Application of long non-coding RP11-499F3.2 in clinical detection of oral squamous cell carcinoma

Abstract

The application belongs to the field of tumor molecular diagnosis and targeted therapy, and specifically finds lncRNA RP11-499F3.2 which is significantly highly expressed in head and neck cancer through bioinformatics analysis of TCGA data; it is verified through clinical samples that the expression level of RP11-499F3.2 in head and neck squamous cell carcinoma is significantly higher than that in paracancerous tissues, and is closely related to the clinical prognosis of patients with head and neck squamous cell carcinoma; it is found through functional experiments that high expression of RP11-499F3.2 can promote the in-vitro proliferation, migration and invasion of head and neck squamous cell carcinoma cells; meanwhile, it is also found that RP11-499F3.2 can promote the drug resistance of head and neck squamous cell carcinoma cells to cetuximab; the disclosed lncRNA RP11-499F3.2 helps to reveal the new pathogenesis of head and neck cancer, provides a new tumor marker for the prognosis monitoring of head and neck cancer, and provides a new idea for the clinical treatment of head and neck cancer.

Description

[0001] This application is a divisional application of the patent application entitled "Application of a long non-coding RP11-499F3.2 in clinical detection of head and neck cancer and in reversing tumor cetuximab resistance treatment", application number: 201811214096.6, application date: October 11, 2018. Technical Field

[0002] This invention belongs to the field of tumor diagnosis and molecular targeted therapy. More specifically, it relates to the application of a long non-coding RNA RP11-499F3.2 in the clinical detection of head and neck cancer and in the reversal of cetuximab resistance in tumor treatment. Background Technology

[0003] Head and neck cancer is the seventh most common type of malignant tumor, accounting for approximately 7% of all malignant tumors. More than 90% of head and neck cancers are head and neck squamous cell carcinoma (HNSCC). Over 500,000 patients are diagnosed with HNSCC each year, with China accounting for about one-fifth of new cases and approximately 56,000 deaths annually. Generally, HNSCC is classified into oral cancer, nasopharyngeal cancer, oropharyngeal cancer, and laryngeal cancer. Furthermore, since about one-quarter of HNSCC patients are associated with human papillomavirus (HPV) infection (especially oropharyngeal cancer patients), HNSCC can also be classified as HPV-negative or HPV-positive tumors. Smoking and excessive alcohol consumption are widely considered contributing factors to HNSCC. Due to poor patient awareness and the prevalence of distant metastasis or recurrence (R / M), early diagnosis and comprehensive treatment of HNSCC remain severely limited, resulting in an average survival of only 5 years for patients.

[0004] Long non-coding RNAs (lncRNAs) are a key focus in current research on tumor disease mechanisms. They are defined as RNA molecules with a length greater than 200 nucleotides (nts) and lacking a complete open reading frame (ORF). lncRNAs exhibit high tissue and cell specificity, and numerous lncRNAs have been reported to have differential expression profiles in tumor diseases. They are considered potential proto-oncogenes and tumor suppressor genes, and hold promise as ideal targets for clinical diagnosis, staging, and treatment. Currently, the involvement of lncRNAs in the development and progression of tumor diseases is widely accepted, but their specific molecular mechanisms still require extensive experimental research. Therefore, this invention analyzes the differential expression profile of lncRNAs in head and neck squamous cell carcinoma to investigate the functional mechanisms of related lncRNAs, hoping to provide a basis for discovering biomarkers for head and neck squamous cell carcinoma.

[0005] Surgical resection, chemotherapy, and radiotherapy are traditional treatment methods commonly used for patients with head and neck squamous cell carcinoma. However, due to the unique location of organs in the head and neck region, these treatments are prone to causing functional damage to these organs, impacting patients' quality of life. Furthermore, for patients diagnosed with advanced head and neck squamous cell carcinoma or exhibiting distant metastases or local recurrence, the efficacy of traditional treatments is often unsatisfactory. With the increasing development of molecular pathology research on malignant tumors, the treatment outcomes of many tumor types have been shown to be related to signaling pathway regulation and target gene mutations. Targeted therapy for head and neck squamous cell carcinoma has also opened up new avenues for clinical treatment.

