CN109880902B - Application of long-chain non-coding RNA RP11-499F3.2 in reversing drug-resistant treatment of tumor cetuximab - Google Patents
Application of long-chain non-coding RNA RP11-499F3.2 in reversing drug-resistant treatment of tumor cetuximab Download PDFInfo
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Abstract
The invention belongs to the field of tumor molecular diagnosis and targeted therapy, and particularly relates to a method for finding lncRNA RP11-499F3.2 which is remarkably and highly expressed in head and neck cancer through TCGA data analysis in bioinformatics, and a clinical sample verifies that the expression level of the RP11-499F3.2 in the head and neck squamous cell carcinoma is remarkably higher than that of tissues beside the head and neck squamous cell carcinoma, and is closely related to the clinical prognosis of a patient with the head and neck squamous cell carcinoma. Functional experiments show that the high expression RP11-499F3.2 can promote the proliferation, migration and invasion of head and neck squamous cell in vitro. Meanwhile, the invention also discovers that RP11-499F3.2 can promote the drug resistance of head and neck squamous cell carcinoma cells to cetuximab. The lncRNA RP11-499F3.2 disclosed by the invention is beneficial to disclosing a new pathogenesis of head and neck cancer, providing a new tumor marker for the prognosis monitoring of the head and neck cancer and simultaneously providing a new idea for the clinical treatment of the head and neck cancer.
Description
Technical Field
The invention belongs to the field of tumor diagnosis and molecular targeted therapy, and particularly relates to application of long-chain non-coding RNA RP11-499F3.2 in clinical detection of head and neck cancer and reversal of drug-resistant therapy of tumor cetuximab.
Background
Head and neck cancer is the seventh ranked malignant tumor type of neoplastic disease, accounting for approximately 7% of the overall human body's systemic malignant tumor disease, with more than 90% of Head and neck cancers belonging to the Head and Neck Squamous Cell Carcinosoma (HNSCC). More than 50 ten thousand patients are diagnosed with head and neck squamous cell carcinoma every year, the number of new cases in China accounts for about 1/5 of the new cases, and the number of death cases is about 5.6 ten thousand cases every year. Generally speaking, head and neck squamous cell carcinoma is classified into oral cancer, nasopharyngeal cancer, oropharyngeal cancer, laryngeal cancer and the like. Meanwhile, since about one-fourth of patients with head and neck squamous cell carcinoma are associated with papillomavirus (HPV) infection in vivo (especially oropharyngeal carcinoma patients), head and neck squamous cell carcinoma can also be classified as HPV-negative and positive tumors. Poor lifestyle habits such as smoking and drinking are generally considered to be the cause of head and neck squamous cell carcinoma. Meanwhile, due to the poor diagnosis consciousness of patients, the prevalence of distant metastasis or recurrence (R/M) in patients, and the like, the clinical early diagnosis and comprehensive treatment of head and neck squamous cell carcinoma diseases are still greatly limited, and the average life cycle of head and neck squamous cell carcinoma patients is only 5 years.
Long non-coding RNA (lncRNA) is the focus of the current research on the mechanism of tumor diseases, and is defined as RNA molecules with nucleotide (nt) length greater than 200 and lacking complete Open Reading Frame (ORF) in non-coding RNA molecules. lncRNA has higher tissue and cell specificity, and a large number of lncRNA is reported to have differential expression profiles in tumor diseases, is considered as a potential proto-oncogene and a tumor suppressor gene, and is expected to be an ideal target for clinical diagnosis, staging and treatment. At present, lncRNA is generally accepted to participate in the development of tumor diseases, but the specific molecular mechanism of lncRNA still needs to be studied in a large amount as an experimental basis. Therefore, the functional mechanism of relevant lncRNA is researched by analyzing the lncRNA differential expression profile of the head and neck squamous cell carcinoma, and a basis is hopefully provided for finding the biomarkers of the head and neck squamous cell carcinoma.
The traditional modes of surgical excision, chemotherapy, radiotherapy and the like are common clinical treatment methods for patients with head and neck squamous cell carcinoma, but because the positions of head and neck organs are special, the treatment is easy to cause functional damage to the organs of the patients and influence the life quality. Meanwhile, the traditional treatment effect is often unsatisfactory for head and neck squamous cell carcinoma patients diagnosed as late stage or having distant metastasis and local recurrence symptoms. With the development of molecular pathology research on malignant tumor diseases, the treatment effects of various tumor types are proved to be related to signal channel regulation and target gene mutation, and the targeted treatment of head and neck squamous cell carcinoma opens a new clinical treatment idea.
Transmembrane glycoprotein Epidermal Growth Factor Receptor (EGFR) can regulate and control life activities such as tumor cell proliferation, invasion, drug resistance generation and the like by activating downstream channels (such as PI3K, ERK-1/2 and the like), is an important target molecule in head and neck squamous cell carcinoma, and 90% of patients with head and neck squamous cell carcinoma overexpress the EGFR. Cetuximab (Cetuximab, trade name Erbitux) became the first FDA-approved targeted therapeutic drug for head and neck squamous cell carcinoma on 7/11/2011, and it was reported that only 10% -20% of head and neck squamous cell carcinoma patients achieved complete remission in long-term Cetuximab treatment. However, the drug resistance generation mechanism is still unclear at present, and the invention hopefully provides reference for clinical treatment by researching the action mechanism of lncRNA related to drug resistance of cetuximab of cervical squamous cell carcinoma.
Disclosure of Invention
1. Object of the invention
In view of the above problems, the object of the present invention is to find a long non-coding RNA RP11-499F3.2 associated with head and neck cancer. The lnc RP11-499F3.2 is closely related to the clinical diagnosis and prognosis of head and neck squamous cell carcinoma, can promote the proliferation and metastasis of head and neck squamous cell carcinoma in vitro, and can be used as a potential biomarker of the head and neck squamous cell carcinoma. The designed locked nucleotide can improve the drug resistance sensitivity of the cetuximab.
2. Technical scheme
In order to realize the purpose, the technical scheme is as follows:
the invention discloses a long-chain non-coding RNA RP11-499F3.2, which is lncRNA which is obviously highly expressed in head and neck cancer tissues and is discovered by comprehensively analyzing lncRNA expression profile chip data of head and neck cancer and normal cancer adjacent tissues in a cancer gene profile (TCGA) database by an inventor. It is located in human chromosome 15: 81,660,482-81,871,125 antisense strand, and the DNA sequence is shown in SEQ ID NO. 1.
The clinical detection method for head and neck cancer is real-time quantitative PCR, and the method comprises the following steps:
obtaining a test sample from a subject having a head and neck cancer;
determining the expression level of long non-coding RNA RP11-499F3.2 contained in the test sample; and
analyzing the expression level to generate a risk score, wherein the risk score can be used to provide a prognosis of the subject.
