolic and nuclear fractions from H1395 cells. RNA was extracted and purified from cell fractions prepared from the same number of cells. The relative expression levels of ACTB (cytoplasmic control), RNU6-1 (control) and LINC00973 RNAs were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); * P <0.001.c is H1395 cells treated with or without EGF (100 ng/ml) for the indicated period of time. The relative expression levels of LINC00973RNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6). * P <0.05, < P <0.001.d is the co-transfection of MS2-GFP with MS12, MS12-LINC00973 or MS12-lncRNA-225205 into H1395 cells. GFP-RIP followed by microRNAqRT-PCR was performed to detect the lncRNA-related microRNAs. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001. e. And f, co-transfecting MS2-GFP with MS12, MS12-LINC00973-mut (miR-216 b) (e), MS12-LINC00973-mut (miR-150) (f) or control MS12-lncRNA-225205 into H1395 cells, and performing GFP-RIP and subsequent microRNAqRT-PCR to detect the lncRNA-related microRNAs. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001.g is H1395 cells were transfected with biotin conjugated LINC00973 or lncRNA-225205, followed by a streptavidin pulldown assay and subsequent microRNAqRT-PCR to detect the relevant endogenous microRNA. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001. h. i is H1395 cells were transfected with biotin conjugated LINC00973, LINC00973-mut (miR-216 b) (H), LINC00973-mut (miR-150) (i) or control lncRNA-225205, followed by a streptavidin pull-down assay followed by microRNAqRT-PCR to detect the relevant endogenous microRNA. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001.

In FIG. 7, the first 200 5 'nucleotides of a luciferase reporter gene expressing H1395 and H322M cells transfected with miR-Control, miR-150-with or without miR-216b or miR-150's binding sequence mutation mimetic or miR-216 b-mimetic of LINC00973 in a. It was shown that in cells expressing the luciferase reporter gene, the relative luciferase activity was normalized to that in the miR-control without fusion of the LINC00973 fragment. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001.B, transfecting H1395 and H322M cells by miR-Control, miR-150-chemicals or miR-216 b-chemicals. The relative expression levels of LINC00973RNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); ns is not significant. c, transfecting H1395 and H322M cells with miR-Control, miR-150-miics or miR-216 b-miics, and then carrying out RIP and subsequent lncRNAqRT-PCR by using antibodies aiming at Ago2 to detect indicated microRNA related lncRNA. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001. In d, H1395 and H322M cells were transfected with LINC00973, LINC00973-mut (miR-216 b), LINC00973-mut (miR-150) or lncRNA-225205. The relative expression levels of LINC00973 and lncRNA-225205RNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.001.e, transfecting H1395 and H322M cells with LINC00973, LINC00973-mut (miR-216 b), LINC00973-mut (miR-150) or lncRNA-225205. Total RNA was extracted from cells. The relative expression levels of miR-216b or miR-150miRNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.01; * P <0.001.f is H1395 and H322M cells treated with or without EGF (100 ng/ml) for 12 hours. Total RNA was extracted from cells. The copy numbers of LINC00973, miR-216b and miR-150 in the indicated cells were quantified using quantitative PCR. Data are expressed as mean ± SD (n=6). * P <0.001. Copy numbers of LINC00973, miR-216b and miR-150 in H1395 cells treated with EGF for 0H, 2H, 4H, 6H and 8H were determined. The stoichiometry of LINC00973 binding to miR-216b and miR-150 was calculated. H1395 and H322M cells were stably transfected with or without LINC00973 shRNA. Cells were treated for 12 hours with or without EGF (100 ng/ml). The relative expression levels of LINC00973RNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); ns, not significant; * P <0.01; * P <0.001. H1395 and H322M cells in i were stably transfected with or without LINC00973 shRNA. Cells were treated with or without EGF (100 ng/ml) for 24 hours. Immunoblot analysis was performed with the indicated antibodies. j. k shows a schematic representation of miR-216 and miR-150 binding elements (miR-216 b/150-BE) on LINC 00973. Genomic DNA was extracted from two separate clones of parental H1395 (j) and H322M (k) cells and H1395 (j) and H322M (k) cells with miR-216b/150-BE knock-in mutations. The PCR products were amplified from the indicated DNA fragments and separated on agarose gel. C1, clone 1; c2, clone 2. Sequencing data of two separate clones of parental H1395 (j) and H322M (k) and H1395 (j) and H322M (k) cells with miR-216b/150-BE knock-in mutations are shown. The red line without arrow indicates Protospacer Adjacent Motif (PAM). The mutated nucleotide is indicated by blue arrows. The position of miR-216b/150-BE with or without a mutant nucleotide is indicated by a filled red box. Silent mutations of specified nucleotides were introduced into the sequence to avoid repeated cleavage by Cas 9. The relative expression levels of CD55 and CD59mRNA in designated clones of parental H1395 and H322M cells and H1395 and H322M cells harboring the miR-216b/150-BE knock-in mutation were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); * P <0.001.WT, wild-type; c1, clone 1; c2, clone 2. Immunoblot analysis was performed with the indicated antibodies in the indicated cells in M.

Long non-coding RNAs (lncrnas) modulate their downstream targets by competitively binding to common micrornas, acting as competitive endogenous RNAs (cernas) and miRNA adsorption. The recognition sequences of miR-216b and miR-150 were analyzed using a miRcode prediction algorithm, and LINC00973 was found to be the only one lncRNA, which contained one and two targeting sequences for miR-216b and miR-150, respectively (FIGS. 5 a-b). The cDNA end Rapid Amplification (RACE) revealed a full-length transcribed sequence of gene LINC00973 (FIGS. 5 c-d), 2039nt in length, comprising 2 exons and 1 intron (http:// asia. Ensembl. Org/index. Html). Analysis of TCGA and genotype tissue expression (GTEx) databases using Gene Expression Profiling (GEPIA) showed that LINC00973 expression was much higher in human lung adenocarcinoma specimens than in their neighboring healthy lung tissues (fig. 5 e)). Fluorescence In Situ Hybridization (FISH) analysis of H1395 cells showed that LINC00973 was localized in the cytosol and nucleus (fig. 6 a), and this distribution was further confirmed by cell fractionation analysis in H1395 (fig. 6 b) cells. Furthermore, EGF treatment did not significantly alter the localization of LINC00973 (fig. 6 a).

Notably, EGF treatment of H1395 and H322M cells enhanced expression of LINC00973 in a time-dependent manner (fig. 6 c). We next performed RNA Immunoprecipitation (RIP) of lysates of H1395 and H322M cells, which co-expressed MS2-GFP fusion protein with LINC00973 or control lncRNA-225205, which did not contain miR-216b or miR-150 binding sites (5H), using anti-GFP antibodies. Subsequent qPCR analysis showed that only LINC00973 was associated with miR-216b and miR-150 (FIG. 6d, FIG. 5 i), and this association was abrogated by mutations in the binding sequence of LINC00973 for miR-216b (FIG. 6 e) or miR-150 (FIG. 6 f) (FIG. 5 j). Similar results were obtained by a pull down assay using biotin conjugated WT or mutated LINC00973 and control lncRNA-225205 (fig. 6 g-i). These results indicate that EGFR activation enhances expression of LINC00973 binding to miR-216b and miR-150.

To further confirm that LINC00973 binds miR-216b and miR-150, we fused the luciferase gene with the first 200 5' nucleotides with or without LINC00973 (Luc-973) (WT or mutation comprising the binding sequences of miR-216b and miR-150). We examined whether expression of miR-216b and miR-150 can reduce luciferase activity by degrading mRNA of the fusion gene. We found that the luciferase activity of H1395 and H322M cells expressing the luciferase gene (fused to WTINC 00973) was reduced. This decrease was exacerbated by overexpression of miR-216b and miR-150 (FIG. 7 a), which did not alter the level of endogenously expressed LINC00973 (FIG. 7 b). Notably, expression of LINC00973 with miR-216b or miR-150 binding site mutations abrogated inhibition induced by miR-216b or miR-150 overexpression, respectively (FIG. 7 a). The interaction between LINC00973 and miR-216b/miR-150 was further confirmed by RNP Immunoprecipitation (RIP) analysis using an Ago2 antibody known to be associated with microRNA. We found that endogenous LINC00973, but not lncRNA-225205, was specifically enriched under conditions of miR-216b and miR-150 overexpression in H1395 and H322M cells (FIG. 7 c). In addition, overexpression of LINC00973 (FIG. 7 d) reduced the levels of free miR-216b and free miR-150 (FIG. 7 e). These results indicate that LINC00973 binds to miR-216b and miR-150.

To quantify the copy numbers of LINC00973, miR-216b and miR-150 in the cells, we performed qPCR and showed that H1395 and H322M cells expressed approximately 80 LINC00973 copies and 150 miR-216b and miR-150 copies. However, EGF treatment significantly increased the copy number of LINC00973 and decreased the expression of miR-216b and miR-150 (extension data FIG. 4 f), indicating that up-regulated LINC00973 was sufficient to adsorb miR-216b and miR-150. Chemometric analysis showed that in H1395 cells treated with EGF for 8 hours, the binding ratio of LINC00973 to miR-216b and miR-150 was 1:0.856:1.314 (FIG. 7 g). Notably, consumption of LINC00973 (fig. 7H) reduced basal and EGF up-regulated levels of CD55 and CD59 in H1395 and H322M (fig. 7 i). Furthermore, we mutated miR-216b and miR-150 binding nucleotides (miR-216 b/150-BE) in LINC00973 in H1395 cells (FIG. 7 j) using CRISPR/Cas9 genome editing knock-in technology (53) (FIG. 5 k). We found that knock-in expression of the miR-216b/150-BE mutation reduced expression of mRNA (FIG. 7 l) and protein (FIG. 7 m) for CD55 and CD59. These results strongly indicate that EGFR activation induces LINC00973 expression to adsorb miR-216b and miR-150, while up-regulating CD55 and CD59.