[0006] The transmembrane glycoprotein epidermal growth factor receptor (EGFR) regulates tumor cell proliferation, invasion, and drug resistance by activating downstream pathways (such as PI3K and ERK-1 / 2), making it an important target molecule in head and neck squamous cell carcinoma (HCC). 90% of HCC patients overexpress EGFR. Cetuximab (trade name Erbitux) became the first FDA-approved targeted therapy for HCC on November 7, 2011. However, it has been reported that only 10%-20% of HCC patients achieve complete remission with long-term cetuximab treatment. The mechanism of drug resistance development remains unclear. This invention investigates the mechanism of action of cetuximab resistance-related lncRNAs in HCC, hoping to provide a reference for clinical treatment. Summary of the Invention

[0007] 1. Purpose of the invention

[0008] To address the aforementioned problems, the purpose of this invention is to discover a long non-coding RNA, RP11-499F3.2, associated with head and neck cancer. This lnc RP11-499F3.2 is closely related to the clinical diagnosis and prognosis of head and neck squamous cell carcinoma, and can promote the in vitro proliferation and metastasis of head and neck squamous cell carcinoma cells, thus serving as a potential biomarker for this cancer. Furthermore, a locked nucleoside nucleotide designed to target RP11-499F3.2 can enhance sensitivity to cetuximab resistance.

[0009] 2. Technical Solution

[0010] To achieve the above objectives, the technical solution is as follows:

[0011] This invention discloses a long non-coding RNA RP11-499F3.2, which was discovered by the inventors through comprehensive analysis of lncRNA expression profile microarray data from head and neck cancer and normal adjacent normal tissues in the Cancer Genome Atlas (TCGA) database. It is a lncRNA that is significantly highly expressed in head and neck cancer tissues. It is located on the antisense strand of human chromosome 15:81,660,482-81,871,125, and its DNA sequence is shown in SEQ ID NO.1.

[0012] The clinical detection method for head and neck cancer is real-time quantitative PCR, which includes the following steps:

[0013] Test samples were obtained from individuals with head and neck cancer.

[0014] Determine the expression level of the long non-coding RNA RP11-499F3.2 in the test sample; and

[0015] The expression levels are analyzed to generate a risk score, which can be used to provide a prognosis for the subject.

[0016] Furthermore, the test samples are tumor tissue and serum preserved in liquid nitrogen.

[0017] Furthermore, the reagents used to detect the expression levels of biomarkers are real-time quantitative PCR detection kits.

[0018] The detection primer sequences for real-time quantitative PCR are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0019] The specific experimental procedures for evaluating the effect of lnc RP11-499F3.2 on the function of head and neck cancer cells are as follows:

[0020] SCC4 cells were infected with lnc RP11-499F3.2 overexpression and silencing lentivirus, respectively, to screen for stable transfected cells. Cell proliferation, migration, invasion, and scratch assays were then performed.

[0021] This invention also discloses a method for establishing a stable cetuximab-resistant cell line: using a concentration gradient screening method to establish an SCC4 / CTX-resistant cell line.

[0022] To investigate the role of lnc RP11-499F3.2 in cetuximab resistance, we designed a method to specifically regulate the expression of RP11-499F3.2 using locked nucleoside. Specifically, this specific locked nucleoside sequence is shown in SEQ ID NO.4, and is: UCUUGGCUCCUCCUCGAAATT, where U is represented by n in the sequence listing.

[0023] Furthermore, using the nonspecific sequence NC as a negative control (SEQ ID NO.5, sequence UUCUCCGAACGUGUCACGUTT, where U is represented by n in the sequence listing), SCC4 / CTX cell lines were transfected. The sensitivity of lnc RP11-499F3.2 to cetuximab-resistant cells was investigated by detecting changes in the in vitro proliferation and metastasis of drug-resistant cells.

[0024] Meanwhile, we investigated the effect of specific locked nucleoside on cetuximab resistance in head and neck squamous cell carcinoma. Specifically, we established a cetuximab resistance PDX model of head and neck squamous cell carcinoma through multiple in vivo passages, and administered a certain dose of lnc RP11-499F3.2 locked nucleoside intratumorally, monitoring changes in tumor volume.

[0025] 3. Beneficial effects

[0026] (1) This invention found that lnc RP11-499F3.2 is significantly highly expressed in head and neck squamous cell carcinoma and is closely related to prognosis, and can be used as a potential biomarker for head and neck squamous cell carcinoma;

[0027] (2) This invention found that the expression level of lnc RP11-499F3.2 in head and neck cancer cells was significantly higher than that in oral epithelial cells. The loss of lnc RP11-499F3.2 expression could significantly inhibit the proliferation, migration, invasion and colony formation of head and neck cancer cells, suggesting its importance to tumor growth and metastasis, and providing a reference for targeted therapy of head and neck cancer.

[0028] (3) This invention establishes a cetuximab-resistant cell line in vitro and a cetuximab-resistant PDX model in vivo. It found that downregulating lnc RP11-499F3.2 expression helps restore the sensitivity of head and neck cancer cells to cetuximab, further proving that lnc RP11-499F3.2 is a key lncRNA for the cetuximab resistance phenotype in head and neck squamous cell carcinoma.

[0029] (4) This invention also attempts to specifically interfere with the role of RP11-499F3.2 in other indications of cetuximab, including colorectal cancer, esophageal cancer, and non-small cell lung cancer drug-resistant cell models, to expand the clinical treatment of cetuximab.

