CN115993455A - Application of RNA binding protein NOVA2 as non-small cell lung cancer metastasis marker - Google Patents
Application of RNA binding protein NOVA2 as non-small cell lung cancer metastasis marker Download PDFInfo
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- CN115993455A CN115993455A CN202211705670.4A CN202211705670A CN115993455A CN 115993455 A CN115993455 A CN 115993455A CN 202211705670 A CN202211705670 A CN 202211705670A CN 115993455 A CN115993455 A CN 115993455A
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- G01N2800/56—Staging of a disease; Further complications associated with the disease
Abstract
The invention discloses application of an RNA binding protein NOVA2 as a non-small cell lung cancer metastasis marker, and relates to the technical field of biological medicines. The invention comprehensively uses means such as transcriptome sequencing, bioinformatics analysis, animal experiments, clinical sample examination and the like to identify a marker which is used for up-regulating expression in NSCLC tissues and promoting NSCLC transfer, namely RNA binding protein NOVA2 for the first time. Further experiments demonstrated that NOVA2 promotes NSCLC metastasis by modulating TGF- β/SMAD mediated EMT. In addition, the present invention also found that the transcription factor ETV4 up-regulates the expression of NOVA2. The invention clarifies the up-regulation of NOVA2 expression and a novel mechanism of promoting TGF-beta/SMAD to mediate epithelial-mesenchymal transition and NSCLC metastasis, enriches the knowledge of RNA binding protein functions and provides a novel strategy and target for the prevention and treatment of NSCLC metastasis.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to application of an RNA binding protein NOVA2 as a non-small cell lung cancer metastasis marker.
Background
Recent statistics show that lung cancer incidence is 12.5% of all tumors and is the first in all tumors with 21% mortality. Non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer, accounting for about 83% of lung cancers, including lung adenocarcinoma, lung squamous carcinoma, and large cell lung carcinoma. NSCLC patients have a poor prognosis with a five year survival rate of only about 15%. Metastasis is the leading cause of death in cancer patients, with more than 90% of NSCLC patients dying clinically being caused by metastasis. Aiming at the severe current situation, the mechanism of NSCLC metastasis is deeply explored, potential molecular intervention targets are searched, and the method has very important significance for improving prognosis of NSCLC patients.
At present, more and more researches reveal the action and mechanism of EGFR, ALK, ROS, KRAS, HER2 and other molecules in the development of NSCLC, and a plurality of targeting drugs developed based on the molecular targets are successfully applied to clinic, so that new hopes and choices are brought to NSCLC patients. However, there is currently no specific targeting drug for the treatment of NSCLC metastasis in the clinic, an important reason for which is the lack of specific molecular targets associated with NSCLC metastasis for downstream drug development and clinical research. Therefore, screening of key regulatory factors for NSCLC metastasis reveals the mechanism of action, provides a new intervention target for clinical treatment of NSCLC metastasis, and is key for improving the prognosis of NSCLC.
RNA Binding Proteins (RBPs) refer to a generic term for proteins that bind RNA and are involved in processes such as splicing, modification, transport, and stability regulation of RNA at the posttranscriptional level. The regulatory role of RBPs in tumor metastasis has been of great interest in recent years, as follows: goodarzi et al for the first time revealed that RBPTARBP2 plays an important role in breast cancer metastasis; recently, yu et al demonstrated that RBPRBMS1 regulates the stability of target RNAs at post-transcriptional levels to inhibit colon cancer metastasis. However, the role and mechanism of RBPs in NSCLC metastasis has been rarely reported.
NOVA2 (neuro-oncological ventral antigen 2) regulates target gene expression at post-transcriptional levels as RBP by recognizing specific YCAY (y=c/U) repeat clusters of target mRNA. NOVA2 was originally thought to be a neuronal specific RBP and Saito et al found that NOVA2 knockout mice exhibited characteristics of callus underdevelopment, and ventral motor neuron axon growth defects. Giampiero et al found that NOVA2 depletion impaired apical distribution of Par polar complexes resulting in an altered polarity of endothelial cells, thereby impeding the formation of vascular lumens in vivo. However, the role of NOVA2 in the development of human malignancy has been studied very rarely, and no report has been seen on the regulation of NSCLC metastasis by NOVA2.
Disclosure of Invention
The invention aims to provide an application of an RNA binding protein NOVA2 as a non-small cell lung cancer metastasis marker, so as to solve the problems in the prior art, and the invention discovers that the RNA binding protein NOVA2 can be used as a biomarker for non-small cell lung cancer metastasis, and the NOVA2 expression up-regulates to promote TGF-beta/SMAD mediated Epithelial Mesenchymal Transition (EMT) and NSCLC metastasis.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a biomarker for non-small cell cancer metastasis, which is an RNA binding protein NOVA2.