Further, the test samples are tumor tissue and serum preserved with liquid nitrogen.
Further, the reagent for detecting the expression level of the biomarker is a real-time quantitative PCR detection kit.
The detection primer sequences for real-time quantitative PCR are shown as SEQ ID NO.2 and SEQ ID NO. 3.
For the evaluation of the effect of lnc RP11-499F3.2 on the function of head and neck cancer cells, the specific experimental steps are as follows:
and respectively infecting SCC4 cells by using lnc RP11-499F3.2 overexpression and silencing lentiviruses, screening stable transgenic cells, and then respectively carrying out cell proliferation, migration invasion and scratch experiment detection.
The invention also discloses a method for establishing the stable cetuximab drug-resistant cell line, which comprises the following steps: and (3) establishing the SCC4/CTX drug-resistant cell strain by adopting a concentration gradient screening method.
To explore the role of lnc RP11-499F3.2 in cetuximab resistance, we specifically regulated the expression of RP11-499F3.2 by designing locked nucleic acids. Specifically, the specific locked nucleotide sequence is shown in SEQ ID NO.4.
Furthermore, the sequence of the non-specific sequence NC is used as a negative control, the sequence is shown in SEQ ID NO.5, SCC4/CTX cell strains are respectively transfected, and the sensitivity change of lnc RP11-499F3.2 to cetuximab-resistant cells is examined by detecting the in-vitro proliferation and transfer capacity change of the resistant cells.
Meanwhile, we investigate the influence of specific locked nucleic acid on cetuximab resistance in head and neck squamous cell carcinoma. Specifically, a head and neck squamous cell carcinoma cetuximab drug-resistant PDX model is established through multiple in vivo passages, a certain dose of lnc RP11-499F3.2 locked nucleotide is given through intratumoral injection, and the change of tumor volume is monitored.
3. Advantageous effects
(1) The invention discovers that lnc RP11-499F3.2 is obviously highly expressed in head and neck squamous cell carcinoma, is closely related to prognosis and can be used as a potential biomarker of the head and neck squamous cell carcinoma;
(2) The invention discovers that the expression level of lnc RP11-499F3.2 in head and neck cancer cells is obviously higher than that of oral epithelial cells, and the lnc RP11-499F3.2 can obviously inhibit the proliferation, migration, invasion and clone formation of the head and neck cancer cells after deletion expression, thereby prompting the importance of the head and neck cancer cells on the growth and metastasis of tumors and providing reference for the targeted therapy of the head and neck cancer;
(3) According to the invention, a cetuximab-resistant cell strain is established in vitro, a cetuximab-resistant PDX model is established in vivo, and it is found that the down-regulation of the expression of lnc RP11-499F3.2 is helpful for recovering the sensitivity of head and neck cancer cells to cetuximab, and the lnc RP11-499F3.2 is further proved to be key lnc RNA generated by the drug-resistant phenotype of head and neck squamous cell carcinoma cetuximab;
(4) The invention also tries to specifically interfere the function of RP11-499F3.2 in the drug-resistant cell models of the cetuximab for other indications, including colon cancer, esophageal cancer and non-small cell lung cancer, and develops the clinical treatment direction of the cetuximab;
(5) The experiment designed by the invention is scientific, reasonable, feasible and effective, and the invention further researches the long-chain non-coding RNA RP 11-499F3.2; based on the findings, the expression level of lnc RP11-499F3.2 can be used as a new biomarker to assist the diagnosis of head and neck cancer and the prediction of malignancy degree, particularly the responsiveness of a patient with drug-resistant head and neck cancer to the therapy of cetuximab is reversed, the therapeutic effect is improved, and the method has good transformation medical prospect.
Drawings
FIG. 1 is a chip cluster diagram of LncRNA differentially expressed in head and neck cancer tissues and paracarcinoma tissues, data from the TCGA database;
FIG. 2 lnc RP11-499F3.2 expression level vs. total survival of HNSCC patients, data from TCGA database;
FIG. 3 lnc RP11-499F3.2 comparison of expression levels in 46 pairs of head and neck cancer tissues and para-cancer tissues, data from the TCGA database;
FIG. 4 Inc RP11-499F3.2 results of qPCR detection in 50 clinical samples of HNSCC (2) -ΔΔct Comparing valuesP<0.05， **P＜0.01)；
FIG. 5 is a graph of the expression levels of lnc RP11-499F3.2 in 50 clinical tissue samples of HNSCC as a function of total survival rate in patients with HNSCC;
FIG. 6 is a graph of the expression levels of lnc RP11-499F3.2 in 30 clinical serum samples of HNSCC as a function of the overall survival rate of patients with HNSCC;
FIG. 7 is a graph showing the comparison between the expression levels of lnc RP11-499F3.2 in HNSCC cells and oral epithelial cells;
FIG. 8 lnc RP11-499F3.2 overexpression and silencing lentiviral vector plasmid map;
FIG. 9 is a graph showing the results of transfection of SCC4 cells with Inc RP11-499F3.2 overexpression and silencing lentiviral vector and qPCR detection;
FIG. 10 is a graph showing the effect of overexpression and silencing of lnc RP11-499F3.2 on the proliferation potency of SCC4 cells;
FIG. 11 is a graph showing the effect of overexpression and silencing of lnc RP11-499F3.2 on the migration ability of SCC4 cells;
FIG. 12 is a graph showing the effect of overexpression and silencing of lnc RP11-499F3.2 on the invasion capacity of SCC4 cells;
FIG. 13 the MTT assay detects the IC50 values of the parent SCC4 response to cetuximab;
FIG. 14 is a graph showing the effect of cetuximab on the proliferation potency of SCC4 and SCC4/CTX cells;
FIG. 15 is a graph showing the results of the effect of cetuximab on the cell cycle distribution of SCC4 and SCC 4/CTX;
FIG. 16 is a graph showing the results of the effects of cetuximab on apoptosis of SCC4 and SCC4/CTX cells;
FIG. 17 is a graph showing the results of the effect of cetuximab on the formation of SCC4 and SCC4/CTX cell clones;
FIG. 18 is a graph showing the results of the effect of cetuximab on tumor growth in SCC4 and SCC4/CTX cells;
FIG. 19 detection of lnc RP11-499F3.2 expression levels in SCC4 and SCC4/CTX cell lines;
FIG. 20 shows the dynamic growth of tumors in a cetuximab drug-sensitive PDX transplantation tumor model;
FIG. 21 is a dynamic growth chart of a tumor model of the head and neck squamous cell carcinoma cetuximab drug-resistant PDX transplantation tumor;
FIG. 22 detection of lnc RP11-499F3.2 expression level in cetuximab sensitive and tolerant HNSCC-PDX model tumor tissue;
FIG. 23 dynamic growth plots of tumors following administration of lnc RP11-499F3.2 targeted therapy in the cetuximab-resistant HNSCC PDX model;
FIG. 24 is a tumor profile of the cetuximab-resistant HNSCC PDX model after administration of lnc RP11-499F3.2 targeted therapy;
FIG. 25 is a graph showing HE staining results of major organs after the drug-resistant HNSCC PDX model was administered with lnc RP11-499F3.2 targeting therapy.