EGFR activation-induced beta-catenin transactivation enhances LINC00973 expression and subsequent up-regulation of CD55 and CD59

To determine the mechanism by which EGF enhances LINC00973 expression, we analyzed the promoter region of LINC 00973. In FIG. 8, a schematic diagram shows three potential LEF/TCF binding elements (TBE; CTTTG (A/T) (A/T)) in the promoter region of LINC 00973. b. c is the extraction of genomic DNA from two separate clones of parental H1395 (b) and H322M (c) cells and H1395 (b) and H322M (c) cells with knock-in mutations (AT to GC) in three TBEs. PCR products were amplified from the indicated DNA fragments and separated on agarose gel. C1, clone 1; c2, clone 2. Sequencing data for two separate clones of parental H1395 (b) and H322M (c) cells and H1395 (b) and H322M (c) cells with knock-in mutations (AT to GC) in three TBEs are shown. The red line with arrow indicates the sgRNA targeting sequence. The red line without arrow indicates Protospacer Adjacent Motif (PAM). The mutated nucleotide is indicated by black arrows. Three TBEs with or without mutated nucleotides are indicated by solid red boxes. Silent mutations of specified nucleotides were introduced into the sequence to avoid repeated cleavage by Cas 9. d is the designated clone (100 ng/ml) of the parental H1395 and H322M cells with or without EGF (100 ng/ml) and H1395 and H322M cells with knock-in mutations (AT to GC) in TBE1 and TBE3 of the LINC00973 promoter for 12 hours. The relative expression levels of LINC00973RNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6); * P <0.001. C1, clone 1; c2, clone 2. e. f is H322M cells stably transfected with or without β -catensinshRNA, and then recombined with designated RNA interference (RNAi) resistance (r) β -catenin (rβ -catenin) or transfected with or without miR-216b inhibitor (e) or miR-150 inhibitor (f). These cells were treated with or without EGF (100 ng/ml) for 24 hours. Immunoblot analysis was performed with the indicated antibodies. g. H is H322M cells were stably transfected with or without LINC00973shRNA#1 or LINC00973shRNA#2, and then transfected with or without miR-216b inhibitor (g) or miR-150 inhibitor (H). These cells were treated with or without EGF (100 ng/ml) for 24 hours. Immunoblot analysis was performed with the indicated antibodies. i is the designated clone of H1395 and H322M cells with or without EGF (100 ng/ml) stimulated parental H1395 and H322M cells with knock-in mutations (AT to GC) in TBE1 and TBE3 for 24 hours. Immunoblot analysis was performed with the indicated antibodies. C1, clone 1; c2, clone 2. j. k is a parental H1395 or H322M cell with or without knock-in mutation (AT to GC) in TBE2 transfected with a LINC00973 promoter with or without miR-216b (j) or miR-150 (k) inhibitors. Cells were treated with or without EGF (100 ng/ml) for 24 hours. Immunoblot analysis was performed with the indicated antibodies. C2, clone 2.l is EGFRL858R stably expressed in H1395 or H322M cells with or without a knock-in mutation (AT to GC) in TBE2 of the LINC00973 promoter. Beta-catenin shRNA is expressed in these cells, followed by recombinant expression of RNA interference (RNAi) resistance (r) beta-catenin (rβ -catenin) or LINC00973shRNA. Immunoblot analysis was performed with the indicated antibodies. M and n are EGFRL858R stably expressed in H1395 or H322M cells. These cells were then transfected with or without miR-216b mimetic (m) or miR-150 mimetic (n). Immunoblot analysis was performed with the indicated antibodies. o is H1395 cells were treated with or without Wnt-7B or Wnt-5A (200 ng/ml) for 12 hours. The relative RNA expression level of LINC00973 and the relative expression levels of CD55 and CD59mRNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6). ns, not significant; * P <0.001.p is the relative mRNA expression levels of the indicated Fzd receptor, LRP5 and LRP6 were measured using quantitative PCR. Data are expressed as mean ± SD (n=4). q immunoblot analysis with the indicated antibodies. H1395 cells in r were treated with or without Wnt-7B (200 ng/ml) for 12 hours. The relative RNA expression level of LINC00973 and the relative expression levels of CD55 and CD59mRNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=4); ns, not significant; * P <0.01; * P <0.001.

In FIG. 9, a is the transfection of H1395 and H322M cells expressing the luciferase reporter fused to the LINC00973 promoter with or without the β -catenin, TCF-4 or truncated TCF-4. DELTA.N mutant. Cells were treated with or without EGF (100 ng/ml) for 24 hours. Shows relative luciferase activity normalized to activity in the group without EGF treatment. Data are expressed as mean ± SD (n=6). ns, not significant; * P <0.001.b is H1395 and H322M cells are stably transfected with or without active beta-catenin (beta-catenin 1-89aa deleted). The relative expression levels of LINC00973RNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6). * P <0.001.c is the designated clone of H1395 and H322M cells with or without EGF (100 ng/ml) stimulation of parental H1395 and H322M cells with knock-in mutation (AT to GC) in TBE2 of LINC00973 promoter for 12 hours. The relative expression levels of LINC00973RNA were measured using quantitative PCR. Data are expressed as mean ± SD (n=6). ns, not significant; * P <0.001. C1, clone 1; c2, clone 2.d is the designated clone of H1395 and H322M cells with or without EGF (100 ng/ml) stimulated parental H1395 and H322M cells with knock-in mutation (AT to GC) in TBE2 of LINC00973 promoter for 12 hours. ChIP analysis was performed using anti-beta-catenin antibodies or anti-TCF 4 antibodies. The scatter plot shows the amount of immunoprecipitated DNA, expressed as a percentage of total input DNA. Data are expressed as mean ± SD (n=6). ns, not significant; * P <0.001. C1, clone 1; c2, clone 2.e is purified bacterial expressed GST or GST-TCF4 on glutathione agarose beads and incubated with the indicated probes or antibodies. EMSA assessed the association of biotin-labeled or unlabeled oligonucleotides of mutant TBE2 containing the WTTBE2 or LINC00973 promoter with purified GST or GST-TCF 4. f. g is H1395 cells stably transfected with or without β -catensinshRNA, and then recombined with expression of designated RNA interference (RNAi) resistance (r) β -catenin (rβ -catenin) or transfected with or without miR-. 216b inhibitor (f) or miR-150 inhibitor (g). Cells were treated with or without EGF (100 ng/ml) for 24 hours. Immunoblot analysis was performed with the indicated antibodies. H. i is H1395 cells were stably transfected with or without LINC00973shRNA#1 or LINC00973shRNA#2, and then transfected with or without miR-216b inhibitor (H) or miR-150 inhibitor (i). Cells were treated with or without EGF (100 ng/ml) for 24 hours. Immunoblot analysis was performed with the indicated antibodies. j. k is H1395 and H322M cells have knock-in mutations (AT to GC) in TBE2 of the LINC00973 promoter, transfected with or without miR-216b inhibitor (j) or miR-150 inhibitor (k). Designated cells were treated with or without EGF (100 ng/ml) for 24 hours. Immunoblot analysis was performed with the indicated antibodies.

This region was found to contain three common LEF/TCF binding elements (TBE; CTTTG (A/T) (A/T)) (FIG. 8 a). Previous reports indicate that EGFR activation results in β -catenin mediated activation of LEF/TCF. Analysis of luciferase reporter genes in H1395 and H322M cells showed that EGF treatment significantly induced luciferase activity driven by the LINC00973 promoter, and that this activity was further enhanced by overexpression of β -catenin or TCF-4 and inhibited by expression of truncated and inactive TCF-4 mutants (FIG. 9 a). Furthermore, overexpression of active β -catenin (deletion of 1-89 amino acids) increased the expression level of endogenous LINC00973 (fig. 9 b). These results strongly indicate that β -catenin/LEF/TCF activation induced by EGFR activation enhances expression of LINC 00973.

To determine the key TBE of LINC00973 responsible for β -catenin modulation, we mutated ATs in TBEs 1-3 in H1395 and H322M to GC cells using CRISPR/Cas9 knock-in technology (fig. 8 b-c). Only TBE2 mutation reduced basal and EGF-induced LINC00973 expression (FIG. 9c, FIG. 8 d). Chromatin immunoprecipitation (ChIP) analysis with anti- β -catenin or anti-TCF 4 antibodies showed that EGF treatment induced binding of β -catenin or TCF4 to the WT promoter of LINC00973, but not to the promoter with TBE2 mutation (fig. 9 d). Furthermore, electrophoretic Mobility Shift Analysis (EMSA) showed that biotin-labeled oligonucleotides containing WT but not mutated TBE2 were able to bind to TCF4 (fig. 9 e). This association is reduced by unlabeled WTs rather than TBE2 mutated oligonucleotides and recognized by anti-TCF 4 antibodies, which results in mobility changes in the gel. These results indicate that the β -catenin/LEF/TCF complex binds to TBE2 of the LINC00973 promoter in response to EGFR activation and induces LINC00973 expression.

Depletion of β -catenin in H1395 and H322M cells inhibited EGF-induced CD55 (fig. 9 f) and CD59 (fig. 9 g); this inhibition was alleviated by recombinant expression of inhibitors of WT beta-catenin and miR-216b (FIG. 9 f) and miR-150 (FIG. 9 g). Similarly, expression of EGF-induced CD55 (FIG. 9 h) and CD59 (FIG. 9 i) by LINC00973 depletion inhibition was abrogated by expression of miR-216b inhibitor (FIG. 9 h) and miR-150 (FIG. 9 i). As expected, TBE2 (fig. 9j, k), but not TBE1 or TBE3 (fig. 8 i), mutant knock-in expression blocked EGF-induced expression of CD55 (fig. 9 j) and CD59 (fig. 9 k), and this effect was abrogated by expression of miR-216b inhibitor (fig. 9 j) and miR-150 (fig. 9 k), respectively. Furthermore, EGFRL858R, which occurs mainly in NSCLC, resulted in an increase in CD55 and CD59 levels in H1395 and H322M cells, and this increase was inhibited by β -catenin consumption or LINC00973 consumption, by TBE2 mutation knocked-in LINC00973 (fig. 8 l), by miR-216b expression (fig. 8M) or miR-150 (fig. 8 n). These results indicate that EGF-induced beta-catenin transactivation enhances expression of LINC00973, resulting in upregulation of CD55 and CD59 by adsorption of miR-216b and miR-150, respectively.