The invention also provides application of the reagent for detecting the expression level of the biomarker in preparation of a product for detecting non-small cell cancer metastasis.
The invention also provides a product for detecting non-small cell cancer metastasis, which comprises a reagent for detecting the expression level of the biomarker.
Further, the product is a kit or a reagent.
The invention also provides application of the inhibitor in preparing a medicament for preventing and/or treating non-small cell cancer metastasis, wherein the inhibitor is a substance for inhibiting expression of the biomarker.
Further, the inhibitor inhibits expression of the biomarker by reducing expression of the transcription factor ETV 4.
The invention also provides a medicament for preventing and/or treating non-small cell cancer metastasis, which comprises a substance for inhibiting the expression of the biomarker.
Further, the medicament also comprises pharmaceutically acceptable auxiliary materials.
The invention discloses the following technical effects:
the invention comprehensively uses means such as transcriptome sequencing, bioinformatics analysis, clinical sample inspection and the like to identify an RBP-NOVA 2 which is used for up-regulating expression in NSCLC tissues and promoting NSCLC transfer for the first time. Further experiments demonstrated that NOVA2 promotes NSCLC metastasis by modulating TGF- β/SMAD mediated EMT. Bioinformatic predictions and pre-experimental analysis prove that the key signal transduction factor SMAD4 of the TGF-beta/SMAD signal path is a potential regulation target of NOVA2. In addition, the present invention also found that the transcription factor ETV4 up-regulates the expression of NOVA2. The invention clarifies the up-regulation of NOVA2 expression and a novel mechanism of promoting TGF-beta/SMAD to mediate EMT and NSCLC transfer, enriches the understanding of RBP function, and provides a novel strategy and target for preventing and treating NSCLC transfer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of a transcriptome sequencing analysis of NSCLC tissue and paracancerous tissue; wherein A:10, generating a differential expression RBPs set by transcriptome sequencing of NSCLC tissues and paracancerous tissues; b: qRT-PCR detection 85 of the expression level of related RBPs mRNA in NSCLC tissues and paracancerous tissues; c: after dividing 85 pairs of NSCLC tissues into two groups, non-metastatic (n=43) and metastatic (n=42), the relevant RBPs mRNA expression levels were analyzed for differences in expression (T/N), T: tumor, cancer tissue; n: normal, paracancerous tissue; * P <0.05; * P <0.01; * P <0.001; d: immunohistochemical analysis of NOVA2 protein expression in non-metastatic and metastatic NSCLC tissues; e: the TCGA database analyzes the relationship between NOVA2 expression and NSCLC patient prognosis;
FIG. 2 is a model of nude mice metastasis demonstrating that overexpression of NOVA2 significantly promotes NSCLC metastasis; wherein A: establishing a flow diagram of a NSCLC nude mouse lung cancer transfer model; b: after 8 weeks of tail vein injection of NSCLC stable transfer cells (A549), dissecting and taking complete lung, observing lung transfer condition by Bouin's dye liquor staining, and analyzing micro transfer pathological condition in the lung by HE staining; c and D are statistics of lung metastases (C) and micro metastases in lung (D) in panel B; * P <0.05; * P <0.01;
FIG. 3 shows that NOVA2 is significantly associated with EMT gene set (A) and TGF-beta/SMAD gene set (B) by GSEA enrichment analysis;
FIG. 4 is a graph showing that overexpression of NOVA2 significantly promotes TGF- β/SMAD mediated EMT, NSCLC cell migration and invasion; wherein A-B is the detection of the effect of over-expression of NOVA2 on TGF-beta/SMAD mediated EMT related molecular marker expression in A549 (A) and H226 (B) cells; C-D is a test for the effect of overexpression of NOVA2 on TGF-beta/SMAD's ability to mediate migration of A549 (C) and H226 (D) cells; E-F is a test for the effect of overexpression of NOVA2 on TGF-beta/SMAD's ability to mediate A549 (E) and H226 (F) cell invasion; * P <0.01; * P <0.001;
FIG. 5 is a graph showing that NOVA2 upregulates its expression by increasing the stability of SMAD4 mRNA; wherein A: NOVA2 has three potential active elements in the SMAD4mRNA3' UTR region; b: RIP experiments demonstrated that NOVA2 can target binding to SMAD4 mRNA; c: overexpression of NOVA2 significantly upregulates SMAD4mRNA and protein expression levels; D-E: overexpression of NOVA2 in a549 (D) and H226 (E) cells significantly prolonged the half-life of SMAD4 mRNA; * P <0.05; * P <0.01;