Detailed Description
The invention is further described with reference to specific examples.
Example 1
And (3) analyzing the sequencing result of lncRNA of the human head and neck cancer tissue and the paired normal tissue.
The tumor Genome map (TCGA) project was initiated by the united states National Cancer Institute (NCI) and National Human Genome Research Institute (NHGRI) in 2006 using large-scale sequencing-based genomic analysis techniques to perform large-scale experiments on 36 cancers, and the TCGA Genome analysis center (GCC) aligned tumor and normal tissues for gene mutations, amplifications, or deletions associated with each Cancer or subtype. The molecular mechanism of cancer is understood, and the scientific understanding of the molecular basis of cancer pathogenesis is improved.
Entering a TCGA (https:// cancer. Nih. Gov /) head and neck squamous carcinoma option page, selecting clinical and RNAseq options, and clicking to enter the page. Selecting a rsem, genes, normalized _ results, txt file from the RNAseq option, selecting all files in METADATA and a clinical directory, and downloading after acquiring addresses; after gene expression data of head and neck squamous carcinoma tissues are obtained, renaming RNA standardized data according to METADATA file information so as to be convenient for comparison with gene expression data files and clinical pathological information; after comparison, the statistics shows that 500 cases of head and neck squamous cell carcinoma tissues and 46 cases of head and neck squamous cell carcinoma tissues are in total, and the corresponding RNA expression data and clinical pathological information are complete.
Analyzing gene expression data of head and neck squamous carcinoma tissues by using R language software (version 3.1) and screening differential expression lncRNA, wherein the R language software is a gene chip expression differential algorithm for correcting the expression abundance and the noise level of a chip; inputting the gene expression data of the head and neck squamous carcinoma tissues as standardized data into Excel software, and taking lncRNA data of which the expressed vacancy value is more than 80% of the sample amount; analyzing the differential expression lncRNA by using R language software, setting the screening condition of the differential expression lncRNA as FDR < 0.05 and Fold change > 2, namely screening lncRNA with the expression level difference P value < 0.05 and the difference value (absolute value) > 2 times in head and neck squamous carcinoma tissue and normal tissue, drawing a Cluster analysis graph by using a Cluster 3.0 software package, and carrying out visual analysis on the high-throughput gene expression data of the head and neck squamous carcinoma tissue.
The results are shown in FIG. 1, and from 13964 candidate lncRNA, 563 differentially expressed lncRNA (fold change) were obtained in total according to the screening conditions> 2，P<0.05 254 were significantly up-regulated and 309 were down-regulated in head and neck squamous carcinoma tissues.
Example 2
Screening of lnc RP11-499F3.2 and relationship of its expression level with total survival rate of HNSCC patients.
Randomly averaging all clinical samples into two groups (a training set group and a test set group) without difference, wherein each group comprises 250 clinical samples; dividing the clinical sample of the training set into a high risk group and a low risk group according to the relative expression level of the screened lncRNA, taking the survival time of a patient as an independent variable, selecting the variable by using a lasso method, and drawing a Kaplan-Meier survival curve; and (4) verifying the lncRNA screened by the training set group by using the test set, wherein the screening method and conditions are the same.
TABLE 1 screening of lncRNA significantly associated with HNSCC survival time by Kaplan-Meier survival Curve method
In order to further verify and screen the lncRNA relevant to head and neck squamous cell carcinoma diagnosis, the invention carries out ROC curve drawing on the 8 screened lncRNA, evaluates the diagnosis prediction capability of the lncRNA on the life cycle of the head and neck squamous cell carcinoma patient and screens an ideal diagnosis molecular marker of the head and neck squamous cell carcinoma.
TABLE 2 screening of lncRNA related to life cycle of head and neck squamous cell carcinoma patients by ROC curve method
After removing lncRNA with AUC <0.6 in the ROC curve by combining the table 1 and the table 2, 4 lncRNA which meet the conditions are screened out: RP11-499F3.2, LINC00460, LINC00958 and ST3GAL4-AS1, and shows that the specificity and the sensitivity are higher. The specific high-expression lnc RP11-499F3.2 in the head and neck squamous carcinoma tissue has the highest AUC area, the AUC of the training set and the AUC of the testing set are respectively 0.714 and 0.748, the sensitivity and the specificity are both higher than 80%, and the target gene can be used as an ideal diagnostic molecular marker for the head and neck squamous carcinoma.
The results are shown in FIG. 1, and the expression level of lnc RP11-499F3.2 in the training set and the test set is significantly related to the prognosis of HNSCC.
Example 3
Expression level analysis of lnc RP11-499F3.2 in HNSCC tumor tissues and paired normal tissues.
Clinical information of cancer tissues and tissues adjacent to the cancer tissues of the HNSCC sample and corresponding lnc RP11-499F3.2 expression data are obtained 43 according to the method in the embodiment 1. As a result, as shown in FIG. 2, the expression level of lnc RP11-499F3.2 in HNSCC is significantly increased compared with that in the matched normal paracarcinoma tissues, and the expression level is considered to be an index for the clinical early diagnosis of head and neck cancer.
Example 4
Expression of lnc RP11-499F3.2 in head and neck cancer patients and tissues adjacent to normal cancer.
(1) Collection of specimens
Collecting head and neck cancer and tissue specimens beside the cancer in the operation under the condition of informed consent of patients, cleaning with normal saline, and storing in liquid nitrogen or refrigerator at-80 deg.C for use.
(2) Primer design
Searching all exon sequence information of the gene in an ensemble database according to the information of lnc RP11-499F3.2, and designing a Primer by using Primer premier5.0 according to the obtained sequence information, wherein the sequence is as follows:
upstream primer (SEQ ID NO. 2)
Downstream primer (SEQ ID NO. 3)
(3) Real-time quantitative PCR detection of the expression of lnc RP11-499F3.2 in head and neck cancer patients and tissues beside normal cancer.
Total RNA from the collected samples was extracted according to the Trizol instructions of life, and then the purity and concentration of the extracted RNA were quantified using a NanoDrop ND-1000 nucleic acid quantification apparatus, and agarose quality testing was performed to ensure the integrity of the extracted RNA. cDNA was synthesized by reverse transcription of the extracted total RNA using TaKaRa Kit PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time). A TaKaRa kit SYBR Premix Ex Taq II (Tli RNaseH Plus) is adopted to carry out qPCR reaction. The reaction system is as follows:
the components are uniformly mixed according to the following procedures: pre-denaturation at 95 ℃ for 30 s for 40 cycles; 95 ℃ for 5 s and 60 ℃ for 30 s.