[0030] (5) The experiments designed in this invention are scientific, reasonable, feasible and effective, and the research on long non-coding RNA RP11-499F3.2 is in-depth and systematic. Based on the above findings, the expression level of lnc RP11-499F3.2 can be used as a new biomarker to assist in the diagnosis of head and neck cancer and the prediction of its malignancy. In particular, it can reverse the responsiveness of drug-resistant head and neck cancer patients to cetuximab treatment and improve the treatment effect, which has good prospects for translational medicine. Attached Figure Description

[0031] Appendix Figure 1 Clustering diagram of differentially expressed LncRNAs in head and neck cancer tissues and adjacent normal tissues, data from TCGA database;

[0032] Appendix Figure 2 The relationship between lnc RP11-499F3.2 expression level and overall survival in HNSCC patients, data from the TCGA database;

[0033] Appendix Figure 3 Comparison of expression levels of lnc RP11-499F3.2 in 46 pairs of head and neck cancer tissues and adjacent normal tissues, data from the TCGA database;

[0034] Appendix Figure 4 qPCR detection results of lnc RP11-499F3.2 in 50 pairs of HNSCC clinical samples (2 -ΔΔct For value comparison, *P<0.05, **P<0.01);

[0035] Appendix Figure 5 A graph showing the relationship between the expression level of lnc RP11-499F3.2 and the overall survival of HNSCC patients in 50 clinical tissue samples;

[0036] Appendix Figure 6 Figure showing the relationship between the expression level of lnc RP11-499F3.2 and the overall survival of HNSCC patients in 30 clinical serum samples;

[0037] Appendix Figure 7 Figure showing the comparison of expression levels of lnc RP11-499F3.2 in HNSCC cells and oral epithelial cells;

[0038] Appendix Figure 8 Map of lentiviral vectors overexpressing and silencing lnc RP11-499F3.2;

[0039] Appendix Figure 9 Figure 1. Results of transfection of SCC4 cells with lnc RP11-499F3.2 overexpression and silencing lentiviral vector and qPCR detection.

[0040] Appendix Figure 10 The effect of overexpression and silencing of lnc RP11-499F3.2 on the proliferation of SCC4 cells (Figure 1).

[0041] Appendix Figure 11 The effect of overexpression and silencing of lnc RP11-499F3.2 on the migration ability of SCC4 cells is shown in the figure.

[0042] Appendix Figure 12 The effect of overexpression and silencing of lnc RP11-499F3.2 on the invasive ability of SCC4 cells;

[0043] Appendix Figure 13 The IC50 value of the parental SCC4 response to cetuximab was determined by the MTT assay.

[0044] Appendix Figure 14 Figure showing the effect of cetuximab on the proliferation of SCC4 and SCC4 / CTX cells;

[0045] Appendix Figure 15 Figure showing the effect of cetuximab on the cell cycle distribution of SCC4 and SCC4 / CTX.

[0046] Appendix Figure 16 Figure showing the effect of cetuximab on apoptosis in SCC4 and SCC4 / CTX cells;

[0047] Appendix Figure 17 Figure showing the effect of cetuximab on colony formation in SCC4 and SCC4 / CTX cells;

[0048] Appendix Figure 18 Figure showing the effect of cetuximab on in vivo tumor growth in SCC4 and SCC4 / CTX cells;

[0049] Appendix Figure 19 Detection of lnc RP11-499F3.2 expression level in SCC4 and SCC4 / CTX cell lines;

[0050] Appendix Figure 20 Establish dynamic tumor growth map of a cetuximab-sensitive PDX xenograft model;

[0051] Appendix Figure 21 Dynamic growth of a cetuximab-resistant PDX xenograft model of head and neck squamous cell carcinoma;

[0052] Appendix Figure 22 Detection of lnc RP11-499F3.2 expression levels in cetuximab-sensitive and cetuximab-tolerant HNSCC-PDX model tumor tissues;

[0053] Appendix Figure 23 Dynamic growth of tumors in a cetuximab-resistant HNSCC PDX model after treatment with lnc RP11-499F3.2;

[0054] Appendix Figure 24Tumor anatomy diagram of a cetuximab-resistant HNSCC PDX model after treatment with lnc RP11-499F3.2 targeted therapy;

[0055] Appendix Figure 25 HE staining results of major organs in a cetuximab-resistant HNSCC PDX model after targeted therapy with lnc RP11-499F3.2. Detailed Implementation

[0056] The present invention will be further described below with reference to specific embodiments.