FIG. 6 is an expression of ETV4 up-regulates NOVA2 at the transcriptional level in NSCLC; wherein A: screening NSCLC related differential expression TFs by combining a plurality of lung cancer expression profile chips; b: ETV4 has two potential binding sites in the NOVA2promoter region; TSS, transcriptional start site, transcription initiation site; c: qRT-PCR detection 85 of expression of ETV 4mRNA in NSCLC tissues and other tissues beside cancer; d: analyzing the correlation between ETV4 and NOVA2 expression in NSCLC tissues; E-F: qRT-PCR (E) and Western blot detection (F) are used for instantly knocking down the influence of ETV4 on the expression of NOVA2 mRNA and protein; * P <0.001;
FIG. 7 is a schematic representation of knockdown of ETV4 significantly inhibiting TGF- β/SMAD mediated EMT and NSCLC cell migration and invasion; wherein, A-B: knocking down ETV4 expression in NSCLC cell a549 (a), detecting its effect on TGF- β/SMAD-mediated expression of an EMT-related molecular marker (B); C-D: knocking down ETV4 expression in NSCLC cell a549, detecting its effect on TGF- β/SMAD mediated ability of NSCLC cell migration (C) and invasion (D); * P <0.01; * P <0.001;
FIG. 8 is a schematic representation of NOVA2 upregulating SMAD4 expression at posttranscriptional levels, thereby promoting TGF- β/SMAD mediated EMT and NSCLC metastasis.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
In the following examples, A549 and H226 were purchased from Wuhanprinus, pLVX-IRES-Neo and pLKO.1-puro were purchased from Ruibo (Guangzhou) Biotech Co.
Example 1
1. Method of
Functional study of NOVA2 to regulate TGF-beta/SMAD mediated EMT to promote NSCLC metastasis
1.1 screening for RBP-NOVA 2 specifically associated with NSCLC metastasis using transcriptome sequencing and clinical sample validation.
(1) Sample preparation: 10 pairs of NSCLC tissues and paracancerous tissues are taken, the tissues with proper sizes are sheared, and the tissues are ground with TRIZol, lysed and RNA is extracted. The total amount of the collected RNA is more than 10 mug, and the concentration is more than 100 mug/mug; RIN is greater than 7.5, 28S/18S is greater than 1.0, and the purity is desired to be OD 260/280 In the interval of 1.8-2.2.
(2) Library establishment and high throughput sequencing: and enriching the RNA sample, establishing a library and controlling quality according to the NEB standard library establishing mode, and finishing high-throughput sequencing on the machine.
(3) Data analysis: carrying out data processing and analysis on the high-throughput sequencing result to obtain a differential expression gene set (up-regulation expression and down-regulation expression) related to NSCLC; the crossing of the above-described differential expression gene set with RBPs library (ATtRACT) allows screening for differential expression RBPs.
(4) Verifying the expression level of the RBPs mRNA in a NSCLC tissue sample, and further analyzing the expression difference of the RBPs mRNA in the transferred NSCLC tissue and the non-transferred NSCLC tissue; immunohistochemistry and survival analysis the expression of RBPs described above in NSCLC tissues and their relationship to NSCLC patient prognosis.
1.2 construction of NSCLC stably transformed cell lines (A549 and H226) for over-expression and knock-down of NOVA2 by RNAi, homologous recombination, lentiviral vector construction, packaging, infection and other techniques.
(1) The invention selects pLVX-IRES-Neo (over-expression vector) and pLKO.1-puro (knock-down vector) plasmid to construct slow virus vector.
(2) Cloning the CDS sequence of the NOVA2 gene to a pLVX-IRES-Neo vector by a homologous recombination technology; and designing an interference shRNA sequence of the targeted NOVA2 through a Thermo Fisher online tool, selecting two shRNA sequences with highest scores, synthesizing and connecting the shRNA sequences into a pLKO.1-puro vector.
The two highest scoring shRNA sequences were as follows:
sequence 1:
NM_002516.4_shrna_1360_top
CACCGCTTAACACGCTGGCAAGTTACGAATAACTTGCCAGCGTGTTAAGC；
NM_002516.4_shrna_1360_bottom
AAAAGCTTAACACGCTGGCAAGTTATTCGTAACTTGCCAGCGTGTTAAGC。
sequence 2:
NM_002516.4_shrna_1916_top
CACCGCCGCTCAATACCTCATCAGTCGAAACTGATGAGGTATTGAGCGGC；
NM_002516.4_shrna_1916_bottom
AAAAGCCGCTCAATACCTCATCAGTTTCGACTGATGAGGTATTGAGCGGC. (3) The constructed vector and the corresponding virus packaging plasmid are co-transfected into 293T cells, viruses are collected 48H after transfection and infected with A549 and H226 cells, and NSCLC stable transfer cell lines which over-express and knock down NOVA2 are respectively screened with G418 (400 mug/mL) and puromycin (2 mug/mL) after 72H.