The specificity of the reaction is judged according to the melting curve, and the relative expression quantity of lnc RP11-499F3.2 is calculated by a formula 2-delta Ct. As a result, as shown in FIG. 4, the expression level of lnc RP11-499F3.2 is significantly higher than that of the normal cancer adjacent tissues in about 75% of the head and neck cancer samples.
Example 5
Correlation of lnc RP11-499F3.2 expression levels with overall survival rates of 50 HNSCC patients.
Dividing 50 cases of HNSCC patients into a high risk group and a low risk group according to the relative expression level of lncRNA, taking the survival time of the patients as an independent variable, selecting the variable by using a lasso method, and drawing a Kaplan-Meier survival curve.
As a result, as shown in FIG. 5, the expression of lnc RP11-499F3.2 has correlation with the survival rate of patients with head and neck cancer, and the overall survival rate of the patients with low expression of lnc RP11-499F3.2 is significantly higher than that of the patients with high expression of TMEM170B, further confirming that lnc RP11-499F3.2 is used as a new index for prognosis of head and neck cancer.
Example 6
Expression levels of lnc RP11-499F3.2 in the serum of patients with head and neck cancer and healthy persons.
(1) Collection of specimens
Serum samples were collected from head and neck cancer patients and healthy volunteers prior to surgery with informed consent and stored in a-80 ℃ freezer for future use.
(2) Real-time quantitative PCR detection of lnc RP11-499F3.2 expression in serum of head and neck cancer patients and healthy volunteers
The primer design and specific detection method were the same as in example 5.
As a result, as shown in FIG. 6, the expression level of lnc RP11-499F3.2 in HNSCC was significantly increased compared with that in healthy volunteers, and it was considered that the expression level was a serological indicator for the early clinical diagnosis of head and neck cancer.
Example 7
Expression of lnc RP11-499F3.2 was assayed in HNSCC cells and normal oral epithelial cells.
Extracting total RNA of head and neck cancer SCC4, SCC9, SCC13, CAL27 and normal oral epithelial cell HIOEC, and detecting lnc RP11-499F3.2 by qPCR, wherein the specific detection method is the same as that in example 5. As a result, as shown in FIG. 7, the expression of the head and neck squamous cell line lnc RP11-499F3.2 was significantly higher than that of HIOEC cells.
Example 8
Preparation of lnc RP11-499F3.2 overexpression and silencing vector and virus transfection efficiency detection.
Synthesizing full-length cDNA aiming at lnc RP11-499F3.2, introducing an overexpression lentiviral vector (figure 8), designing three small interfering RNAs (the sequences are respectively SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8) aiming at lnc RP11-499F3.2 exons according to the shRNA design rule, introducing a silencing lentiviral vector (figure 8), co-transferring the plasmid co-packaging plasmid DR8.9 and the envelope plasmid VSVG.2 into 293T cells to generate viruses, collecting virus supernatant of the cells after transfection for 48 h, and infecting SCC4 cells. After infection for 24 h, puromycin is added for screening to obtain a stable lnc RP11-499F3.2 overexpression and silencing cell strain. Total RNA from the transfected cells was collected and the change in expression of lnc RP11-499F3.2 was detected by qPCR (as described in example 5).
As a result, as shown in FIG. 9, according to the observation that transfected cells overexpressing lentiviral plasmid (LRC RP 11-499F3.2) showed significant GFP expression under fluorescence, the expression of the LRC RP11-499F3.2 was significantly up-regulated compared with that of the control group CTRL (CTRL)P<0.01). Meanwhile, according to GFP expression and qRT-PCR experiments, results show that sh-c interference lentivirus plasmid transfection efficiency is highest.
Example 9
Effect of overexpression and silencing of lnc RP11-499F3.2 on the proliferative capacity of SCC4 cells.
Detection by MTT methodThe effect on the activity of SCC4 cell proliferation after overexpression and silencing of lnc RP11-499F3.2 was examined. The head and neck cancer cells were digested and collected with trypsin when cultured in an incubator containing 5% CO2 at 37 ℃ at a density of 90% or more, the cells were resuspended in a culture medium and counted under a microscope, and the cell concentration was adjusted to 3.0X 10 4 Cell suspension was seeded into 96-well plates at 100. Mu.L per well and cultured in a 5% CO2 incubator at 37 ℃. After 0 h, 24 h, 48 h and 72 h of incubation, 20. Mu.L of 5 mg/mL MTT was added to each well of the 96-well plate and incubation was continued for 4 h. The medium was aspirated off and dissolved by adding 100. Mu.L DMSO per well. The absorbance was measured at a detection wavelength of 570 nm and a reference wavelength of 630 nm with a microplate reader, and the growth inhibition ratio (PI) was calculated.
The test was independently repeated 3 times, the results obtained from the test were expressed as mean ± SD, and statistical T-test was performedP< 0.05 significant differencesPA very significant difference is < 0.01.
As a result, as shown in FIG. 10, the group overexpressing lnc RP11-499F3.2 produced a very significant upregulation in cell proliferation capacity, while silencing lnc RP11-499F3.2 reduced the proliferation capacity of head and neck squamous cell carcinoma cells. This shows that lnc RP11-499F3.2 has the capability of promoting the proliferation of head and neck squamous cancer cells in vitro.
Example 10
Effect of overexpression and silencing of lnc RP11-499F3.2 on the migratory capacity of SCC4 cells.
The head and neck cancer cell SCC4 was inoculated into a transwell chamber at 100. Mu.L per well, and 0.6 mL of complete medium containing 10% FBS was added to the lower chamber of the transwell to stimulate cell migration, followed by culture at 37 ℃ for 24 h in 5% CO 2. Removing culture solution from the wells, fixing with 90% ethanol at room temperature for 30 min, dyeing with 0.1% crystal violet at room temperature for 10 min, rinsing with clear water, slightly wiping off non-migrated cells on the upper layer with a cotton swab, observing under a microscope, and selecting four fields for photographing and counting. Mobility Inhibition Rate (MIR) was calculated according to the formula:
where Ntest is the cell migration number of the test group and Ncontrol is the cell migration number of the blank control group. The test is independently repeated for 3 times, mean + -SD is calculated from the test results, and statistical t-test is carried out, wherein P < 0.05 is significant difference, and P < 0.01 is very significant difference.
As a result, it was shown in FIG. 11 that the number of successful migration of SCC4 cells was significantly increased after overexpression of lnc RP11-499F3.2, whereas SCC4 cells knocked down in lnc RP11-499F3.2 had significantly reduced the number of migrated cells after 48 h of culture. This indicates that lnc RP11-499F3.2 can promote cell migration in the head and neck squamous cell line SCC 4.