It is well known that β -catenin can be activated by WNT signaling. Human lung adenocarcinoma cells have been previously shown to express mainly Wnt-5A and Wnt-7B. Treatment of H1395 cells with these Wnt ligands showed that Wnt-7B, but not Wnt-5A, enhanced expression of LINC00973, CD55 and CD59 (fig. 8 o). Furthermore, quantitative PCR analysis of 10 Fzd receptors, LRP5 and LRP6 indicated that Fzd6 was highly expressed in H1395 cells (fig. 8 p). Frizzled6 consumption (fig. 8 q) reduced Wnt-7B-induced expression of LINC00973, CD55, and CD59 (fig. 8 r). These results indicate that Wnt signaling induces β -catenin transactivation-mediated upregulation of LINC00973, similar to EGFR activation, CD55 and CD59 in a frizzled 6-dependent manner.

EGFR/beta-catenin activation inhibits complement activation by upregulating miR-216b and miR-150 mediated CD55 and CD59 adsorbed by LINC00973

We next examined the effect of EGFR- β -catenin-LINC00973-miR-216b/miR-150 signaling-upregulated CD55 and CD59 on complement activation. In FIG. 10, a-c are H1395 cells with or without EGF (100 ng/ml) stimulation or H1395 cells with or without β -catenin hRNA expression or with knock-in mutation (AT to GC) in TBE2 of LINC00973 promoter for 24 hours. d-f is miR-216 b-micrometers, miR-150-micrometers, miR-216b-inhibitor, miR-150-inhibitor or shRNA aiming at CD55 and CD59 is expressed in H1395 cells. These cells and the corresponding parental cells were treated with or without EGF (100 ng/ml) for 24 hours. a. d is H1395 cells were incubated with 25% human serum and 25 μg/mllepirudin in DMEM medium (10% FBS) for 3H. Expression of C3b and C5b-9 on the surface of tumor cells was determined by flow cytometry with the indicated antibodies. Gray represents isotype control. Data are expressed as mean ± SD (n=6); ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. b. e is H1395 cells were incubated with 25% v/v human serum and 25. Mu.g/ml pellpirudin in DMEM medium (10% FBS) for 3 hours. The anaphylatoxins (C3 a and C5 a) in the supernatant were determined by ELISA. Data are expressed as mean ± SD (n=6); ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. c. f is H1395 cells co-cultured with human PBMC. The expression of the cytokines indicated in the medium was examined. Data are expressed as mean ± SD of seven independent experiments using different human donors; ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2.

In FIG. 11, a-c are H322M cells expressing or not expressing β -catenin shRNA in TBE2 of LINC00973 promoter or H322M cells with knock-in mutation (AT to GC) stimulated with or without EGF (100 ng/ml) for 24 hours. d-f is miR-216 b-micrometers, miR-150-micrometers, miR-216b-inhibitor, miR-150-inhibitor or shRNA aiming at CD55 and CD59 is expressed in H322M (d-f) or H1395 (d, e) cells. Cells and corresponding parental cells were treated with or without EGF (100 ng/ml) for 24 hours. a. d is the incubation of the indicated cells with DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25. Mu.g/ml pirudin for 3 hours. Expression of C3b and C5b-9 on the surface of tumor cells was determined by flow cytometry with the indicated antibodies. Gray represents isotype control. Data are expressed as mean ± SD (n=6); ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. b. e is the incubation of the indicated cells with DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25. Mu.g/ml pirudin for 3 hours. The anaphylatoxins (C3 a and C5 a) in the supernatant were determined by ELISA. Data are expressed as mean ± SD (n=6); ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. c. f cells were co-cultured with human PBMC. The expression of the indicated cytokines was detected. Data are expressed as mean ± SD of seven independent experiments using different human donors. ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2.g-i H1395 cells and EGFRL858R expression H1395 cells were constructed that expressed or not expressed β -catenin shRNA or knock-in mutation (AT to GC) in TBE2 of the LINC00973 promoter. j-l is shRNA expressed or not expressed in TBE2 expressing LINC00973 promoter in H1395 cells and EGFR 858R expressed H1395 cells with knock-in mutation (AT to GC) or CD55 and CD 59. The miR-216b inhibitor and miR-150 inhibitor are expressed in the indicated cells. g. j is the incubation of the indicated cells with DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25. Mu.g/ml pirudin for 3 hours. Expression of C3b and C5b-9 on the surface of tumor cells was determined by flow cytometry with the indicated antibodies. Gray represents isotype control. Data are expressed as mean ± SD (n=6); ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. h. k is the incubation of the indicated cells with DMEM medium (10% fbs) supplemented with 25% v/v human serum and 25 μg/ml pirudin for 3 hours. The anaphylatoxins (C3 a and C5 a) in the supernatant were determined by ELISA. Data are expressed as mean ± SD (n=6); ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. i. l cells were co-cultured with human PBMCs. The expression of the indicated cytokines was detected in the medium. Data are expressed as mean ± SD of seven independent experiments using different human donors; ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2.

We found that β -catenin knockdown and TBE2 mutant knock-in expression abrogated EGF-induced inhibition and triggered complement activation, which was reflected in enhanced deposition of C3b and C5b-9 on the tumor cell surface (FIG. 10 a), release of C3a and C5a (FIG. 10 b), and secretion of IFN- γ, TNF- α, IL-6, IL-1β and IL-17 (FIG. 10C). Furthermore, consumption of miR-216b or miR-150 reduced complement activation and abrogated EGF-induced complement regulation, whereas overexpression of miRNA-150 or miR-216b or consumption of CD55 and CD59 enhanced complement activation, which was resistant to EGF-dependent inhibition (FIG. 10 d-f). Similar to EGF treatment, expression of EGFRL858R in H1395 cells resulted in similar inhibition of complement activation, depending on β -catenin and TBE2 mediated up-regulation of LINC00973 (fig. 11 g-i). This inhibition was abrogated by the depletion of CD55 and CD59, while depletion of miR-216b and miR-150 inhibited TBE2 mutation-enhanced complement activation (FIGS. 11j, 11k and 11 l). These results indicate that EGFR activation-induced β -catenin transactivation in NSCLC cells inhibits complement activation by upregulating CD55 and CD59 by LINC 00973-adsorbed miR-216b and miR-150.

EGFR/beta-catenin activation inhibits immune cell function by upregulating CD55 and CD59

To determine the effect of EGFR-inhibited complement activation on immune cell-mediated tumor cell survival, we expressed nanoluciferases in H1395 and H322M cells, which were co-cultured with PBMCs in human serum.

In FIG. 12, a, b are H1395 cells expressing or not expressing β -catenin hRNA or knock-in mutation (AT to GC) in TBE2 of LINC00973 promoter stimulated with or without EGF (100 ng/ml) for 24 hours. C1, clone 1; c2, clone 2. c. d is miR-216 b-micrometers, miR-150-micrometers, miR-216b-inhibitor, miR-150-inhibitor or shRNA aiming at CD55 and CD59 is expressed in H1395 cells. These cells and the corresponding parental cells were treated with or without EGF (100 ng/ml) for 24 hours. a. c the indicated cells were stably transfected with vector expressing nano-luciferase before 24 hours treatment with or without EGF (100 ng/ml). These cells were co-cultured with human PBMCs. The CARIA assay was performed using the nano-luciferase release method. Data are expressed as mean ± SD of seven independent experiments using different human donors. ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. b. d is incubation of the indicated cells with DMEM medium (10% fbs) supplemented with 25% v/v human serum and 25 μg/ml pirudin for 3 hours, followed by co-culture with human PBMC. MTS assay was performed. Data are expressed as mean ± SD of seven independent experiments using different human donors; ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2.E is H1395 cells with or without β -catenin hRNA expression in TBE2 of LINC00973 promoter or H1395 cells with knock-in mutation (AT to GC) were stimulated for 24 hours with or without EGF (100 ng/ml). Representative images of live cell microscopy of phagocytic pHrodored+ tumor cells (t=4h) are shown; these images represent six donors and six experimental replicates (left panel). Normalized phagocytosis rate of tumor cells (n=6 donors) was specified. Data are expressed as mean ± SD of six independent experiments using different human donors; ns, not significant; * P <0.001 (right panel). F is miR-216 b-micrometers, miR-150-micrometers, miR-216b-inhibitor, miR-150-inhibitor or shRNA aiming at CD55 and CD59 is expressed in H1395 cells. These cells and the corresponding parental cells were treated with or without EGF (100 ng/ml) for 24 hours. Representative images of phagocytosis assays using a live cell microscope of the pHrodored+ tumor cells (t=4h) are shown; these images represent six donors and six experimental replicates (left panel). Normalized phagocytosis rate of tumor cells (n=6 donors) was specified. Data are expressed as mean ± SD of six independent experiments using different human donors; ns, not significant; * P <0.01 (right panel). G is H1395 cells expressing or not expressing β -catenin hRNA or knock-in mutation (AT to GC) in TBE2 of LINC00973 promoter were stimulated with or without EGF (100 ng/ml) for 24 hours. H is miR-216b-mimics and miR-150-mimics are expressed in H1395 cells. These cells and the corresponding parental cells were treated with or without EGF (100 ng/ml) for 24 hours. g. h is incubation of designated cells for 3 hours with DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25 μg/ml pirudin, followed by co-culture with human PBMC. The expression of granzyme B and perforin in cd8+ T cells was detected by flow cytometry with the indicated antibodies. Data are expressed as mean ± SD of seven independent experiments using different human donors; ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. H1395 cells were constructed in I, with or without expression of shRNA or knock-in mutations (AT to GC) for-catenin or CD55 and CD59 in TBE2 of the LINC00973 promoter. Designated cells were incubated with 25% v/v human serum and 25 μg/ml pirudin in DMEM medium (10% fbs) for 3 hours and then co-cultured with human PBMCs in medium with or without anti-CD 8 monoclonal antibodies. MTS assay was performed. Data are expressed as mean ± SD of seven independent experiments using different human donors; ns, not significant; * P < 0.001. C1, clone 1.