[0057] Example 1

[0058] Analysis of lncRNA sequencing results from human head and neck cancer tissues and paired normal tissues

[0059] The Tumor Genome Atlas (TCGA) project, jointly launched in 2006 by the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI), utilizes large-scale sequencing-based genomic analysis technologies to conduct large-scale experiments on 36 types of cancer. The TCGA Genome Analysis Center (GCC) compares tumors with normal tissues to identify gene mutations, amplifications, or deletions associated with each cancer or subtype. This contributes to understanding the molecular mechanisms of cancer and improving our scientific understanding of the molecular basis of cancer pathogenesis.

[0060] Access the TCGA (https: / / cancergenome.nih.gov / ) head and neck squamous cell carcinoma option page, select the clinical and RNAseq options, and click to enter the page. In the RNAseq option, select the rsem.genes.normalized_results.txt file, and select all files in the METADATA and clinical directories. Obtain the download address and download them. After obtaining the gene expression data of head and neck squamous cell carcinoma tissues, rename the RNA normalized data according to the METADATA file information to facilitate comparison with gene expression data files and clinicopathological information. After comparison, a total of 500 head and neck squamous cell carcinoma tissues and 46 adjacent tissues of head and neck squamous cell carcinoma were found, with complete corresponding RNA expression data and clinicopathological information.

[0061] R software (version 3.1) was used to analyze gene expression data from head and neck squamous cell carcinoma tissues and screen for differentially expressed lncRNAs. R software employs a gene chip expression differential algorithm that corrects for expression abundance and noise levels. Gene expression data from head and neck squamous cell carcinoma tissues were standardized and entered into Excel. lncRNAs with more than 80% of the sample size showing missing values ​​were extracted. Differentially expressed lncRNAs were analyzed using R software. The screening criteria for differentially expressed lncRNAs were set as FDR < 0.05 and Fold change > 2, meaning lncRNAs with a p-value < 0.05 and an absolute difference > 2 between head and neck squamous cell carcinoma tissues and normal tissues were selected. Cluster analysis was performed using the Cluster 3.0 software package to visualize the high-throughput gene expression data from head and neck squamous cell carcinoma tissues.

[0062] See results Figure 1 From 13,964 candidate lncRNAs, this study obtained 563 differentially expressed lncRNAs (fold change>2, P<0.05) based on the screening criteria. Among them, 254 lncRNAs were significantly upregulated in head and neck squamous cell carcinoma tissues, and 309 lncRNAs were downregulated.

[0063] Example 2

[0064] Screening of lnc RP11-499F3.2 and its relationship with overall survival in HNSCC patients

[0065] All clinical samples were randomly and equally divided into two groups (training set and test set), with 250 clinical samples in each group. The clinical samples in the training set were divided into high-risk and low-risk groups based on the relative expression level of the screened lncRNAs. The Kaplan-Meier survival curves were plotted using the lasso method with patient survival time as the independent variable. The lncRNAs screened in the training set were validated using the test set, with the same screening methods and conditions.

[0066] Table 1. Kaplan-Meier survival curve method for screening lncRNAs significantly associated with HNSCC survival.

[0067]

[0068] To further validate and screen lncRNAs related to the diagnosis of head and neck squamous cell carcinoma, this invention plotted ROC curves for the eight screened lncRNAs, evaluated the diagnostic predictive ability of these lncRNAs for the survival of patients with head and neck squamous cell carcinoma, and screened ideal diagnostic molecular markers for head and neck squamous cell carcinoma.

[0069] Table 2. Screening of survival-related lncRNAs in head and neck squamous cell carcinoma patients using ROC curve method.

[0070]

[0071] After removing lncRNAs with AUC < 0.6 from the ROC curves based on Tables 1 and 2, a total of four lncRNAs meeting the criteria were screened: RP11-499F3.2, LINC00460, LINC00958, and ST3GAL4-AS1, showing high specificity and sensitivity. Among them, lncRNA RP11-499F3.2, which is specifically highly expressed in head and neck squamous cell carcinoma tissues, had the highest AUC area, with AUCs of 0.714 and 0.748 in the training and test sets, respectively. Its sensitivity and specificity were both above 80%, making it an ideal diagnostic molecular marker for head and neck squamous cell carcinoma.

[0072] See results Figure 1 The expression level of lnc RP11-499F3.2 was significantly associated with the prognosis of HNSCC in both the training and test sets.

[0073] Example 3

[0074] Analysis of expression levels of lnc RP11-499F3.2 in HNSCC tumor tissues and paired normal tissues

[0075] Clinical information and corresponding lnc RP11-499F3.2 expression data of 43 pairs of HNSCC samples, including cancerous and adjacent tissues, were obtained according to the method in Example 1. Results are shown in […]. Figure 2 Compared with paired normal adjacent normal tissue, the expression level of lnc RP11-499F3.2 was significantly increased in HNSCC, which may be considered as an indicator for early clinical diagnosis of head and neck cancer.