Example 11
Effect of overexpression and silencing of lnc RP11-499F3.2 on the invasive potential of SCC4 cells.
10 mg/mL Matrigel was diluted with medium at 1. Head and neck cancer cells cultured to logarithmic growth phase were trypsinized, collected, washed twice with PBS and resuspended in blank medium. Adjusting the cell concentration to 1X 10 5 one/mL. Cells were seeded into transwell chambers at 100. Mu.L per well, and 0.6 mL of complete medium containing 10% FBS was added to the lower chamber of the transwell to stimulate cell invasion, and cultured at 5% CO2 at 37 ℃ for 24 h. Discarding culture solution in the hole, fixing with 90% alcohol at room temperature for 30 min, dyeing with 0.1% crystal violet at room temperature for 10 min, rinsing with clear water, slightly wiping off uninfected cells on the upper layer with a cotton swab, observing under a microscope, and selecting four fields for photographing and counting. The Invasion Inhibition Rate (IIR) was calculated according to the formula:
where Ntest is the number of cell invaders in the test group and Ncontrol is the number of cell invaders in the blank group. The test was repeated 3 times independently, and mean ± SD was calculated from the results obtained from the test and statistical t-test was performed, with significant differences of P < 0.05 and very significant differences of P < 0.01.
As a result, as shown in FIG. 12, SCC4 cells showed a significant increase in the number of successful attacks after overexpression of lnc RP11-499F3.2, while SCC4 cells knocked down in lnc RP11-499F3.2 showed a significant decrease in the number of invaded cells after 48 h of culture. This suggests that lnc RP11-499F3.2 can promote cell invasion in the head and neck squamous cell line SCC 4.
Example 12
The MTT method detects the IC50 values of the parent sensitive SCC4 in response to cetuximab.
Cetuximab concentrations of 10 nM, 20 nM, 40 nM, 80 nM, 160 nM and 320 nM were set, docetaxel (10 μ g/ml) was used as a positive drug, and MTT was used to detect zengzhi conditions of SCC4 cells at different drug concentrations, as described in example 9.
The result is shown in fig. 13, the proliferation inhibition effect of the cetuximab on the SCC4 cells is remarkably increased along with the increase of the drug concentration, the cell strain is sensitive to the cetuximab drug, and the IC50 value of the SCC4 cells under the action of the cetuximab drug is 80 nM, which is used as the initial concentration for screening the cetuximab drug-resistant cell strain.
Example 13
Effect of cetuximab on the proliferative capacity of SCC4 and SCC4/CTX cells.
(1) Recovering and culturing SCC4 cells, when the SCC4 cells grow to 70% density, removing a culture medium in a culture bottle, replacing the culture medium with a DMEM complete culture medium with the final drug concentration of 80 nM, culturing for 48 h at 37 ℃ in an incubator containing 5% CO2, and stably subculturing for 3 times or more by using a drug-free culture medium;
(2) When the cells recover to stably grow and the cell density reaches 70%, starting to multiply the drug concentration of the cetuximab in the culture medium, and repeating the steps, wherein each drug dosage keeps the cell culture for 15-20 days;
(3) And (3) regularly freezing the SCC4 cells induced by each drug concentration, measuring the IC50 value of the induced cells, finally obtaining the SCC4 cells which can tolerate 1280 nM cetuximab, and naming the drug-resistant cells as SCC4/CTX.
(4) The MTT method is used for detecting the influence of different concentrations of cetuximab on the proliferation capacity of SCC4 and SCC4/CTX cells, and the specific method is shown in example 9.
The results are shown in fig. 14, and compared with the parent SCC4 drug-sensitive cells, the cell proliferation level of the drug-resistant cells SCC4/CTX established in the invention within the cetuximab-resistant dose is still significantly increased.
Example 14
Effect of cetuximab on SCC4 and SCC4/CTX cell cycle distribution.
(1) When SCC4 and SCC4/CTX grow to logarithmic phase, adding 0.25% trypsin solution for digestion, rotating to an EP tube, centrifuging for 5 min at 1000 rpm, resuspending and paving to a six-hole cell culture plate for culture, when the cell density grows to 60%, discarding the culture medium, respectively adding DMEM serum-free culture medium containing 0 nM, 40 nM and 320 nM cetuximab drug concentration, placing the six-hole plate at 37 ℃, and culturing for 48 h in a 5% CO 2-containing culture box;
(2) Adding 0.25% trypsin solution for digestion, transferring to centrifuge tube, centrifuging at 900 rpm for 5 min, re-suspending and washing cells with PBS, calculating cell amount with cell counter, and adjusting cell suspension concentration to 1 × 10 6 Per ml;
(3) Taking 1 ml of cell suspension, adding 500 mu l of 70% precooled ethanol solution, fixing for 2 h to overnight at 4 ℃, centrifuging for 3 min at 1000 rpm, discarding supernatant, washing cells twice by PBS, and washing away residual fixative;
(4) According to the RNase: preparing a dyeing working solution by using a PI working solution in a volume ratio of 1;
(5) Detecting the cell cycle by adopting a flow cytometer, selecting and recording red fluorescent signals of each cell sample at the 488 nm excitation wavelength;
(6) Data analysis the distribution of sample cells in each cell cycle was counted using the Dean-Jett-Fox fitting model with cell number as ordinate and PI red fluorescence signal intensity as abscissa using FlowJo v5.7.3 software.
The results are shown in FIG. 15, the cell distribution ratio of SCC4 cells in G1 phase is significantly increased with the increase of drug concentration (P<0.05 ); the distribution ratio of S-phase cells is obviously reduced along with the increase of the concentration of the medicine (P<0.05 Cetuximab retards cell growth in G1 phase, inhibiting SCC4 cell proliferation viability. Under the action of different drug concentrations of cetuximab, G1, S and G of SCC4/CTX cellsNo significant difference was found in the 2/M cell distribution.
Example 15
Effect of cetuximab on apoptosis of SCC4 and SCC4/CTX cells.