In FIG. 13, a, b are H322M cells expressing or not expressing β -catenin shRNA in TBE2 of LINC00973 promoter or H322M cells with knock-in mutation (AT to GC) stimulated with or without EGF (100 ng/ml) for 24 hours. c. d is miR-216b mimetic, miR-150 mimetic, miR-216 b-inhibitor, miR-150-inhibitor or shRNA targeting CD55 and CD59 is expressed in H322M cells. These cells and the corresponding parental cells were treated with or without EGF (100 ng/ml) for 24 hours. e. f is H1395 cells and EGFRL858R expression H1395 cells were constructed that expressed or not expressed β -catenin shRNA or knock-in mutation (AT to GC) in TBE2 of the LINC00973 promoter. g. H is H1395 cells and EGFRL858R expression H1395 cells were constructed that expressed or not expressed shRNA or knock-in mutation (AT to GC) for CD55 and CD59 in TBE2 of the LINC00973 promoter. The miR-216b inhibitor and miR-150 inhibitor are expressed in the indicated cells. a. c, the designated cells were stably transfected with the vector expressing the nano-luciferase before 24 hours of treatment with or without EGF (100 ng/ml). Cells were then co-cultured with human PBMCs. The CARIA assay was performed using the nano-luciferase release method. Data are expressed as mean ± SD of seven independent experiments using different human donors. ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. b. d designated cells were incubated with 25% v/v human serum and 25 μg/ml DMEM medium (10% fbs) with 25 μg/ml human serum for 3h after 24 hours treatment with or without EGF (100 ng/ml) and then co-cultured with human PBMCs. MTS assay was performed. Data are expressed as mean ± SD of seven independent experiments using different human donors; ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. e. g is the cell stably transfected with the vector expressing the nano-luciferase. Cells were then co-cultured with human PBMCs. The CARIA assay was performed using the nano-luciferase release method. Data are presented as mean ± standard deviation of seven independent experiments using different human donors. ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. f. h is incubation of designated cells for 3 hours with DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25 μg/ml pirudin, followed by co-culture with human PBMC. MTS assay was performed. Data are expressed as mean ± SD of seven independent experiments using different human donors. ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2.

In fig. 14, a is the normalized phagocytosis of H1395 and H322M cells by M0, M1 and M2 macrophages (n=4 donors). Data are expressed as mean ± SD of four independent experiments using different human donors. * P <0.05; * P <0.001.b is H322M cells with or without beta-catenin shRNA expression or H322M cells with knock-in mutation (AT to GC) in TBE2 of LINC00973 promoter stimulated with or without EGF (100 ng/ml) for 24 hours. C1, clone 1; c2, clone 2. Representative images of the phagocytosis of the pHrodored+ tumor cells (t=4h) were obtained by live cell microscopy; these images represent six donors and six experimental replicates (left panel). Normalized phagocytosis rate of tumor cells (n=6 donors) was specified. Data are expressed as mean ± SD of six independent experiments using different human donors. ns, not significant; * P <0.001 (right panel). C is miR-216 b-mimetic, miR-150-mimetic, miR-216 b-inhibitor, miR-150-inhibitor or shRNA for CD55 and CD59 is expressed in H322M cells. Cells and corresponding parental cells were treated with or without EGF (100 ng/ml) for 24 hours. Representative images of the phagocytosis of the pHrodored+ tumor cells (t=4h) were obtained by live cell microscopy; these images represent six donors and six experimental replicates (left panel). Normalized phagocytosis rates of designated tumor cells (n=6 donors) are shown. Data are expressed as mean ± SD of six independent experiments using different human donors. ns, not significant; * P <0.001 (right panel). d is a gating strategy for in vitro phagocytosis assays. After removal of the fragments and doublets, phagocytosis was assessed as the frequency of Zombioviolet-CD11b+ FITC+ events normalized to all Zombioviolet-CD11b+ events. The number indicates the frequency of the previous off-door event. These figures represent at least 12 experimental replicates. e H1395 cells and EGFRL858R expression H1395 cells were constructed that expressed or not expressed β -catenin shRNA or knock-in mutation (AT to GC) in TBE2 of the LINC00973 promoter. C1, clone 1; c2, clone 2, represents a flow cytometry graph showing phagocytosis of H1395 cells. These figures represent six donors. FITC, fluorescein isothiocyanate (left panel). Normalized phagocytosis rates of the indicated cells (n=6 donors) are shown. Data are expressed as mean ± SD of six independent experiments using different human donors. ns, not significant; * P <0.01 (right panel). f is H1395 cells and EGFRL858R expression H1395 cells were constructed that expressed or not expressed shRNA or knock-in mutation (AT to GC) for CD55 and CD59 in TBE2 of the LINC00973 promoter. The miR-216b inhibitor and miR-150 inhibitor are expressed in the indicated cells. Normalized phagocytosis rates of the indicated cells (n=6 donors) are shown. Data are expressed as mean ± SD of six independent experiments using different human donors. ns, not significant; * P < 0.01. C1, clone 1.g is H1395 cells and EGFRL858R expression H1395 cells were constructed that expressed or not expressed β -catenin shRNA or knock-in mutation (AT to GC) in TBE2 of the LINC00973 promoter. H is miR-216 b-chemicals and miR-150-chemicals express H1395 cells in H1395 cells and EGFR 858R-expressing cells. g. h is the incubation of the indicated cells with DMEM medium (10% FBS) supplemented with 25% v/v human serum and 25. Mu.g/ml pirudin for 3 hours, followed by co-incubation with human PBMC. The expression of granzyme B and perforin in cd8+ T cells was detected by flow cytometry with the indicated antibodies. Data are expressed as mean ± SD of seven independent experiments using different human donors. ns, not significant; * P <0.001. C1, clone 1; c2, clone 2. H322M cells were constructed in i, with or without shRNA or knock-in mutations (AT to GC) for-catenin, CD55 and CD59 expressed in TBE2 of LINC00973 promoter. Designated cells were incubated with 25% v/v human serum and 25 μg/ml pirudin in DMEM medium (10% fbs) for 3 hours and then co-cultured with human PBMC in medium with or without anti-CD 8 monoclonal antibodies for 30 hours. MTS assay was performed. Data are expressed as mean ± SD of seven independent experiments using different human donors. ns, not significant; * P <0.001. C1, clone 1.

Complement activation mediated immune cell attack (CARIA) analysis showed that EGF treatment inhibited complement activation induced cytotoxicity, as reflected by inhibition of nano-luciferase release from tumor cells (fig. 12 a) and enhanced tumor cell survival (fig. 12 b). This modulation was abolished by β -catenin consumption or knock-in expression of TBE2 mutants of LINC00973, which greatly enhanced CARIA and tumor cell death (fig. 12 a-b). Similar enhancement is caused by overexpression of miRNA-150 and miR-216b or depletion of CD55 and CD 59; in contrast, consumption of miR-216b and miR-150 reduced CARIA and increased cell viability (FIGS. 12 c-d). Notably, the immune cell response mediated by EGFR/β -catenin transactivation was summarized by expression of EGFR L858R in these cells (FIGS. 13 e-f). Furthermore, the EGFR 858R-inhibited CARIA (FIG. 13 g) and cell death (FIG. 13 h) were eliminated due to the consumption of CD55 and CD59, while the consumption of miR-216b and miR-150 inhibited TBE2 mutant-enhanced CARIA and cell death (FIG. 13 g-h).

C3b produced by complement activation coats the pathogen surface and interacts with the macrophage's complement receptor 3 (CR 3 or macrophage 1 antigen) to promote phagocytosis of the pathogen by macrophages. We labeled tumor cell membranes with borored succinimidyl ester, which does not fluoresce outside the cell in a neutral environment and exhibits red fluorescence in the acid phagosome of macrophages. Incubation of labeled tumor cells with non-activated (M0), pro-inflammatory/anti-tumor (M1) or anti-inflammatory/pro-tumor (M2) macrophages indicated that M1 macrophages phagocytose tumor cells much more active than M0 and M2 macrophages (fig. 14 a). Notably, EGF treatment protected H1395 cells from M1 macrophage-mediated phagocytosis (fig. 12 e). Knock-in expression of TBE2 mutant of LINC00973 (fig. 12 e), overexpression of miRNA-150 and miR-216b or deletion of CD55 and CD59 (fig. 12 f) abrogated EGF-induced inhibition and greatly enhanced phagocytosis of macrophages. In contrast, consumption of miR-216b and miR-150 decreased phagocytosis (FIG. 12 f). Notably, a similar modulation was observed by FACS-based measurement of phagocytosis of tumor cells expressing EGFRL858R (fig. 14 d) (fig. 14 e). Furthermore, EGFRL858R inhibited phagocytosis was eliminated by CD55 and CD59 depletion, while miR-216b and miR-150 depletion inhibited TBE2 mutant-enhanced macrophage phagocytosis (fig. 14 f). These results indicate that EGFR activation-induced beta-catenin transactivation inhibits macrophage phagocytosis by miR-216b and miR-150-mediated upregulation of CD55 and CD59 adsorbed by LINC 00973.

EGFR activation inhibited CD8+ T cell function, either by β -catenin depletion, knock-in expression of the TBE2 mutant of LINC00973 (FIG. 12 g) or by over-expression of miR-216b and miRNA-150 (FIG. 12 h). Notably, β -catenin knockdown, expression of TBE2 mutant of LINC00973, or knockdown of CD55 and CD59 resulted in enhanced tumor cell death, which was greatly reduced by the deletion of cd8+ T cells (fig. 12 i). In summary, EGFR/β -catenin activation up-regulates CD55 and CD59 to promote immune escape of tumor cells by inhibiting the complement system and the activity of macrophages and cd8+ T cells.