1.3 detection of the effect of NOVA2 on TGF-beta/SMAD mediated expression of EMT-related epithelial and mesenchymal markers. The effect of NOVA2 on TGF- β/SMAD mediated NSCLC cell migration and invasion capacity was also examined.
(1) NSCLC stably transformed cell lines that overexpress NOVA2 are divided into two groups: NOVA2 over-expression group (pLVX-NOVA 2) and control group (pLVX-Vector); NSCLC cell lines that stably knocked down NOVA2 were divided into two groups: NOVA2 knockdown group (sh-NOVA 2) and control group (sh-NC).
(2) The cells were induced to develop EMT by stimulation with 5ng/mL TGF-beta 1 for 24h, and Western blot was used to detect expression of EMT-related epithelial markers (E-cadherein) and interstitial markers (N-cadherin, snail).
(3) And the migration and invasion capacities of NSCLC cells are detected by using a Transwell experimental technology. A24-well Transwell chamber was selected for the Transwell migration and invasion assay, wherein the Transwell chamber used for the invasion assay also required a layer of Matrigel gel (Corning) to migrate or invade the relative number of cells under the membrane of the Transwell chamber to evaluate the migration and invasion capacity of NSCLC cells.
1.4A nude mouse tumor metastasis model was established to determine the effect of NOVA2 on TGF-beta/SMAD mediated metastasis of NSCLC.
(1) A NSCLC transfer nude mouse model is established by using 4-6 week old BALB/c nude mice, and the study of the in vivo transfer capacity of NSCLC cells is carried out.
(2) NSCLC stably transformed cells a549 were grouped as follows: stable over-expressed NOVA2 group (pLVX-NOVA 2) and control group (pLVX-Vector); stable overdamped NOVA2 group (sh-NOVA 2) and control group (sh-NC).
(3) Each of the above groups of NSCLC stably transformed cells (2×10 6 cells/mouse) were tail intravenously injected into BALB/c nude mice and TGF- β1 (4 μg/kg BW) was intraperitoneally injected every 5 days (1, 6, 11, and 16 days) the next day, body weights were periodically weighed and NSCLC cell in vivo metastasis progression was monitored using in vivo imaging.
(4) Euthanized nude mice after 8 weeks, dissected to obtain complete lung tissues, counted the number of metastasis nodules on the surface of the lung, and subjected to paraffin section on part of the lung tissues, and subjected to HE staining to observe pathological changes of lung metastasis; and (3) placing part of lung tissues with tumor metastasis into liquid nitrogen for freezing, extracting proteins, and detecting the expression levels of NOVA2, SMAD4 and EMT related molecular markers (E-cadherin, N-cadherin and Snail) by Western blot.
Nova2 regulates SMAD4mRNA stability at post-transcriptional levels, thereby facilitating molecular mechanism studies of TGF- β/SMAD mediated EMT and NSCLC metastasis.
2.1 study of the regulation of SMAD4 by NOVA2 at posttranscriptional levels.
(1) In NSCLC stably transformed cell lines (A549 and H226) which overexpress and knock down NOVA2, the effect of NOVA2 on SMAD4mRNA and protein expression levels was examined using qRT-PCR and Westernblot methods.
(2) Interactions between NOVA2 (protein) and SMAD4mRNA were detected using RNA co-immunoprecipitation (RNAimmunoprecipitation, RIP) experiments. Relevant RIP experiments are carried out in NSCLC cell lines by using an EZ-Magna RIP kit (Millipore company), after NSCLC cells are grown to a proper density (90%), the cells are lysed and the products are collected, the cell lysate and magnetic beads combined with NOVA2 or IgG antibodies are incubated, RNA is extracted and purified, specific RIP-PCR primers (comprising a potential binding sequence cluster of NOVA2 in the 3' UTR region of the SMAD4 mRNA) are designed for SMAD4mRNA, and qRT-PCR is used for detecting the enrichment of SMAD4mRNA in the NOVA2-RIP product.