(1) When SCC4 and SCC4/CTX grow to logarithmic phase, digesting with 0.25% trypsin solution, centrifuging at 1000 rpm for 5 min after transferring to an EP tube, resuspending and laying to a six-hole cell culture plate for culture, when the cell density grows to 60%, discarding the culture medium, respectively adding DMEM serum-free culture medium containing cetuximab drug concentration of 0 nM, 40 nM and 320 nM, placing the six-hole plate in a culture box at 37 ℃ and containing 5% CO2 for culture for 48 h;
(2) Adding 0.25% trypsin solution for digestion, transferring to a centrifuge tube, centrifuging at 900 rpm for 5 min, re-suspending and rinsing cells with precooled PBS buffer solution twice, removing residual pancreatin in cell suspension, calculating cell amount with a cell counter, adding 400 μ l Annexin V binding solution, re-suspending cells, adjusting cell concentration to 1 × 10 6 Per ml;
(3) Adding 5 μ l of Annexin V-FITC staining solution into the cell suspension, mixing, incubating at 4 deg.C in dark for 15 min, adding 10 μ l of PI staining solution, mixing, and incubating at 4 deg.C in dark for 5 min;
(4) Detecting apoptosis by using a flow cytometer, selecting and recording fluorescence signals of cell samples at 488 nm excitation wavelength and 530 nm emission wavelength, detecting Annexin V-FITC single positive tubes of cells which are not treated by drugs and PI single positive tubes, and determining fluorescence compensation values and positions of a cross quadrant gate;
(5) The data analysis adopts FlowJo v5.7.3 software, takes the intensity of PI red fluorescence signal as ordinate, and the intensity of Annexin V-FITC green fluorescence signal as abscissa. In the experimental results, the upper right Q2 quadrant belongs to late apoptotic cells, the lower right Q3 quadrant belongs to early apoptotic cells, the lower left Q4 quadrant belongs to normal cells, and the Q2 quadrant and the Q3 quadrant are adopted when the apoptosis rate is counted.
The results are shown in figure 16, the apoptosis rate of SCC4 cells is obviously increased under the action of 40 nM and 320 nM cetuximab drugs; however, the apoptosis rate of SCC4/CTX is increased significantly only under the drug concentration pressure of 320 nM, and the apoptosis rate of SCC4/CTX is significantly reduced compared with that of SCC4 under the same drug concentration. Therefore, it is believed that SCC4/CTX has a stronger apoptosis resistance function than SCC4 under cetuximab drug stress.
Example 16
Effect of cetuximab on the formation of SCC4 and SCC4/CTX cell clones.
(1) Weighing 0.56 g of low-melting-point agarose powder, adding 10 ml of double distilled water to prepare 5.6% agarose gel mother liquor, autoclaving at 121 ℃ for 30 min, and placing in a 50 ℃ water bath kettle to keep the temperature constant;
(2) Preparing 0.8% of lower-layer agarose gel: taking 1.5 ml of 5.6% agarose gel mother liquor, maintaining the temperature at about 40 ℃ to prevent solidification, adding 9 ml of DMEM complete culture medium, uniformly blowing and beating, subpackaging 0.5 ml/hole to 12-hole cell culture plates, paving 18 holes in total, and cooling and solidifying for 5 min at 4 ℃;
(3) Respectively preparing 45.7 nM DMEM complete medium containing cetuximab and 365.7 nM DMEM complete medium containing cetuximab, digesting and collecting SCC4 and SCC4/CTX cells in an exponential growth phase, equally dividing the two cells into three parts, adopting the DMEM complete medium, the 45.7 nM drug-added DMEM medium and the 365.7 nM drug-added DMEM medium for resuspending, counting the cells, and adjusting the cell concentration to 1 x 10 4 Per ml;
(4) Preparing 0.7% of upper agarose gel: sucking 1.2 ml of 5.6% agarose gel mother liquor, evenly dividing into 6 centrifuge tubes of 5 ml, respectively adding 1.4 ml of prepared cell suspension of the SCC4 and the SCC4/CTX, slowly blowing and beating uniformly, subpackaging 0.5 ml/hole into a twelve-hole cell culture plate paved with lower layer gel, paving three multiple holes with the same drug concentration of the same cell, paving 9 holes for each cell suspension, paving 5 multiplied cells in each hole, and cooling and solidifying for 5 min at 4 ℃;
(5) After the agarose gel is solidified, adding corresponding DMEM complete culture medium, 40 nM dosing DMEM culture medium and 320 nM dosing DMEM culture medium into each hole according to the clone formation experiment group, wherein each hole is 0.5 ml;
(6) Placing the 12-hole plate in an incubator at 37 ℃ and containing 5% CO2 for 15-20 days, replacing the corresponding culture medium on the upper layer every three days, and stopping cell culture when macroscopic cell clones exist in the culture plate;
(7) Absorbing and removing the upper culture medium, adding 1 ml of 0.01% crystal violet staining solution into each hole, staining for 1 h in a dark place, decoloring by PBS buffer solution until the clone is clear, placing the cell culture plate in an inverted microscope, and observing and counting the cell clone quantity of SCC4 and SCC4/CTX.
The results are shown in FIG. 17, with the increase of drug concentration, the cell clone formation number of SCC4 cells is reduced significantly, while with the pressure of different concentrations of drug, the cell clone formation amount of SCC4/CTX is not reduced significantly, and has no significant difference, which indicates that the capability of the cetuximab for inhibiting the formation of SCC4/CTX cells is weaker.
Example 17
Tumor growth effects of cetuximab on SCC4 and SCC4/CTX cell in vivo transplanted tumor models.
(1) Culturing large amount of SCC4 and SCC4/CTX cells, digesting with 0.25% pancreatin solution, centrifuging the cell suspension at 1000 rpm for 5 min after digestion is terminated, re-suspending the cells with serum-free DMEM medium, counting, and adjusting the cell concentration to 5 × 10 7 Per ml;
(2) Each nude mouse (24 female BALB/c nude mice with age of 4-6 weeks and weight of 14-16 g are ordered and adaptively raised in SPF-grade animal feeding room for 1 week) is inoculated with 200. Mu.l of cell suspension of the corresponding group in left axilla, and injected with 1X 10 cells 7 A plurality of;
(3) Closely observing the growth condition of the tumor of the inoculated part of the nude mice after inoculation, wherein on the 7 th day after inoculation, white nodules appear on the inoculated part, the white nodules can move subcutaneously after touch, the inoculated part gradually forms hard tumor masses along with the growth of tumor tissues, the average volume of the tumor tissues reaches 100 mm3 in about 14 days, the BALB/c nude mice are randomly divided into four groups, each group comprises 6 mice, and the weight of the animals is 16-18 g when administration is started;
(4) The volume of the transplanted Tumor was measured and recorded every two days, and the Tumor Volume (TV) was calculated as follows:
tumor volume =0.5 × a × b ^2
Wherein a is the graft length and b is the graft width.
The results are shown in fig. 18, scc4 graft tumor model,cetuximab group (20 mg kg) -1 ) Has a significant reduction in tumor volume and has a very significant difference of (A)P<0.001 ); cetuximab group (20 mg kg) in SCC4/CTX transplantation tumor model -1 ) Under the continuous pressure of the cetuximab drug, obvious tumor growth slowing does not occur, and the tumor growth curve is similar to that of a negative group. This indicates that the SCC4/CTX cell transplantation tumor model developed a resistant phenotype to cetuximab, and that SCC4/CTX cells have cetuximab resistance in vivo.