[0076] Example 4

[0077] Expression of lnc RP11-499F3.2 in head and neck cancer patients and normal adjacent tissues.

[0078] (1) Specimen collection

[0079] With the patient's informed consent, specimens of head and neck cancer and adjacent tissues are collected during the operation, washed with physiological saline, and then stored in liquid nitrogen or a -80°C refrigerator for later use.

[0080] (2) Primer design

[0081] Based on the information from lnc RP11-499F3.2, all exon sequence information for this gene was searched in the ensemble database. Primers were then designed using Primer Premier 5.0 based on the obtained sequence information. The sequences are as follows:

[0082] Upstream primer (SEQ ID NO.2)

[0083] Downstream primer (SEQ ID NO.3)

[0084] (3) Real-time quantitative PCR was used to detect the expression of lnc RP11-499F3.2 in head and neck cancer patients and normal adjacent tissues.

[0085] Total RNA was extracted from the collected samples according to the Trizol instructions from Life. The purity and concentration of the extracted RNA were then quantified using a NanoDrop ND-1000 nucleic acid quantification instrument. Agarose gel electrophoresis was performed to ensure the integrity of the extracted RNA. cDNA was synthesized from the extracted total RNA using the TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time). The TaKaRa kit was used. Premix Ex Taq TM qPCR was performed using Tli RNaseH Plus. The reaction system is shown in the table below:

[0086] Table 3 PCR reaction system

[0087]

[0088] After mixing the above components evenly, follow the procedure below: pre-denaturation at 95℃ for 30s, 40 cycles; 95℃ for 5s, 60℃ for 30s.

[0089] The specificity of the reaction was determined based on the melting curve, and the relative expression level of lnc RP11-499F3.2 was calculated using formula 2-ΔΔCt. The results are shown below. Figure 4 In approximately 75% of head and neck cancer samples, the expression level of lnc RP11-499F3.2 was significantly higher than that of normal adjacent normal tissue.

[0090] Example 5

[0091] Relationship between lnc RP11-499F3.2 expression level and overall survival in 50 patients with HNSCC

[0092] Fifty HNSCC patients were divided into high-risk and low-risk groups based on the relative expression level of lncRNA. With patient survival time as the independent variable, the lasso method was used to select variables and Kaplan-Meier survival curves were plotted.

[0093] See results Figure 5The expression of lncRP11-499F3.2 was correlated with the survival rate of patients with head and neck cancer. Patients with low expression of lncRP11-499F3.2 had a significantly higher overall survival rate than patients with high expression of TMEM170B, further confirming lncRP11-499F3.2 as a new prognostic indicator for head and neck cancer.

[0094] Example 6

[0095] Expression levels of lnc RP11-499F3.2 in the serum of patients with head and neck cancer and healthy individuals

[0096] (1) Specimen collection

[0097] With the informed consent of the patients, serum samples were collected from head and neck cancer patients and healthy volunteers before the operation and stored in a -80°C freezer for later use.

[0098] (2) Real-time quantitative PCR detection of lnc RP11-499F3.2 expression in serum of head and neck cancer patients and healthy volunteers.

[0099] The primer design and specific detection method are the same as in Example 5.

[0100] See results Figure 6 Compared with healthy volunteers, the expression level of lnc RP11-499F3.2 was significantly increased in HNSCC, which is considered as a serological marker for the early clinical diagnosis of head and neck cancer.

[0101] Example 7

[0102] Expression of lnc RP11-499F3.2 in HNSCC cells and normal oral epithelial cells was detected.

[0103] Total RNA was extracted from head and neck cancer cells SCC4, SCC9, SCC13, CAL27 and normal oral epithelial cells HIOEC. lnc RP11-499F3.2 was detected by qPCR, using the same method as in Example 5. Results are shown below. Figure 7 The expression of the head and neck squamous cell carcinoma cell line lnc RP11-499F3.2 was significantly higher than that of HIOEC cells.

[0104] Example 8

[0105] Preparation of lnc RP11-499F3.2 overexpression and silencing vectors and detection of viral transfection efficiency.

[0106] Synthesize full-length cDNA targeting lnc RP11-499F3.2 and introduce it into an overexpression lentiviral vector. Figure 8Following the design rules for shRNA, three small interfering RNAs (sequences SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8) targeting exon RP11-499F3.2 were designed and introduced into a silencing lentiviral vector ( ). Figure 8 The above plasmids, along with packaging plasmid DR8.9 and envelope plasmid VSVG.2, were co-transfected into 293T cells to generate virus. Forty-eight hours after transfection, the viral supernatant was collected and used to infect SCC4 cells. Twenty-four hours after infection, puromycin was added to select for stable lnc RP11-499F3.2 overexpression and silenced cell lines. Total RNA from stable cells was collected, and changes in lnc RP11-499F3.2 expression were detected by qPCR (specific method as in Example 5).