Enhanced expression of CD55 and CD59 by EGFR/beta-catenin transactivation promotes tumor growth by inhibiting mouse complement activation and immune cell activation

To obtain immunocompetent syngeneic mice for animal studies, we identified mouse MLINC00973 (accession sequence ID: NC_000082.7; chromosome 16) as a homolog of LINC00973, with 75.96% identity. In FIG. 15, a is the 5 'and 3' sequences of MLINC00973 obtained using RACE. PCR products amplified from known regions of the 5 'and 3' sequences of MLINC00973 and the MLINC00973 sequences of the national center for Biotechnology information database were separated on agarose gels. The size of the sequence is expressed according to the sequencing result of the PCR product. M, marking; l1, lane 1. The complete sequence of MLINC00973 in b was obtained using RACE. The 5 'and 3' sequences of MLINC00973 were sequenced using the PCR products obtained with the primers. The resulting MLINC00973 full-length sequence is shown. c is a LEF/TCF binding element (TBE; CTTTG (A/T) (A/T)) map in the promoter region of MLINC 00973. d is genomic DNA extracted from two separate clones of LA795 cells with knock-in mutations in the two TBEs (AT to GC or AA to GG as shown). PCR products were amplified from the indicated DNA fragments and separated on agarose gel. C1, clone 1; c2, clone 2. The parent cell and the sequences of two separate clones of LA795 cells with knock-in mutations in both TBEs (AT to GC or AA to GG as shown) are shown. The red line with arrow indicates the sgRNA targeting sequence. The red line without arrow indicates Protospacer Adjacent Motif (PAM). Two TBEs with or without mutated nucleotides are indicated by red solid boxes. Silent mutations of specified nucleotides were introduced into the sequence to avoid repeated cleavage by Cas 9. e parental or MLINC00973TBE mutant knock-in expressing LA795 cells were stably transfected with or without EGFRL 858R. The relative expression level of MLINC00973RNA was measured using quantitative PCR. Data are expressed as mean ± SD (n=6). ns, not significant; * P < 0.001. C1, clone 1; c2, clone 2. A graph of the binding sites of mmu-miR-216b and mma-miR-150 on lncRNAMLINC00973 is shown in f. The LA795 cells in g were stably transfected with miR-Control, mmu-miR-216b or mmu-miR-150 with or without EGFR 858R. Immunoblot analysis was performed with the indicated antibodies. h is LA795 cells stably transfected with or without beta-catenin shRNA with or without EGFR 858R expression in the presence or absence of mma-miR-216 b/mma-miR-150 adsorption expression. Immunoblot analysis was performed with the indicated antibodies. i is parental or MLINC00973TBE4 mutant knock-in expressing LA795 cells stably transfected with or without EGFR L858R in the presence or absence of mma-miR-216 b/mma-miR-150 adsorption. Immunoblot analysis was performed with the indicated antibodies. C1, clone 1; c2, clone 2.

In FIG. 16, a-g are LA795 cells (1X 10) expressing GFP ⁶ ) Stable transfection with or without EGFRL858R in the presence or absence of MLINC00973 knock-in TBE4 mutant (AT to GC) or CD55 and CD59shRNA expression. Cells were subcutaneously injected into immunocompetent isogenic "615" mice. The resulting tumor was resected 23 days after injection. Tumors were measured in a (n=6/group) (left panel). Excised tumors were weighed (right panel). Data are expressed as mean ± SD (n=6). ns, not significant; * P:<0.001. c1, clone 1; c2, clone 2.b, overall survival of mice (n=10/group) was analyzed by Kaplan-Meier plot. P-values were calculated using a log rank test (two-tailed). Number of digitsExpressed as mean ± SD (n=10); ns, not significant; * P:<0.001. c1, clone 1; c2, clone 2.c is IHC and ISH analysis of tumors from mice (n=6/group) with the indicated antibodies. Representative images are displayed. The area in the red frame is displayed at a higher magnification. The significance of the expression differences between the groups was analyzed by a non-parametric test. Data are expressed as mean ± SD (n=6). * P:<0.001. c1, clone 1; c2, clone 2.d is a single cell suspension obtained from tumor fragments of mice (n=8/group) by digestion with collagenase IV. Tumor fragments were homogenized and filtered through a 70 μm cell filter. Tumor cells expressing GFP were obtained by flow cytometry. Expression of the tumor cell surfaces C3b and C5b-9 was determined by flow cytometry with the indicated antibodies. Data are expressed as mean ± SD (n=8). * P (P) <0.05，***P<0.01，***P<0.001. C1, clone 1; c2, clone 2.e is the collection and lysis of 100 mg of minced tumor tissue from mice. The tissue was homogenized and adjusted to 10mg/ml. The CARIA assay was performed using the nano-luciferase release method. Data are expressed as mean ± SD (n=10). * P:<0.001. c1, clone 1; c2, clone 2. The frequency of phagocytosis events in total TAMs in tumors is specified in f. Data are expressed as mean ± SD (n=10). * P<0.01；***P<0.001. C1, clone 1; c2, clone 2.g is the single cell suspension obtained from the tumor fragments of the mice by digestion with collagenase D. Tumor fragments were homogenized and filtered through a 100 μm cell filter. The percent of granzyme b+ or perforin+ cells in tumor-infiltrating cd8+ T cells was analyzed by flow cytometry. Data are expressed as mean ± SD (n=6). * P (P)<0.05，***P<0.001. C1, clone 1. TUNEL analysis was performed on designated tumor samples from mice (n=10/group) in h. Apoptotic cells were stained green (left panel) and quantified in microscopic fields (right panel). Data are expressed as mean ± SD (n=10). * P:<0.001. c1, clone 1; c2, clone 2.i is LA795 cells stably transfected with EGFRL858R 4 weeks after virus injection (1×10) with or without the presence of MLINC00973 knock-in TBE4 mutant (AT to GC) or expression of CD55 and CD59shRNA ⁶ ) Tumors (n=6/group) were measured (left panel) and resected 17 days after injection. For a pair ofExcised tumors were weighed (right panel). ns, not significant; * P:<0.001. c1, clone 1.j-l is the stable transfection of H1395 cells with or without EGFR 858R in the presence or absence of LINC00973 knock-in TBE2 mutant (AT to GC) or expression of CD55 and CD59shRNA (4X 10) ⁶ ). These cells were injected subcutaneously into humanized NOG mice with recovered human complement system by intraperitoneal injection of Human Serum (HS). These mice were treated or not with anti-PD-1 or anti-CD 55/CD59 antibodies. j is the tumor measured (n=6/group) (left panel) and resected 26 days after injection. Excised tumors were weighed (right panel). Data are expressed as mean ± SD (n=6). ns, not significant; * P:<0.05. c1, clone 1.k is the overall survival of mice (n=10/group) analyzed by Kaplan-Meier plot. P-values were calculated using a log rank test (two-tailed). Data are expressed as mean ± SD (n=10). ns, not significant; * P:<0.001. c1, clone 1.l is a single cell suspension obtained from a tumor fragment of a mouse by digestion with collagenase D. Tumor fragments were homogenized and filtered through a 100 μm cell filter. The percent of granzyme b+ or perforin+ cells in tumor-infiltrating cd8+ T cells was analyzed by flow cytometry. Data are expressed as mean ± SD (n=6). ns, not significant; * P (P) <0.05，**P<0.01，***P<0.001. C1, clone 1.

In fig. 17, a is an analysis of immunofluorescence with a designated antibody. b. c is LA795 cells expressing GFP (1×10 ⁶ ) Stable transfection with or without EGFRL858R in the presence or absence of MLINC00973 or knock-in TBE4 mutant (ATtoGC) expression of CD55 and CD59 shRNA. Cells were subcutaneously injected into immunocompetent isogenic "615" mice. The resulting tumor was resected 23 days after injection. 100 mg of minced tumor tissue from mice was collected and lysed. The tissue was homogenized and adjusted to 10mg/ml. The release of anaphylatoxins (C3 a and C5 a) in the Tumor Microenvironment (TME) was determined by ELISA. Data are expressed as mean ± SD (n=9). b the cytokine protein level of tumor lysate in TME was detected by ELISA. Data are expressed as mean ± SD (n=6). P in c<0.05，**P<0.01，***P<0.001. C1, clone 1; c2, clone 2.d is the gate of TAM phagocytosis in LA795 cellsAnd (5) controlling strategies. After debris removal and bimodal TAM phagocytosis was assessed as the frequency of zombie violet-cd11b+f4/80+gfp+ events normalized to the total number of zombie violet-cd1b+f4/80+ events. The number indicates the frequency of the previous off-door event. These figures represent eight experimental replicates. e is a representative flow cytometry pattern demonstrating TAM phagocytosis in the indicated gfp+ LA795 tumors. Numbers represent the frequency of phagocytosis events normalized to all TAMs. C1, clone 1; c2, clone 2.f is a representative result of granzyme B and perforin expression in tumor-infiltrating cd8+ T cells. g-o is LA795 cells expressing GFP (1X 10) ⁶ ) Adsorption in the presence or absence of MLINC00973 or mmu-miR-216 b/mmu-miR-150. These cells were subcutaneously injected into immunocompetent isogenic "615" mice. The resulting tumor was resected 23 days after injection. ns is not significant. * P (P)<0.05，***P<0.01，***P<0.001. C1, clone 1. Tumors were measured in g (n=6/group) (left panel). Excised tumors were weighed (right panel). Data are expressed as mean ± SD (n=6). h, overall survival of mice (n=10/group) was analyzed by Kaplan-Meier plot. P-values were calculated using a log rank test (two-tailed). Data are expressed as mean ± SD (n=10). i is IHC and ISH analysis of tumors from mice (n=6/group) with the indicated antibodies and probes. Representative images are displayed. The area in the red frame is displayed at a higher magnification. The significance of the expression differences between the groups was analyzed by a non-parametric test. Data are expressed as mean ± SD (n=6). j is a single cell suspension obtained from tumor fragments of mice (n=8/group) by digestion with collagenase IV. Tumor fragments were homogenized and filtered through a 100 μm cell filter. Tumor cells expressing GFP were obtained by flow cytometry. Expression of C3b and C5b-9 on the surface of tumor cells was determined by flow cytometry with the indicated antibodies. Data are expressed as mean ± SD (n=8). k. l, m are 100 mg of minced tumor tissue collected and lysed from mice. The tissue was homogenized and adjusted to 10mg/ml. The release of anaphylatoxins (C3 a and C5 a) in the Tumor Microenvironment (TME) was determined by ELISA. Data are expressed as mean ± SD (n=9) (k). Cytokine protein levels of lysates in TME were detected by ELISA. Data are expressed as mean ± SD (n=6) (l). Use of nano-luciferases The release method was used for the CARIA assay. Data are expressed as mean ± SD (n=10) (m). n is the frequency of phagocytosis events normalized to all TAMs in a given tumor. Data are expressed as mean ± SD (n=9). o is TUNEL analysis was performed on designated tumor samples from mice (n=10/group). Apoptotic cells were stained green (left panel) and quantified in microscopic fields (right panel). Data are expressed as mean ± SD (n=10). p-r is "615" mice (n=6/group) express non-targeted shRNA (AAV 8-shrrol) or adeno-associated virus 8 (AAV 8) of complement C3shRNA and C5shRNA (AAV 8-C3/C5 shRNA) by tail vein injection data expressed as mean ± SD (n=6). * P:<0.001. mice were euthanized 4 weeks after plasmid injection. 100 mg of crushed liver tissue from mice was collected. The relative expression levels of C3 and C5mRNA in liver tissue were measured using quantitative PCR (p). Liver tissue was lysed, homogenized and adjusted to 10 μg/ml. The lysates were assayed for mouse complement C3 and C5 protein levels by ELISA (q). Peripheral blood was collected from mice and mouse complement C3 and C5 protein levels were probed by ELISA (r).