Example 18
Detecting the expression level of lnc RP11-499F3.2 after the in vivo tumor administration of the SCC4 and SCC4/CTX cells is finished;
after the administration of the SCC4 and SCC4/CTX cell in vivo transplantation tumor models is finished, the tumor tissue is cut and subjected to qRT-PCR monitoring, the experimental result is shown in figure 19, and the pressure of the cetuximab drug (20 mg kg) -1 ) Next, the expression level of lnc RP11-499F3.2 of SCC4 cell-transplanted tumor tissue appeared to be significantly reduced (P<0.05 And the expression of the SCC4/CTX cell line is not obviously changed, and the expression level of lnc RP11-499F3.2 is still very significantly different compared with that of the SCC4 cell (the expression level is very different from that of the SCC4 cell line by the method of the invention)P<0.01). The lnc RP11-499F3.2 expression is positively correlated with the cetuximab drug-resistant phenotype, and the expression can be a target molecule for reversing the drug resistance of the head and neck squamous cell carcinoma cetuximab.
Example 19
Establishing the dynamic growth condition of the tumor of the cetuximab drug-sensitive PDX transplantation tumor model.
(1) Directly obtaining a fresh tissue sample of the head and neck squamous carcinoma after operation, quickly putting the sample into a sterile vessel after the sample is separated, and conveying the sample to a sterile operation table;
(2) The fresh tumor tissue is quickly cleaned by using physiological saline containing antibiotics, is immediately transferred to a 10 cm cell culture dish containing a DMEM culture medium containing antibiotics, necrotic tumor tissue, fibrous tissue and adipose tissue in tumor are cut off, and is cleaned again;
(3) The remaining viable tumor tissue was sheared into tumor tissue pieces of 2X 2 mm3 size. Taking 2-3 BALB/c nude mice, disinfecting forelimb back and abdomen skin by 75% ethanol, carrying out isoflurane anesthesia on the mice, shearing a skin laceration of 2 mm at the axillary position of the forelimb of the mouse, filling a 18-gauge cannula puncture needle into a tumor tissue block, puncturing to the subcutaneous part of the forelimb shoulder and back of the mouse to form a subcutaneous 10 mm long migration sinus, pushing a needle core of the cannula puncture needle to inoculate the tumor tissue block to the subcutaneous part of the shoulder and back, inoculating two sites to each mouse, inoculating 2-3 mice (determined according to the amount of the tumor sample) to each sample, ensuring that the process from the sample in vitro to the transplantation of the tumor tissue is not more than 4 h, and naming the PDX transplantation tumor model of the head and neck squamous cell carcinoma as G1 generation;
(4) The average tumor volume of the PDX transplanted tumor model in each experimental group grows to 150 mm 3 And the number of PDX model mice with the same algebra meets grouping requirements (not less than 8), in-vivo cetuximab administration is started, and the sensitivity of the PDX parental model mice to cetuximab drugs is verified;
(5) The experimental groups of PDX model mice are randomly divided into two groups, each group comprises 4 mice, G1 negative control (normal saline) and G2 cetuximab (20 mg. Kg < -1 >) are respectively arranged in each group, and the administration period is 21 days.
The results are shown in fig. 20, and the PDX group in which the tumor volume of nude mice decreased by more than 50% after drug treatment was defined as the drug-sensitive group, the PDX group in which the tumor volume increased by more than 35% after drug treatment was defined as the drug-resistant group, and the remaining groups were defined as the drug-stable group. 2 cases of PDX models of a drug sensitive group are obtained, and a moderate sensitive PDX model (C2 group, oral squamous cell carcinoma patient) is selected as a parental model for establishing a cetuximab-resistant head and neck squamous cell carcinoma PDX transplantation tumor model to carry out an in vivo administration experiment.
Example 20
Dynamic growth condition of head and neck squamous carcinoma cetuximab drug-resistant PDX transplantation tumor model tumor.
After the screened head and neck squamous carcinoma medicament sensitive PDX transplantation tumor is subcultured, the tumor volume grows to 150 mm 3 And carrying out in vivo administration of cetuximab to induce the generation of drug-resistant phenotype of the drug-sensitive PDX transplantation tumor model. After three rounds of in vivo administration of cetuximab and tumor tissue passage, the C2 group PDX model shows a remarkable drug resistance phenotype, and the tumor volume still generates remarkable increase under the pressure of cetuximab drugs. The results are shown in FIG. 21, where the Cetuximab sensitive oral squamous cell carcinoma PDX model (OSCC) was administered in CetuximabAfter administration, the tumor volume is significantly reduced compared with that of a negative control group (physiological saline group), while the tumor growth volume of the negative control group of a cetuximab-induced oral squamous cell carcinoma PDX model (OSCC-CR) generated by multiple in vivo administration is significantly higher than that of a drug sensitive model (OSCC) (OSCC)P<0.01 The tumor volume is increased by more than 35 percent, and the obvious drug tolerance phenotype is shown, which accords with the definition of a drug tolerance group, namely the head and neck squamous cell carcinoma cetuximab drug-resistant PDX transplantation tumor model is successfully established.
Example 21
The expression level of lnc RP11-499F3.2 in the tumor tissue of a cetuximab-sensitive and tolerant HNSCC-PDX model is detected.
After the in vivo administration of the cetuximab-sensitive and resistant HNSCC-PDX model is finished, qRT-PCR monitoring is carried out on a tumor dissecting tissue, an experimental result is shown in figure 22, and the expression level of lnc RP11-499F3.2 of the cetuximab drug-resistant OSCC-CR PDX model is very significantly different from that of a drug-sensitive model (the expression level of lnc RP11-499F3.2 of the cetuximab drug-resistant OSCC-CR PDX model is very significantly different from that of the drug-sensitive model: (the expression level of cetuximab drug-sensitive and the expression level of cetuximab drug-resistant HNSCC-PDX model are very significantly different from that of the drug-sensitive model)P<0.01). Meanwhile, under the pressure of the cetuximab drug, the relative expression of lnc RP11-499F3.2 of the tumor tissue of the drug sensitive model is shown to be remarkably reduced (P<0.05 However, the expression of the OSCC-CR model tumor tissue is not obviously changed, the expression level of lnc RP11-499F3.2 is still higher than that of the drug sensitive model, and the expression level has very significant difference (A)P<0.01). The above results are consistent with the expression of lnc RP11-499F3.2 in SCC4/CTX (example 18), and clinical tumor tissues prove that the expression level of lnc RP11-499F3.2 is positively correlated with the drug resistance of cetuximab, and meanwhile, the successful establishment of the cetuximab-resistant head and neck squamous cell carcinoma PDX model in the invention is verified.