[0107] See results Figure 9 Observations showed that transfected cells overexpressing the lentiviral plasmid lncRP11-499F3.2 exhibited significant GFP protein expression under fluorescence, with lncRP11-499F3.2 expression significantly upregulated compared to the control group CTRL (P<0.01). Furthermore, based on GFP expression and qRT-PCR experiments, the results indicated that the sh-c interference lentiviral plasmid achieved the highest transfection efficiency.

[0108] Example 9

[0109] Effects of overexpression and silencing of lnc RP11-499F3.2 on the proliferation of SCC4 cells

[0110] The effects of overexpression and silencing of lnc RP11-499F3.2 on the proliferation of SCC4 cells were detected using the MTT assay. Head and neck cancer cells were cultured at 37°C in a 5% CO2 incubator until confluence with the target cell line. Cells were then collected by trypsin digestion, resuspended in culture medium, and counted under a microscope. The cell concentration was adjusted to 3.0 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates and incubated at 37°C in a 5% CO2 incubator. After 0 h, 24 h, 48 h, and 72 h of incubation, 20 μL of 5 mg / mL MTT was added to each well, and the plates were incubated for another 4 h. The culture medium was then removed, and 100 μL of DMSO was added to each well to dissolve the cells. The absorbance was measured using a microplate reader at a detection wavelength of 570 nm and a reference wavelength of 630 nm, and the proliferation inhibition rate (PI) was calculated.

[0111] The experiment was independently repeated 3 times. The results were expressed as mean ± SD and statistical t-tests were performed. *P < 0.05 was considered statistically significant, and **P < 0.01 was considered highly statistically significant.

[0112] See results Figure 10 Overexpression of lnc RP11-499F3.2 significantly upregulated cell proliferation, while silencing lnc RP11-499F3.2 reduced the proliferation of head and neck squamous cell carcinoma cells. This indicates that lnc RP11-499F3.2 has the ability to promote the proliferation of head and neck squamous cell carcinoma cells in vitro.

[0113] Example 10

[0114] Effects of overexpression and silencing of lnc RP11-499F3.2 on the migration ability of SCC4 cells

[0115] Head and neck cancer cells (SCC4) were seeded into transwell chambers at a density of 100 μL per well. 0.6 mL of complete culture medium containing 10% FBS was added to the lower chamber to stimulate cell migration. Cells were incubated at 37°C with 5% CO2 for 24 h. The culture medium was discarded, and the cells were fixed with 90% ethanol at room temperature for 30 min, stained with 0.1% crystal violet at room temperature for 10 min, rinsed thoroughly with water, and the supernatant of unmigrated cells was gently wiped away with a cotton swab. The cells were observed under a microscope, and four fields of view were photographed for cell counting. The migration inhibition rate (MIR) was calculated using the following formula:

[0116]

[0117] Where Ntest ​​represents the number of migrating cells in the test group and Ncontrol represents the number of migrating cells in the blank control group. The experiment was independently repeated three times. The results were calculated as mean ± SD and subjected to statistical t-tests. *P < 0.05 was considered statistically significant, and **P < 0.01 was considered highly statistically significant.

[0118] See results Figure 11 Overexpression of lnc RP11-499F3.2 significantly increased the number of successfully migrating SCC4 cells, while knockdown of lnc RP11-499F3.2 significantly reduced the number of migrating SCC4 cells after 48 hours of culture. This indicates that lnc RP11-499F3.2 can promote cell migration in the head and neck squamous cell carcinoma line SCC4.

[0119] Example 11

[0120] Effects of overexpression and silencing of lnc RP11-499F3.2 on the invasive ability of SCC4 cells

[0121] 10 mg / mL Matrigel was diluted 1:3 with culture medium and spread onto Transwell membranes, then air-dried at room temperature. Head and neck cancer cells cultured to the logarithmic growth phase were digested with trypsin, collected, washed twice with PBS, and resuspended in blank culture medium. The cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 5 Cells / mL. Seed 100 μL of cells into each well of a transwell chamber. Add 0.6 mL of complete culture medium containing 10% FBS to the lower chamber of the transwell to stimulate cell invasion. Incubate at 37°C with 5% CO2 for 24 h. Discard the culture medium in the wells, fix with 90% ethanol at room temperature for 30 min, stain with 0.1% crystal violet at room temperature for 10 min, rinse thoroughly with water, gently wipe away the supernatant of non-invading cells with a cotton swab, observe under a microscope, and photograph and count four fields of view. Calculate the invasion inhibition rate (IIR) according to the formula:

[0122]

[0123] Where Ntest ​​represents the number of invasive cells in the test group and Ncontrol represents the number of invasive cells in the blank control group. The experiment was independently repeated three times. The results were calculated as mean ± SD and statistically analyzed using t-tests. *P < 0.05 was considered statistically significant, and **P < 0.01 was considered highly statistically significant.