(3) The interaction relationship between NOVA2 protein and SMAD4mRNA was further verified using RNApull-down experiments. A related experiment was performed in NSCLC cell lines using a Pierce TM Magnetic RNA-Protein Pull-Down kit (Thermo Fisher Co.) to synthesize biotin-labeled wild-type and mutant SMAD4 single-stranded oligonucleotides (containing a cluster of potential binding sequences of NOVA2 in the 3' UTR region of SMAD4 mRNA) in vitro, incubating them with magnetic beads, then incubating the magnetic beads with NSCLC cell extracts, extracting the proteins, and Westernblot to examine whether SMAD4mRNA specifically binds to NOVA2 Protein.
(4) The effect of NOVA2 on the fluorescent activity of the 3' UTR of SMAD4mRNA was examined using the Dual fluorescent reporter gene (Dual-luciferase report assay) technique. Analysis using the RBPs prediction database revealed that NOVA2 had three potentially binding sequence clusters in the 3' UTR region of SMAD4 mRNA. Construction of a double fluorescent reporter vector psiCHECK2-SMAD4-3'-UTR-wild type containing three potential binding sequence clusters (about 500 bp) of NOVA2 and a vector psiCHECK2-SMAD4-3' -UTR-wild type-mutant containing mutated sequences of the corresponding NOVA2 binding sequence clusters. The above vectors were transfected into NSCLC stably transformed cells that had been constructed to overexpress and knock down NOVA2, and the effect of NOVA2 on the fluorescent activity of the 3' UTR of SMAD4mRNA was examined (specific method reference "Wang S, tong X, li C, et al, rating 5 supporting TGF-. Beta. -induced EMT and cell invasion in lung adenoca. EMBO Rep.2021;22 (6): e 52079").
(5) The effect of NOVA2 on SMAD4mRNA stability was examined using an mRNA half-life assay. In NSCLC stably transformed cell lines (A549 and H226) which overexpress NOVA2 and knock down NOVA2, cells 0, 2, 4 and 6H are treated with Actinomycin D (actD) respectively to inhibit the synthesis process of RNA; extracting total RNA of cells, and detecting the attenuation change condition of SMAD4mRNA along with time by using a qRT-PCR method.
(6) The stability of SMAD4mRNA was improved by detecting which key molecules regulating RNA metabolism were recruited by NOVA2 using co-Immunoprecipitation combined mass spectrometry (IP-MS) analysis (immunopreprint-Mass spectrometry). Constructing a NOVA2 expression plasmid of FLAG-tag, transfecting the NOVA2 expression plasmid into an NSCLC cell line, capturing protein interacted with NOVA2 in cell lysate by using FLAG antibody, performing mass spectrometry analysis, and detecting and verifying the captured interacted protein by using Western blot; and screening important proteins with RNA metabolic regulation function in a mass spectrum result by combining a letter generation method to carry out subsequent research.
2.2 establishment of a stable cell line (A549 and H226) of NSCLC knockdown SMAD4, detection of EMT-related molecular markers, cell migration and invasion capacity and in vivo transfer capacity, and verification of important roles of SMAD4 in promoting NSCLC transfer by TGF-beta/SMAD mediated EMT.
(1) Referring to study method 1.2, NSCLC stably transformed cell lines knocked down SMAD4 were established (a 549 and H226).
(2) Referring to study method 1.3, the effect of knockdown SMAD4 on TGF- β/SMAD mediated EMT, NSCLC cell migration and invasiveness was examined using Western blot, transwelll migration and invasiveness experiments.
(3) Referring to study method 1.4, a nude mouse tumor metastasis model was established to analyze the effect of knockdown SMAD4 on TGF- β/SMAD-mediated NSCLC metastasis.
2.3 confirmation by a back-filling experiment that NOVA2 promotes TGF- β/SMAD mediated EMT and NSCLC transfer by modulating SMAD4 expression.
(1) Referring to study method 1.2, SMAD4 was further knocked down in NSCLC stably transformed cell lines that overexpressed NOVA2, and the experiments were divided into four groups: control (vector+sh-NC), NOVA2 over-expression (sh-NC+NOVA 2), SMAD4 knockdown (vector+sh-SMAD 4), and NOVA2 over-expression & SMAD4 knockdown (NOVA 2+sh-SMAD 4).
(2) With reference to study method 1.3, a Westernblot, transwelll migration and invasion assay was used to analyze whether NOVA2 regulated TGF- β/SMAD mediated EMT, promoting NSCLC cell migration and invasion was dependent on SMAD4.
(3) Referring to study method 1.4, a nude mouse tumor metastasis model was established and analyzed for whether NOVA2 promoted TGF- β/SMAD-mediated NSCLC metastasis was dependent on SMAD4.