In FIG. 18, a, b are stable transfections of H322M cells with or without EGFR 858R in the presence or absence of LINC00973 knock-in TBE2 mutant (AT to GC) or CD55 and CD59shRNA expression (6X 10) ⁶ ). These cells were injected subcutaneously into humanized NOG mice with recovered human complement system by intraperitoneal injection of Human Serum (HS). These mice were treated or not with anti-PD-1 or anti-CD 55/CD59 antibodies. ns is not significant. * P<0.01，***P<0.001. C1, clone 1.a is the tumor measured (n=6/group) (left panel) and resected 26 days after injection. Excised tumors were weighed (right panel). Data are expressed as mean ± SD (n=6). b is the overall survival of mice (n=10/group) analyzed by Kaplan-Meier plot. P-values were calculated using a log rank test (two-tailed). Data are expressed as mean ± SD (n=10). c is treatment of designated cells with or without Wnt-7B (200 ng/ml) with or without XAV939 (10 μm) for 12 hours. The relative expression levels of CD55 and CD59 were measured for mRNA using quantitative PCR. Data are expressed as mean ± SD (n=4). ns, not significant; * P:<0.001.d is treatment of designated cells with or without Wnt-7B (200 ng/ml), with or without XAV939 (10. Mu.M) for 24 hours. By means of the indicated antibodiesImmunoblot analysis. e. f is H1395 cells (4X 10) by intraperitoneal injection of Human Serum (HS) ⁶ ) Subcutaneous injection into humanized NOG mice with recovered human complement system. These mice were treated or untreated with anti-PD-1 antibodies and/or XAV 939. * P (P) <0.05；***P<0.001.e is the tumor measured (n=6/group) (left panel) and resected 26 days after injection. Excised tumors were weighed (right panel). Data are expressed as mean ± SD (n=6). ns is not significant. f is the overall survival of mice (n=10/group) analyzed by Kaplan-Meier plot. P-values were calculated using a log rank test (two-tailed). Data are expressed as mean ± SD (n=10). ns is not significant. g is MC38 cells (2X 10) expressing APCshRNA ⁶ ) Stably transfected with or without CD55 and CD59 shRNA. These cells were subcutaneously injected into C57Bl/6 mice. These mice were treated or untreated with anti-PD-1 antibodies. Tumors (n=6/group) (left panel) were measured and resected 13 days after injection. Excised tumors were weighed (right panel). Data are expressed as mean ± SD (n=6). * P:<0.001.h is the injection of H1395 cells (4X 10) by intraperitoneal injection of Human Serum (HS) ⁶ ) Subcutaneously into humanized NOG mice with recovered human complement system. These mice were treated or untreated with anti-PD-1 antibodies or anti-CD 55 and anti-CD 59 antibodies. Tumors (n=6/group) (left panel) were measured and resected 26 days after injection. Excised tumors were weighed (right panel). Data are expressed as mean ± SD (n=6). * P <0.01；***P<0.001. i. j, k are H1395 cells expressing EGFRL858R (4X 10) ⁶ ) The human complement system was recovered by intraperitoneal injection of Human Serum (HS) subcutaneously into humanized NOG mice. These mice received gefitinib treatment or not on day 11. Tumors were resected 26 days after injection. i is the tumor measured (n=6/group) (left panel). Excised tumors were weighed (right panel). Data are expressed as mean ± SD (n=6). * P:<0.001.j is immunoblot analysis with the indicated antibodies. k is immunohistochemical staining with the indicated antibodies.

RACE analysis (FIG. 15 a) revealed the full-length sequence of the MLINC00973 transcript (FIG. 15 b). The promoter region of MLINC00973 has two TBEs (TBE 4 and TBE 5) (FIG. 15 c). Knocking AT into TBE of LA795 mouse lung adenocarcinoma cells using CRISPR/Cas9 technology either GC-to-GC or AA-to-GG (fig. 15 d) showed that only TBE4 mutation reduced basal and EGFRL 858R-induced MLINC00973 expression (fig. 15 e).

Analysis of the recognition sequences by the miRcode prediction algorithm showed that MLINC00973 contained two single sequences for mouse mmi-miR-216 b and mmi-miR-150, respectively (fig. 15 f). As expected, EGFR 858R expression enhanced proteins in CD55 and CD59 expressing LA795 cells, and this enhancement was inhibited by overexpression of mmu-miR-216b and mmu-miR-150, respectively (FIG. 15 g). Notably, EGFRL 858R-enhanced CD55 and CD59 expression was abolished by β -catenin depletion (fig. 15 h) or knock-in expression of TBE4 mutant of MLINC00973 (fig. 15 i); these inhibitory effects were alleviated by stable transfection of vectors expressing oligonucleotides directed against both mmu-miR-216b and mmu-miRNA-150 (mmi-miR-216 b/150-spike) (FIGS. 15h, 15 i). These results indicate that LA795 mouse lung adenocarcinoma cells share the same EGFR activation up-regulation mechanism for CD55 and CD59 as human NSCLC cells.

EGFRL858R expression significantly increased tumor growth (FIG. 16 a), shortened mice survival time (FIG. 16B), enhanced expression and activity of CD55, CD59, MLINC00973 beta-catenin (FIG. 16C), reduced accumulation of C3B and C5B-9 (FIG. 16C, 16 d) on tumor cell surfaces (FIG. 17 a) and release of anaphylatoxins (17B), reduced secretion of IFN-gamma, TNF-alpha, IL-6, IL-1 beta and IL-17 (17C), inhibited CARIA in tumor tissue (FIG. 16 e), reduced phagocytosis of tumor cells by infiltrated tumor-associated macrophages (TAM) (FIG. 16 f), and reduced expression of granzyme B and perforin in tumor infiltrating CD8+ T cells (FIG. 16 g). Knock-in mutations in TBE4 of MLINC00973 abrogated these changes, and depletion of CD55 and CD59 abrogated these changes (fig. 16 a-g), which also increased cell death detected by TUNEL detection (fig. 16 h). Notably, the effects caused by the TBE4 mutation of MLINC00973 were offset by the depletion of mmu-miRNA-216b and mmu-miR-150 (FIG. 17 g-o).

C3 and C5 are synthesized mainly in the liver and circulate in the blood, usually as inactive precursors. AAV8 viruses expressing C3shRNA and C5shRNA (AAV 8-C3/C5 shRNAs) by tail intravenous injection reduced expression of C3 and C5 mRNAs (fig. 17 p) and proteins (fig. 17 q) in mouse liver and reduced levels of C3 and C5 in mouse peripheral blood (fig. 17 r). Notably, depletion of C3 and C5 in mice promoted tumor growth and abrogated tumor suppression induced by expression of TBE4 mutant of MLINC00973 or depletion of CD55 and CD59 (fig. 16 i). These results indicate that beta-catenin transactivation enhanced CD55 and CD59 expression promotes tumor immune evasion and tumor growth by inhibiting mouse complement activation and cd8+ T cell activation.

Given the limited therapeutic efficacy of programmed death 1 (PD-1) antibody treatment on human NSCLC (66-68) with EGFR activation, we next examined the effect growth on tumors of the combined inhibition of CD55 and CD59 expression and immune checkpoint blockade. We subcutaneously injected H1395 or H322M cells into humanized NOG mice to restore the human immune system and human complement system by transplanting human cd34+ hematopoietic stem cells and intraperitoneally injecting Human Serum (HS). Treatment with antibodies against CD55 and CD59 resulted in similar tumor growth inhibition (fig. 16j, fig. 18 a), prolonged mice survival (fig. 16k, fig. 18B), and enhanced expression of granzyme B and perforin in tumor infiltrating cd8+ T cells (fig. 16 l), whereas treatment with anti-PD-1 antibodies had limited efficacy compared to the results resulting from expression of TBE2 mutant of LINC00973 or consumption of CD55 and CD 59. Notably, the combination of antibodies against CD55, CD59 and PD-1 resulted in significantly enhanced synergistic inhibition of tumor growth (FIG. 16j, FIG. 18 a) and significantly prolonged survival of mice (FIG. 16k, FIG. 18B), accompanied by a greatly increased expression of granzyme B and perforin in tumor-infiltrating CD8+ T cells (FIG. 16 l).

Consistently, inhibition of β -catenin with the tankyrase inhibitor XAV939, elimination of Wnt-7B-induced mRNA (fig. 18 c) and protein (fig. 18 d) expression of CD55 and CD59, inhibition of tumor growth (fig. 18 e) and prolonged survival of mice (fig. 18 f). Furthermore, combined treatment with XAV939 and anti-PD-1 antibodies resulted in synergistic inhibition of tumor growth (fig. 18 e) and greatly prolonged survival of mice (fig. 18 f). Consistent with these findings, CD55/CD59 depletion reduced tumor growth from APC-depleted MC38 mouse colorectal cancer (CRC) cells and showed a synergistic effect on tumor growth inhibition in combination with anti-PD-1 treatment (fig. 18 g), which revealed an improvement in anti-tumor effects by targeting complement and immune checkpoints in EGFR-activated NSCLC and β -catenin-activated CRC. Given that CD55 and CD59 expression can be regulated by EGFR or Wnt, CD55/CD59 antibody treatment also inhibited the growth of tumors derived from H1395 cells that did not express EGFRL858R (fig. 18H). As expected, gefitinib treatment with the EGFR inhibitor slowed tumor growth (fig. 18 i), inhibited CD55 and CD59 expression (fig. 18j, 18 k).

Taken together, these results demonstrate that reducing EGFR/Wnt/beta-catenin/LINC 00973 mediated inhibition of complement activation combined with immune checkpoint blockade significantly improves the anti-tumor effects of these treatments in mice.