[0124] See results Figure 12 Overexpression of lnc RP11-499F3.2 significantly increased the number of SCC4 cells that successfully invaded the cell line. Conversely, knockdown of lnc RP11-499F3.2 significantly reduced the number of invading SCC4 cells after 48 hours of culture. This indicates that lnc RP11-499F3.2 can promote cell invasion in the head and neck squamous cell carcinoma line SCC4.

[0125] Example 12

[0126] IC50 value of parental sensitive SCC4 response to cetuximab using the MTT assay

[0127] Cetuximab concentrations of 10 nM, 20 nM, 40 nM, 80 nM, 160 nM, and 320 nM were set, with docetaxel (10 μg / ml) as the positive control. The MTT assay was used to detect the growth of SCC4 cells at different drug concentrations. The specific method is described in Example 9.

[0128] See results Figure 13The inhibitory effect of cetuximab on the proliferation of SCC4 cells increased significantly with increasing drug concentration. This cell line was sensitive to cetuximab. The IC50 value of SCC4 cells under the action of cetuximab was 80 nM, which was used as the starting concentration for screening cetuximab-resistant cell lines.

[0129] Example 13

[0130] Effects of cetuximab on the proliferation of SCC4 and SCC4 / CTX cells

[0131] (1) Resuscitate and culture SCC4 cells. When SCC4 cells grow to 70% density, discard the culture medium in the culture flask and replace it with DMEM complete medium with a final drug concentration of 80 nM. Culture in an incubator at 37°C with 5% CO2 for 48 h, and then use drug-free medium to stably passage 3 or more times.

[0132] (2) Once the cells have recovered stable growth and the cell density has reached 70%, start doubling the concentration of cetuximab in the culture medium and repeat the above steps. Each drug dose is used to maintain cell culture for 15-20 days.

[0133] (3) SCC4 cells induced by various drug concentrations were frozen periodically, and the IC50 value of the induced cells was measured. Finally, SCC4 cells resistant to 1280 nM cetuximab were obtained, and the drug-resistant cells were named SCC4 / CTX.

[0134] (4) The effect of different concentrations of cetuximab on the proliferation ability of SCC4 and SCC4 / CTX cells was detected by the MTT assay. The specific method is described in Example 9.

[0135] See results Figure 14 Compared to the parental SCC4 drug-sensitive cells, the drug-resistant SCC4 / CTX cells established in this invention still showed a significant increase in cell proliferation at cetuximab-tolerant doses.

[0136] Example 14

[0137] Effects of cetuximab on SCC4 and SCC4 / CTX cell cycle distribution

[0138] (1) When SCC4 and SCC4 / CTX grow to the logarithmic growth phase, add 0.25% trypsin solution to digest them, transfer them to EP tubes, centrifuge at 1000 rpm for 5 min, resuspend them and plate them into six-well cell culture plates. When the cell density grows to 60%, discard the culture medium and add DMEM serum-free culture medium containing 0 nM, 40 nM and 320 nM cetuximab drug concentrations respectively. Place the six-well plates in an incubator at 37°C with 5% CO2 for 48 h.

[0139] (2) Digest with 0.25% trypsin solution, transfer to centrifuge tubes, centrifuge at 900 rpm for 5 min, resuspend and wash cells with PBS, count cell mass using a cell counter, and adjust cell suspension concentration to 1×10⁻⁶. 6 pcs / ml;

[0140] (3) Take 1 ml of cell suspension, add 500 μl of 70% pre-cooled ethanol solution, fix at 4°C for 2 h to overnight, centrifuge at 1000 rpm for 3 min and discard the supernatant, wash the cells twice with PBS to remove residual fixative;

[0141] (4) Prepare staining working solution according to the volume ratio of RNase:PI working solution of 1:9. Add 500 μl of staining working solution to each sample and incubate at 4°C in the dark for 30 min.

[0142] (5) Cell cycle was detected by flow cytometer, and the red fluorescence signal of each cell sample at an excitation wavelength of 488 nm was selected and recorded.

[0143] (6) Data analysis was performed using FlowJo v5.7.3 software and the Dean-Jett-Fox fitting model. The cell number was used as the vertical axis and the intensity of the PI red fluorescence signal was used as the horizontal axis to statistically analyze the distribution of sample cells in each cell cycle.

[0144] See results Figure 15 The proportion of SCC4 cells in the G1 phase increased significantly with increasing drug concentration (P<0.05); the proportion in the S phase decreased significantly with increasing drug concentration (P<0.05). Cetuximab arrested cell growth in the G1 phase and inhibited the proliferation of SCC4 cells. However, there were no significant differences in the distribution of SCC4 / CTX cells in the G1, S, and G2 / M phases under different concentrations of cetuximab.