Molecular mechanism study of the up-regulated NOVA2 expression of the transcription factor ETV4 in NSCLC
3.1 study of the effect of ETV4 on NOVA2 expression at the transcriptional level. ETV4 was overexpressed and knocked down in NSCLC cell lines (A549 and H226), and the effect of ETV4 on NOVA2 mRNA and protein expression was detected using qRT-PCR and Western blot methods.
3.2 study of the regulatory effect of ETV4 on NOVA2 at the transcriptional level.
(1) Interaction between ETV4 (protein) and NOVA2DNA promoter was analyzed using chromatin co-immunoprecipitation (Chromatin Immunoprecipitation, chIP) and PCR techniques. The experiment is selected from
The relevant ChIP experiments were performed in NSCLC cell lines using the kit (Active Motif). After the cells grow to a proper density (90%), the cells are fixed and lysed, the cell lysate (DNA) is broken to about 300bp under ultrasound, the ultrasound product is incubated with ETV4 antibody which can be used for performing a ChIP experiment, specific ChIP-PCR primers are designed for the NOVA2promoter, and the enrichment of NOVA2 in the ETV4-ChIP product is detected by PCR.
(2) The interaction relationship between the transcription factors ETV4 and NOVA2 (DNA promoter) was further verified using gel migration or electrophoretic mobility Experiments (EMSA) experiments. The experiment selects the LightShift TM Chemiluminescent EMSA kit (Thermo Fisher Co.) relevant EMSA experiments were performed in NSCLC cell lines, double-stranded oligonucleotide probes were synthesized which were biotin-labeled with the specific sequence of the NOVA2DNA promoter, and corresponding wild-type and mutant double-stranded unlabeled with biotinOligonucleotide competitive probes (comprising the potential binding sequence of ETV4 in the NOVA2promoter region) were incubated with NSCLC nuclear extracts, EMSA effect was assessed by adding anti-ETV 4 and IgG antibodies, and interaction of ETV4 and NOVA2DNA was judged by detecting the presence or absence of specific Supershift bands (probe DNA-protein complexes).
(3) The effect of ETV4 on the fluorescent activity of the NOVA2DNA promoter was examined using the Dual fluorescent reporter gene (Dual-luciferase report assay) technique. The potential binding sequence of ETV4 in the NOVA2DNA promoter region was analyzed by using transcription factor prediction software to construct a NOVA2promoter dual fluorescent reporter vector pGL3-NOVA2promoter-wild type containing the ETV4 binding site, and pGL3-NOVA2promoter-mutant containing the mutant sequence of the corresponding ETV4 binding site. The above vectors were transfected into NSCLC cell lines (A549 and H226) that overexpressed and knocked down ETV4, and the effect of altered ETV4 expression on the fluorescent activity of the NOVA2DNA promoter was examined (specific method reference "Wang S, tong X, li C, et al, quick 5 supporting TGF-. Beta. -induced EMT and cell invasion in lung adenocarpcinoma. EMBO Rep.2021;22 (6): e 52079").
3.3 study of the effect of ETV4 on TGF- β/SMAD mediated EMT, NSCLC cell migration and invasion.
(1) ETV4 was overexpressed and knocked down in NSCLC cell lines (a 549 and H226), and the effect of ETV4 on TGF- β/SMAD-mediated EMT-associated epithelial and mesenchymal marker expression was examined using Western blot.
(2) And the migration and invasion capacity of NSCLC cells is detected by using a Transwell migration and invasion experiment.
5. Statistical analysis
In the invention, all data were counted and analyzed using SPSS and GraphPad Prism software. According to the differences of experimental grouping and data types, the invention adopts different inspection methods: for comparison between two sets of data involved in cell, animal and clinical sample analysis, unpaired t-test (double tailed) was used; for analysis among three sets of data involved in multi-factor analysis of clinical samples, the Kruskall-Wallis test was used; for analysis of correlation of two sets of data in NSCLC clinical samples, pearson's correlation test was used. Survival curves were calculated using the Kaplan-Meier method and their differences were evaluated by the Log-rank test. Experimental data are expressed as "Means ± SEM", differences are considered statistically significant as P < 0.05.
2. Results
1. Identification of RBP-NOVA 2 that up-regulates expression in NSCLC and is specifically related to metastasis
The present invention performed transcriptome sequencing analysis (RNA-seq) on NSCLC tissue and paracancerous tissue (10 pairs) found that 3 RBPs were significantly up-regulated and 1 RBPs were significantly down-regulated in NSCLC tissue (fig. 1 a).