EGFR/beta-catenin transactivation, LINC00973 expression, and CD55 and CD59 levels are positively correlated with each other in human NSCLC specimens, and with clinical aggressiveness of the disease

To determine the clinical relevance of EGFR/β -catenin transactivation-regulated expression of LINC00973, CD55 and CD59, we analyzed protein and LINC00973 levels expression in 200 human NSCLC specimens using IHC and Locked Nucleic Acid (LNA) probes. In fig. 19, a-f are 200 human NSCLC samples analyzed by IHC assay using the indicated antibodies and In Situ Hybridization (ISH) assay using the indicated Locked Nucleic Acid (LNA) probes. a is a representation image which is an enlarged area in the image of the expanded data fig. 12 a. b is a Kaplan-Meier plot of total survival of 200 NSCLC patients grouped according to high (staining score 5-8) and low (staining score 1-4) expression levels of the indicated protein or LINC 00973. P-values were calculated using a log rank test (two-tailed). c shows a representative image. d is correlation analysis using a two-tailed Pearson correlation test (n=200). Note that the scores of some samples overlap. The intensity of blue represents the number of human NSCLC samples (darker blue represents a greater number of human NSCLC samples). e shows a representative image. f is a Kaplan-Meier plot of total survival of 200 NSCLC patients grouped according to high (staining score 5-8) and low (staining score 1-4) deposition levels of C4d, C3b, and C5 b-9. P-values were calculated using a log rank test (two-tailed). g is a waterfall plot of the optimal percent change from baseline of the sum of the longest diameters of target lesions determined according to RECIST version 1.1 (n=24 patients). The dashed line represents a 20% increase from baseline (determining the cutoff value for PD) and a 30% decrease (determining the cutoff value for PR according to recistv1.1 criteria). PD, progressive disease; SD, stable disease; and (5) customs, partially responding. h-k are 24 tumor specimens from NSCLC patients who received sintillimab treatment prior to surgery and were analyzed by IHC with the indicated antibodies. PD, progressive disease; SD, stable disease; and (5) customs, partially responding. h shows a representative image. i is correlation analysis using a two-tailed Pearson correlation test (n=24). Note that the scores of some samples overlap. The intensity of red represents the number of human NSCLC specimens (darker blue represents a greater number of human NSCLC specimens). j is the correlation between sintillimab treatment response and C3b, C4d and C5b-9 expression levels. PD, progressive disease; SD, stable disease; customs, partial response; IHC, immunohistochemistry. * P <0.05, P <0.01.k is a Kaplan-Meier plot of Progression Free Survival (PFS) time for 24 NSCLC patients grouped according to high (staining scores 5-8) and low (staining scores 1-4) deposition levels of C3b, C4d, and C5 b. P-values were calculated using a log rank test (two-tailed). l is the mechanism of EGFR activation induction and beta-catenin transactivation dependent complement inhibition, immune cell function inhibition and subsequent tumor growth promotion. CR3 complement receptor 3; EGFR, epidermal Growth Factor (EGF) receptor.

In fig. 20, a, b, c are 200 human NSCLC specimens with adjacent normal tissues analyzed by IHC with the indicated antibodies and assayed by In Situ Hybridization (ISH) of LINC00973 levels of Locked Nucleic Acid (LNA) probes. a is a representational image is redisplayed. The area in the indicated red box is enlarged and shown in fig. 8 a. b is correlation analysis using a two-tailed pearson correlation test (n=200). Note that the scores of some samples overlap. The intensity of red represents the number of human NSCLC samples (darker red or blue represents a greater number of human NSCLC samples). c shows representative images of the expression of the indicated proteins (left panel) and LINC00973 (right panel). U6 expression was used as a control. The area in the red frame is displayed at a higher magnification. The expression levels of the indicated proteins and LINC00973 were scored by a non-parametric test (right panel) and compared between tumor tissue and paired adjacent normal tissue. * P <0.001.d is a univariate and multivariate total survival analysis of the correlation of indicator protein and LINC00973 expression levels in non-small cell lung cancer patients. HR, risk ratio; 95% ci,95% confidence interval.

The results showed that EGFR phosphorylation levels and nuclear activity β -catenin, LINC00973, CD55 and CD59 expression were positively correlated with each other, inversely correlated with tumor infiltration of M1 macrophages (CD 80, CD86 and CD64 expression) and CD8+ T cells (CD 8 expression) in human NSCLC specimens (FIGS. 19a, 20 a-b) and significantly higher than corresponding levels in paired adjacent normal tissue samples (FIG. 20 c). Furthermore, the expression levels of active β -catenin, LINC00973, CD55 and CD59 were inversely correlated with patient survival time (fig. 19 b). According to the Cox multivariate model, all of these levels were independent predictors of NSCLC patient survival after adjustment of patient age and tumor lymph node metastasis (TNM) stage, which were relevant clinical covariates (fig. 20 d).

In addition, the expression levels of miR-216b and miR-150, as determined by In Situ Hybridization (ISH) analysis, indicate. The expression levels of miR-216b and miR-150 were inversely correlated with the expression levels of LINC00973, CD55 and CD59 in human lung cancer tissues (FIGS. 19 c-d). Analysis of 200 human NSCLC specimens by immunohistochemistry (fig. 19 e) showed that the expression levels of C3b, C4d and C5b-9 correlated with a good prognosis for the patient (fig. 19 f).

Notably, in our sintillimab treatment cohort, the majority of evaluable patients had reduced target lesions (75%; FIG. 19 g); IHC analysis of resected 24 NSCLC specimens showed that high expression of C3b, C4d and C5b-9 correlated inversely with the expression levels of CD55 and CD59 (fig. 19h, 19 i) and correlated positively with the anti-therapeutic good response of singeing Li Shan (fig. 19 j) and prolonged Progression Free Survival (PFS) of NSCLC patients (fig. 19 k). These results support the key role of EGFR induction and β -catenin transactivation mediated expression of LINC00973, CD55 and CD59 in human NSCLC clinical aggressiveness and immune checkpoint blockade treatment resistance.

EGFR activation may up-regulate Lnc1574203 expression by beta-catenin transcription

In FIG. 21, a and b are qPCR results under the conditions shown. C is the result of the ChIPqPCR with the beta-catenin antibody under the indicated conditions. As can be seen from the figure, EGF treatment of human lung adenocarcinoma cells H1395, H322M and human lung squamous carcinoma cell H226, with increasing expression of Lnc1574203 in the cells over time (fig. 21 a), while knock-down of β -catenin blocked this upregulation (fig. 21 b). Experiments with chromatin immunoprecipitation (ChromatinImmunoprecipitation, chIP) revealed that β -catenin was bound to the promoter of Lnc1574203 after EGF treatment, but not to the promoter of CD73 (fig. 21 c). These results indicate that EGFR signaling might up-regulate expression of Lnc1574203 by beta-catenin transcription.

Knock-down of beta-catenin inhibits the up-regulation of CD73 expression by EGFR activation

In FIG. 22, a is the qPCR result under the conditions shown. b is the Westernblotting result of the indicated conditions. It can be seen from the figure that knock-down of β -catenin inhibited the up-regulation of expression of mRNA (fig. 22 a) and protein (fig. 22 b) of CD73 by EGF treatment.

Inhibition of Lnc1574203 blocked the upregulation of CD73 expression by EGFR activation

FIG. 23 shows qPCR results for the indicated conditions. As can be seen from the figure, knockdown Lnc1574203 inhibited the up-regulation of CD73 expression by EGF treatment.

Inhibition of Dicer blocked down-regulation of CD73 expression by Lnc1574203 knockdown

FIG. 24 shows the result of Westernblotting under the conditions shown. From the figure, it can be seen that inhibition of Dicer by siRNA technology can block down-regulation of CD73 expression by Lnc1574203 knockdown.

Taken together, it can be seen from FIGS. 21-24 that EGFR signaling pathway may transcriptionally up-regulate a novel lncRNALnc1574203 by activating β -catenin in non-small cell lung cancer cells, and that up-regulated Lnc1574203 reduces mRNA degradation of CD73 by miR-590-3p by adsorbing miRNA (possibly miR-590-3 p), thereby enhancing CD73 expression.

According to the above experiments of the present invention, immune checkpoint blockade using PD-1 antibodies has a moderate effect on the treatment of human NSCLC to which EGFR mutated or activated patients show resistance. In view of the complexity of the immune system, it is important to understand the interactions between cancer cells and all components of the immune response (including the complement system) in the tumor microenvironment. mCRPs play an important role in immune response to tumors; overexpression of mCRPs, including CD55 and CD59, is reported in many primary cancers and is associated with poor prognosis in cancer patients (14, 15). However, it is not clear how these proteins are regulated by oncogenic signals to regulate complement activation to evade tumor immunity. We demonstrate here that EGF or Wnt treatment or EGFRL858R expression, which frequently occurs in lung cancer, increases expression of CD55 and CD59, caused by up-regulating LINC00973 adsorption of miR-216b and miR-150, respectively. EGFR activation induces transactivation of the beta-catenin/TCF/LEF complex binding to TBE2 in the LINC00973 promoter region, resulting in enhanced LINC00973 transcription. Depletion of β -catenin or CD55/CD59 or mutation of TBE2 in human NSCLC cells enhances complement activation, which is reflected by deposition of C3b and C5b-9 on tumor cell surfaces; anaphylatoxin release; the increased expression of cytokines IFN-gamma, TNF-alpha, IL-6, IL-1 beta and IL-17, which may be due to C5a mediated activation of antigen presenting cells and TH1/TH17 differentiation; and phagocytosis by CARIA and macrophages. Activation of complement and subsequent activation of cd8+ T cells results in tumor growth inhibition. Notably, the use of anti-PD-1 antibodies to activate complement or disrupt EGFR/β -catenin up-regulated CD55/CD59 binding by anti-CD 55/CD59 antibody treatment elicits synergistic tumor suppression, revealing an attractive therapeutic strategy for the treatment of EGFR-activated NSCLC in combination with anti-CD 55/CD59 antibodies and immune checkpoint inhibitors.