Example 22
Dynamic growth monitoring of tumors following the administration of lnc RP11-499F3.2 targeted therapy in the cetuximab-resistant HNSCC PDX model.
In order to verify the function of lnc RP11-499F3.2 in forming a head and neck squamous cell carcinoma cetuximab-resistant PDX transplantation tumor model in vivo, LNA targeting LNAs of lnc RP11-499F3.2 are designed for intratumoral injection.
(1) Ordering 20 female BALB/c nude mice with age of 4-6 weeks and weight of 14-16 g, adaptively feeding in an SPF animal feeding room for 1 week, recovering and inoculating OSCC-CR cetuximab drug-resistant oral squamous cell carcinoma model tissue blocks, and closely observing the growth condition of transplanted tumors at the inoculation part of the nude mice;
(2) When the average volume of tumor tissues reaches 150 mm3, randomly dividing BALB/c nude mice into four groups, wherein each group comprises 5 mice, and the groups are respectively a G1 negative control group (0.2 ml/20G), a G2 cetuximab group (20 mg.kg-1), a G3 LNA group (5 mg.kg-1), a G4 cetuximab + LNA group (cetuximab: 20 mg.kg-1 LNA;
(3) The administration period is 21 days, the PDX model mouse is observed for one week after the administration period is finished, and the change of the tumor volume of the PDX model is measured and recorded;
(4) After the experiment, the mice were killed by dislocation, tumor masses were peeled off, tumor volumes were calculated and photographed, and tumor tissue masses of each PDX model group were sampled and subjected to liquid nitrogen quick freezing and cryopreservation.
The results are shown in FIG. 23, and the tumor volumes of the group C2 of the saline solution group, the cetuximab group, the LNA group, and the cetuximab group + LNA group of the cetuximab-resistant oral squamous cell carcinoma PDX model (OSCC-CR) are (1959.08 + -79.09) mm after the end of the administration period 3 、(832.08±92.08）mm 3 、(419.03±73.38）mm 3 And (97.05 +/-35.04) mm 3 (see FIG. 24). The OSCC-CR model has the advantages that the tumor volume is increased by more than 35% under the pressure of the cetuximab, and the obvious drug tolerance phenotype is shown, so that the OSCC-CR model is proved to have the drug resistance of the cetuximab; LNA alone significantly reduced tumor growth volume compared to control group (P<0.001 Proves that the LNA targeting down-regulation of lnc RP11-499F3.2 can inhibit the growth of the drug-resistant model tumor of cetuximab; after the dosing period of the cetuximab group and the LNA group is over, the tumor volume is increased negatively, namely the OSCC-CR model can reverse the original drug resistance symptom phenotype to a remarkable drug treatment effect under the cetuximab drug pressure by simultaneously carrying out targeted down-regulation on lnc RP11-499F3.2 expression in the tumor, which shows that the LNA targeted down-regulation on lnc RP11-499F3.2 can cause the drug-resistant head and neck squamous cell carcinoma of the cetuximab to be desensitized.
Example 23
HE staining results of major organs after cetuximab-resistant HNSCC PDX model given lnc RP11-499F3.2 targeted therapy.
After the target treatment of the cetuximab-resistant HNSCC PDX model on lnc RP11-499F3.2 is finished, heart, liver, spleen, lung and kidney tissues of the model mouse are taken and fixed by 10 percent of formalin, and the section is cut after paraffin embedding and subjected to HE staining to observe the histopathology. Collecting tissues of each experimental group targeted by the head and neck squamous cell carcinoma OSCC-CR PDX model lnc RP11-499F3.2, carrying out HE staining, and observing pathological conditions of each tissue.
The results are shown in figure 25, the heart, liver, spleen and kidney organs of each group have no obvious pathological symptoms, and the G3 cetuximab administration group has lung infiltration, which shows that the group has tumor metastasis phenomenon, namely, the lung metastasis to a certain degree occurs in the process of establishing the head and neck squamous cell carcinoma cetuximab drug-resistant PDX transplantation tumor model.
Example 24
The target interference of lnc RP11-499F3.2 on the proliferation capacity of colon cancer parents and drug-resistant cells HT29 (CTX-R), esophageal squamous carcinoma cells TE13 (CTX-R) and non-small cell lung cancer A549 (CTX-R).
The method for establishing HT-29, TE13 and A549 drug-resistant cell strains by adopting a cetuximab concentration gradient is shown in an example 13, and the MTT method is used for detecting the influence of lnc RP11-499F3.2 locked nucleotide on the proliferation capacity of HT29, TE13 and A549 sensitive and drug-resistant cells respectively is shown in an example 9.
The results are shown in Table 4, compared with the control group, the lonc RP11-499F3.2 locked nucleotide designed by the invention can obviously reduce the proliferation capacity of HT-29, TE13 and A549 tolerant cells under the action of single nucleotide and has extremely obvious reduction after combining cetuximab, which shows that the targeted specificity reduces the expression of the lonc RP11-499F3.2, and can enable the cetuximab-resistant colon cancer, the esophageal squamous cell carcinoma and the non-small cell lung cancer to be desensitized.
The test is independently repeated for 3 times, the result obtained by the test is expressed by mean +/-SD, and a statistical T test is carried out，*P< 0.05 significant differencesPA very significant difference is < 0.01.
Claims (6)
1. The application of the long-chain non-coding RP11-499F3.2 in preparation of drugs for reversing drug resistance of tumor cetuximab is characterized in that the sequence of the long-chain non-coding RP11-499F3.2 is shown as SEQ ID NO.1 in a sequence table.
2. The use of a long chain non-coding RP11-499f3.2 as claimed in claim 1 for the preparation of a therapeutic agent for reversing cetuximab resistance to tumors, wherein the expression of lnc RP11-499f3.2 is regulated by the synthesis of specific locked nucleic acids.
3. The use of the long chain non-coding RP11-499f3.2 as claimed in claim 1 for the preparation of a therapeutic agent for reversing the drug resistance of the tumor cetuximab, wherein said tumor includes colon cancer, esophageal cancer, non-small cell lung cancer.
4. The use of a long chain non-coding RP11-499f3.2 as claimed in claim 2 for the preparation of a drug for reversing cetuximab resistance to tumors, wherein the locked nucleotide sequence is SEQ ID No.4 of the sequence listing.
5. The use of the long-chain non-coding RP11-499F3.2 as claimed in claim 4, wherein the synthesis mode of the locked nucleic acid is chemical synthesis and is used as gene therapy medicine.
6. The use of a long chain non-coding RP11-499f3.2 as claimed in claim 4 for the preparation of a therapeutic agent for reversing cetuximab resistance to tumors, wherein the in vivo administration of the locked nucleotide of lnc RP11-499f3.2 is intratumoral in situ, tail vein, subcutaneous or intramuscular injection.
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