Thereafter, the present invention performed an expanded sample validation of the 4 RBPs described above in 85 pairs of NSCLC tissue and paracancerous tissue, the results of which were consistent with the results of transcriptome sequencing (FIG. 1B). Surprisingly, when the present invention analyzed NSCLC tissues into two groups, metastatic and non-metastatic, it was found that only NOVA2 was differentially expressed (up-regulated) in the NSCLC tissues in which metastasis occurred (C in fig. 1), suggesting that high NOVA2 expression might be involved in NSCLC metastasis process.
Next, the present invention examined the expression of NOVA2 protein in metastatic and non-metastatic NSCLC tissues, and found that NOVA2 protein was highly expressed in the metastatic NSCLC tissues (D in fig. 1), whereas TCGA database analysis showed that NOVA2 high expression was significantly correlated with poor prognosis in NSCLC patients (E in fig. 1).
The above results indicate that RBPNOVA2 is up-regulated in metastatic NSCLC tissue, and that its high expression is significantly correlated with NSCLC metastasis and prognosis, suggesting that NOVA2 may play an important role in NSCLC metastasis.
2. Nude mice transfer model demonstrated that overexpression of NOVA2 significantly promoted NSCLC transfer
To demonstrate the role of NOVA2 in NSCLC metastasis, the present invention constructs a NSCLC stably transformed cell line over-expressed by NOVA2 and injected into nude mice with control cells via tail vein to construct a NSCLC nude mice metastasis model (a in fig. 2). The result shows that: after 8 weeks of NSCLC cell inoculation, significantly visible lung metastasis nodules appear on the lung surface of nude mice, but the number of lung metastasis nodules is significantly greater in nude mice in the NOVA2 group over-expressed than in the control group (B and C in fig. 2). After the lung tissue was sectioned, HE staining was performed to observe pathological changes in the lung tissue, and the results showed that the nude mice with the novo va2 group overexpressed had significantly more micrometastases formed in the lung tissue than the control group (B and D in fig. 2). The results show that the overexpression of NOVA2 significantly promotes NSCLC metastasis, and a better bedding is made for the subsequent intensive discussion of the molecular mechanism of NOVA2 for promoting NSCLC metastasis.
3. Overexpression of NOVA2 significantly promotes TGF-beta/SMAD mediated EMT, NSCLC cell migration and invasion
In order to search for the potential mechanism by which NOVA2 promotes NSCLC metastasis, the present invention performed gene enrichment analysis (GSEA) analysis of the pre-transcriptome sequencing results (10 pairs of NSCLC tissues and paracancestral tissues in 1.1) found that NOVA2 high expression was significantly correlated with the epithelial-to-mesenchymal transition (EMT) gene set and TGF- β/SMAD gene set (fig. 3 a and B). To further confirm whether NOVA2 promotes NSCLC metastasis by modulating TGF- β/SMAD mediated EMT, the present invention conducted the following studies: treatment of NOVA2 overexpressing NSCLC stably transformed cells and their control cells with TGF-beta 1 (activating TGF-beta/SMAD signaling pathway) induced EMT, and detection of expression of EMT-associated epithelial (E-cadherein) and mesenchymal (N-cadherein and Snail) markers, as well as cell migration and invasion capacity. The results show that: overexpression of NOVA2 significantly promoted the down-regulation of TGF- β1-induced expression of the epithelial marker E-cadherin and up-regulation of expression of the interstitial markers N-cadherin and Snail (a and B in fig. 4); meanwhile, the invention discovers that the over-expression of NOVA2 promotes the migration and invasion of NSCLC cells induced by TGF-beta 1 (C-F in FIG. 4). The above results indicate that overexpression of NOVA2 significantly promotes TGF- β/SMAD-mediated EMT, NSCLC cell migration and invasion, suggesting that NOVA2 is an important regulator of TGF- β/SMAD-mediated EMT.
Nova2 upregulates its expression by increasing SMAD4mRNA stability
To confirm whether NOVA2 promotes TGF- β/SMAD mediated EMT and NSCLC metastasis by posttranscriptionally regulating the expression of a key factor of the TGF- β/SMAD signaling pathway. The key signal transduction factors of the TGF-beta/SMAD signal channels are analyzed one by utilizing the RBPs prediction database, and only three repeated sequence clusters ((YCAY) n, Y=C/U) with potential recognition and binding of NOVA2 exist on the 3' UTR of SMAD4mRNA, so that SMAD4 is a potential regulation target of NOVA2 (A in fig. 5).