Both the potential anti-tumor and pro-tumor effects of the complement system under certain conditions have been reported (combined blockade of PD-1/PD-L1 and C5a can reduce tumor growth and metastasis, which is also observed in C3 deficient mice, and is associated with increased numbers of cd4+ and cd8+ T cells.) the expression of mCRP in tumor cells or other cells in the tumor microenvironment (such as T cells and macrophages) may elicit a unique complement response to target cells, thereby affecting tumor growth.

The present invention illustrates a previously unknown mechanism by which oncogenic EGFR or Wnt signaling inhibits the complement system through LINC 00973-mediated upregulation of CD55 and CD 59. This was the first report that demonstrates that oncogenic signaling inhibits cytotoxic cd8+ T cells in a complement inhibition-dependent manner, revealing a new type of intrinsic cell regulation between complement and cd8+ T. Importantly, we also provide preclinical evidence for the first time, suggesting that the combined blockade of mCRP function and PD-1/PD-L1 checkpoints may promote complement and cd8+ T cell activation may be a rational strategy for the treatment of human NSCLC. The clinical significance of this modulation was demonstrated by positive correlation of EGFR activation with the expression levels of active β -catenin, LINC00973, CD55 and CD59 in human NSCLC specimens, which is associated with clinical aggressiveness of the tumor. These findings reveal that the unknown mechanism of oncogenic signal-dependent inhibition of the complement system and subsequent cd8+ T cells (fig. 19 l) is activated by tumor cells and underscores the importance of EGFR/Wnt/β -catenin transactivation-mediated upregulation of CD55 and CD59 for tumor immune evasion.

We analyzed the activation of EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in lung adenocarcinoma, lung squamous carcinoma, cholangiocarcinoma, liver cancer, pancreatic cancer and gastric adenocarcinoma by public database Gene Expression Profiling Interactive Analysis (GEPIA), and further found and confirmed that activation of EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibited complement activation, and thus anti-tumor immune response.

FIG. 25 shows expression of LINC00973 and C5 in lung adenocarcinoma and normal tissues. Analysis of the expression of LINC00973, CD55, CD59 and C5 in pancreatic cancer (PAAD) (179) and normal tissue (171) by means of the Gene Expression Profiling Interactive Analysis (GEPIA) database revealed that expression of LINC00973, CD55, CD59 was higher in tumor tissue than in normal tissue and that of C5 was lower in tumor tissue than in normal tissue. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

Fig. 26 shows a correlation analysis of CD55 and CD59 expression in lung adenocarcinoma. The expression of CD55, CD59 in lung adenocarcinoma (LUAD) (483 cases) was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) database and found to be positively correlated. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

FIG. 27 shows survival analysis of LINC00973 expression in lung adenocarcinoma patients. Analysis of LINC00973 expression in lung adenocarcinoma (LUAD) (518 cases) by Gene Expression Profiling Interactive Analysis (GEPIA) database found that expression of LINC00973 in tumor tissue correlated with poor prognosis. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

FIG. 28 shows the expression of C3 and C5 in lung squamous carcinoma and normal tissue. Analysis of C3 and C5 expression in lung squamous carcinoma (luc) (486) and normal tissue (338) by Gene Expression Profiling Interactive Analysis (GEPIA) database revealed that C3 and C5 were expressed in tumor tissue less than normal tissue. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

Fig. 29 shows a correlation analysis of CD55 and CD59 expression in lung squamous carcinoma. The expression of CD55, CD59 in lung squamous carcinoma (luc) (486) was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) database and found to be positively correlated with CD55 and CD59 expression. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

Figure 30 shows survival analysis of CD59 expression in lung adenocarcinoma patients. CD59 expression in lung squamous carcinoma (LUSC) (290 cases) was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) database and found to be associated with poor prognosis in tumor tissue. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

Fig. 31 shows the expression of CD55, CD59, C3 and C5 in cholangiocarcinoma and normal tissues. Analysis of CD55, CD59, C3 and C5 expression in Cholangiocarcinoma (CHOL) (36 cases) and normal tissue (9 cases) by the Gene Expression Profiling Interactive Analysis (GEPIA) database revealed that CD55 and CD59 were expressed higher in tumor tissue than in normal tissue, and that C3 and C5 were expressed lower in tumor tissue than in normal tissue. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

Fig. 32 shows a correlation analysis of expression of CD55 and CD59 in cholangiocarcinoma. The expression of CD55, CD59 in Cholangiocarcinoma (CHOL) (36 cases) was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) database and found to be positively correlated with CD55 and CD59 expression. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

FIG. 33 shows the expression of CD55, CD59 and C5 in liver cancer and normal tissues. The expression of CD55, CD59 and C5 in liver cancer (LIHC) (369 cases) and normal tissues (160 cases) was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) database, and it was found that the expression of CD55 and CD59 in tumor tissues was higher than that in normal tissues, and the expression of C5 in tumor tissues was lower than in normal tissues. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

Fig. 34 shows a correlation analysis of the expression of CD55 and CD59 in liver cancer. The expression of CD55 and CD59 in liver cancer (LIHC) (369 cases) was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) database, and it was found that the expression of CD55 and CD59 were positively correlated. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

Fig. 35 shows survival analysis of C3 expression in liver cancer patients. Analysis of C3 expression in liver cancer (LIHC) (292 cases) by Gene Expression Profiling Interactive Analysis (GEPIA) database found that C3 expression in tumor tissue correlated with good prognosis. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

FIG. 36 shows expression of LINC00973, CD55, CD59 and C5 in pancreatic cancer and normal tissues. Analysis of the expression of LINC00973, CD55, CD59 and C5 in pancreatic cancer (PAAD) (179) and normal tissue (171) by means of the Gene Expression Profiling Interactive Analysis (GEPIA) database revealed that expression of LINC00973, CD55, CD59 was higher in tumor tissue than in normal tissue and that of C5 was lower in tumor tissue than in normal tissue. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

Fig. 37 shows a correlation analysis of CD55, CD59 and LINC00973 expression in pancreatic cancer. Analysis of CD55, CD59 and LINC00973 expression in pancreatic cancer (PAAD) (179) by the Gene Expression Profiling Interactive Analysis (GEPIA) database revealed positive correlation of CD55 and CD59 expression and positive correlation of LINC00973 and CD59 expression. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

Fig. 38 shows survival analysis of LINC00973 and CD59 expression in pancreatic cancer patients. Analysis of LINC00973 and CD59 expression in pancreatic cancer (PAAD) by Gene Expression Profiling Interactive Analysis (GEPIA) database found that expression of LINC00973 and CD59 in tumor tissue correlated with poor prognosis. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

FIG. 39 shows the expression of CD55 and C5 in gastric adenocarcinoma and normal tissues. CD55 and C5 were analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) database for expression in gastric adenocarcinoma (STAD) (408 cases) and normal tissue (211 cases), and it was found that CD55 was expressed higher in tumor tissue than in normal tissue and that C5 was expressed lower in tumor tissue than in normal tissue. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

Fig. 40 shows a correlation analysis of CD55 and CD59 expression in gastric adenocarcinoma. The expression of CD55, CD59 in gastric adenocarcinoma (STAD) (408 cases) was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) database and found to be positively correlated. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

Fig. 41 shows survival analysis of CD59 expression in gastric adenocarcinoma patients. Analysis of LINC00973 and CD59 expression in gastric adenocarcinoma (STAD) (462) by Gene Expression Profiling Interactive Analysis (GEPIA) database found that CD59 expression in tumor tissue correlated with poor prognosis. This result further demonstrates that activation of the EGFR/beta-catenin/LINC00973/CD55/59 signaling pathway in tumor tissue inhibits complement activation, and thus inhibits anti-tumor immune responses.

The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.

Claims (9)

1. The application of a marker in preparing a detection reagent or a kit for cancer diagnosis and prognosis prediction is characterized in that the marker is one or more of EGFR, wnt, beta-cateni, long non-coding RNA LINC00973, LNC1574203, CD55, CD59, CD73, C4d, C3a, C3b, C5a and C5 b-9.

2. The use of claim 1, wherein increased EGFR activation level, wnt pathway activation level, activated β -catenin level, long non-coding RNA LINC00973 expression level, LNC1574203 expression level, protein and mRNA expression level of CD55, protein and mRNA expression level of CD59, protein and mRNA expression level of CD73, and various combinations of these six elevated levels, as compared to a reference level, predicts that the patient has an aggressive cancer, has an aggressive cancer in the progressive stage, or has a poor prognosis.

3. The use of claim 1, wherein reduced C4d expression levels, C3a expression levels, C3b expression levels, C5a expression levels, C5b-9 expression levels, and combinations thereof, as compared to a reference level, predicts that the patient has invasive cancer, has invasive cancer in the progressive stage, or has a poor prognosis.

4. The use according to claim 2 or 3, wherein the reference level is a level in blood from non-cancerous cells or early cancer cells or healthy individuals, or early cancer patients.

5. The use according to claim 1, wherein the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, renal cancer, biliary system cancer, pheochromocytoma, islet cell cancer, li-frao Mei Niliu, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

Use of an egfr inhibitor, a Wnt inhibitor, a β -catenin inhibitor, a LINC00973 inhibitor, an LNC1574203 inhibitor, a protein and mRNA inhibitor of CD55, a protein and mRNA inhibitor of CD59, a protein and mRNA inhibitor of CD73 or a pharmaceutical composition comprising the inhibitor for the preparation of a product for the treatment of cancer.

7. The use of claim 6, wherein the EGFR inhibitor, wnt inhibitor, β -catenin inhibitor, LINC00973 inhibitor, LNC1574203 inhibitor, protein and mRNA inhibitor of CD55, protein and mRNA inhibitor of CD59, protein and mRNA inhibitor of CD73, including small molecule inhibitors, polypeptides, complementary inhibitory oligonucleotides or neutralizing antibodies to EGFR, wnt, β -catenin, LINC00973, LNC1574203, protein and mRNA of CD55, protein and mRNA of CD59, protein and mRNA of CD 73.

Use of a C4d agonist, a C3a agonist, a C3b agonist, a C5a agonist, a C5b-9 agonist, or a pharmaceutical composition comprising the agonist, in the manufacture of a product for the treatment of cancer.

9. The use of claim 8, wherein the C4d agonist, C3a agonist, C3b agonist, C5a agonist, C5b-9 agonist comprises a small molecule agonist for C4d, C3a, C3b, C5a, C5 b-9.