Next, the present invention demonstrates that NOVA2 can target binding to SMAD4mRNA in NSCLC cells by RNA-protein co-precipitation (RIP) experiments (method references "Wang S, tong X, li C, et al, rating 5 support TGF-. Beta. -induced EMT and cell invasion in lung adenocarcinoma. EMBORep.2021;22 (6): e 52079"); overexpression of NOVA2 significantly upregulated SMAD4mRNA and protein expression levels (C in fig. 5); actinomycin D (ActD) treatment found that overexpression of NOVA2 significantly prolonged the half-life of SMAD4mRNA (D and E in fig. 5). The above results suggest that NOVA2 upregulates its expression by increasing SMAD4mRNA stability, thereby promoting TGF- β/SMAD mediated EMT and NSCLC transfer.
ETV4 up-regulates expression of NOVA2 at the transcriptional level in NSCLC
To confirm whether upregulation of NOVA2 expression in NSCLC is due to a transcription factor with an abnormal expression, the present invention performed an integrated analysis of multiple lung cancer expression profiling chip data (TCGA, GEO and GSE datasets) to preliminarily identify 4 NSCLC-related differentially expressed Transcription Factors (TFs): ETV4, KLF4, TBX3 and TFAP2A (a in fig. 6), whereas the transcription site prediction results indicate that only the transcription factor ETV4 has two potential binding sites on the NOVA2promoter (B in fig. 6). Further studies have found that ETV4 expression is significantly up-regulated in NSCLC tissues and that ETV4 is significantly positively correlated with NOVA2 expression (C and D in fig. 6).
Furthermore, knockdown of ETV4 expression significantly down the expression of NOVA2 mRNA and protein in NSCLC cell a549 (E and F in fig. 6). The above results suggest: high expression of ETV4 up-regulates the expression of NOVA2 at the transcriptional level is a potential mechanism for its up-regulation in NSCLC.
6. Knock-down ETV4 significantly inhibited TGF- β/SMAD-mediated EMT, NSCLC cell migration and invasion
The present inventors have initially demonstrated that ETV4 up-regulates NOVA2 expression at the transcriptional level. Then, in modulating TGF- β/SMAD-mediated EMT, ETV4 exerts a similar promoting effect as NOVA 2? The present invention treats NSCLC cell A549 with TGF-beta 1 to induce EMT, and the knockdown ETV4 is found to significantly inhibit the down regulation of TGF-beta 1 induced E-cadherein expression and the up regulation of N-cadherein and Snail expression (A and B in FIG. 7). The present invention also found that knock-down ETV4 inhibited TGF- β1-induced migration and invasion of NSCLC cells (C and D in fig. 7). The above results indicate that ETV4 is involved in the regulation of TGF- β/SMAD mediated EMT.
Taken together, the present invention demonstrates that RNA Binding Protein (RBP) NOVA2 upregulates SMAD4 expression at posttranscriptional levels, thereby promoting TGF- β/SMAD mediated EMT and NSCLC transfer; ETV4 up-regulates NOVA2 expression at the transcriptional level (fig. 8).
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
1. A biomarker for non-small cell cancer metastasis, wherein the biomarker is the RNA binding protein NOVA2.
2. Use of an agent that detects the expression level of the biomarker of claim 1 in the manufacture of a product for detecting non-small cell cancer metastasis.
3. A product for detecting metastasis of non-small cell carcinoma comprising an agent that detects the expression level of the biomarker of claim 1.
4. A product according to claim 3, wherein the product is a kit or reagent.
5. Use of an inhibitor for the manufacture of a medicament for the prevention and/or treatment of metastasis of non-small cell cancer, wherein the inhibitor is a substance that inhibits the expression of the biomarker of claim 1.
6. The use according to claim 5, wherein the inhibitor inhibits the expression of the biomarker by reducing the expression of the transcription factor ETV 4.
7. A medicament for preventing and/or treating metastasis of non-small cell cancer, comprising a substance that inhibits the expression of the biomarker of claim 1.
8. The medicament of claim 7, further comprising a pharmaceutically acceptable adjuvant.
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| CN202211705670.4A CN115993455A (en) | 2022-12-29 | 2022-12-29 | Application of RNA binding protein NOVA2 as non-small cell lung cancer metastasis marker |
| PCT/CN2023/072255 WO2023165281A2 (en) | 2022-12-29 | 2023-01-16 | Application of rna-binding protein nova2 as non-small cell lung cancer metastasis marker |
| LU505384A LU505384B1 (en) | 2022-12-29 | 2023-01-16 | Application of rna-binding proteins nova2 as non-small-cell lung cancer metastasis marker |
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| WO2023165281A3 (en) | 2023-11-23 |
| WO2023165281A2 (en) | 2023-09-07 |
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