[0145] Example 15

[0146] Effects of cetuximab on apoptosis in SCC4 and SCC4 / CTX cells

[0147] (1) When SCC4 and SCC4 / CTX grow to the logarithmic growth phase, digest them with 0.25% trypsin solution, transfer them to EP tubes, centrifuge at 1000 rpm for 5 min, resuspend them and plate them into six-well cell culture plates. When the cell density grows to 60%, discard the culture medium and add DMEM serum-free culture medium containing 0 nM, 40 nM and 320 nM cetuximab drug concentrations respectively. Place the six-well plates in an incubator at 37°C with 5% CO2 for 48 h.

[0148] (2) Digest with 0.25% trypsin solution, transfer to centrifuge tubes, centrifuge at 900 rpm for 5 min, resuspend in pre-cooled PBS buffer and wash twice to remove residual trypsin from the cell suspension. Count the cell count using a cell counter, add 400 μl of Annexin V binding solution, resuspend the cells and adjust the cell concentration to 1 × 10⁻⁶. 6 pcs / ml;

[0149] (3) Add 5 μl of Annexin V-FITC staining solution to the cell suspension, mix gently, incubate at 4°C in the dark for 15 min, then add 10 μl of PI staining solution, mix gently, and incubate at 4°C in the dark for 5 min.

[0150] (4) Immediately use a flow cytometer to detect cell apoptosis, select and record the fluorescence signal of cell samples with an excitation wavelength of 488nm and an emission wavelength of 530nm, and at the same time detect Annexin V-FITC single positive tubes and PI single positive tubes of untreated cells to determine the fluorescence compensation value and the position of the cross quadrant gate.

[0151] (5) Data analysis was performed using FlowJo v5.7.3 software. The intensity of PI red fluorescence signal was used as the ordinate, and the intensity of Annexin V-FITC green fluorescence signal was used as the abscissa. In the experimental results, the upper right Q2 quadrant belonged to late apoptotic cells, the lower right Q3 quadrant belonged to early apoptotic cells, and the lower left Q4 quadrant belonged to normal cells. The Q2 and Q3 quadrants were used to calculate the cell apoptosis rate.

[0152] See results Figure 16 In SCC4 cells, the apoptosis rate significantly increased under treatment with cetuximab at both 40 nM and 320 nM concentrations. However, the apoptosis rate of SCC4 / CTX cells only significantly increased under the 320 nM concentration, and at the same concentration, the apoptosis rate of SCC4 / CTX cells was significantly lower than that of SCC4 cells. Therefore, it can be concluded that under cetuximab treatment, SCC4 / CTX cells exhibit stronger apoptosis resistance than SCC4 cells.

[0153] Example 16

[0154] Effects of cetuximab on SCC4 and SCC4 / CTX cell colony formation

[0155] (1) Weigh 0.56g of low melting point agarose powder, add 10ml of double distilled water to prepare a 5.6% agarose gel stock solution, autoclave at 121℃ for 30min, and place in a 50℃ water bath for constant temperature.

[0156] (2) Preparation of 0.8% lower layer agarose gel: Take 1.5 ml of 5.6% agarose gel stock solution, keep the temperature at about 40℃ to prevent solidification, add 9 ml of DMEM complete culture medium, blow evenly, dispense 0.5 ml / well into 12-well cell culture plate, and lay a total of 18 wells. Cool and solidify at 4℃ for 5 min.

[0157] (3) Prepare 45.7 nM and 365.7 nM cetuximab-containing DMEM complete medium, respectively. Digest and collect SCC4 and SCC4 / CTX cells in the logarithmic growth phase. Divide both cell types into three equal portions and resuspend them in DMEM complete medium, 45.7 nM cetuximab-containing DMEM medium, and 365.7 nM cetuximab-containing DMEM medium. Count the cells and adjust the cell concentration to 1 × 10⁻⁶. 4 pcs / ml;

[0158] (4) Preparation of 0.7% upper agarose gel: Take 1.2 ml of 5.6% agarose gel stock solution and divide it into 6 5 ml centrifuge tubes. Add 1.4 ml of the prepared cell suspensions of SCC4 and SCC4 / CTX to each tube, and gently pipette to mix. Dispense 0.5 ml / well into a 12-well cell culture plate that has been plated with the lower gel. Set up three replicates for the same drug concentration for the same cell type. Plate a total of 9 wells for each cell suspension. The cell amount per well is 5 × 103 cells. Cool and solidify at 4°C for 5 min.

[0159] (5) After the agarose gel solidifies, add the corresponding DMEM complete medium, 40 nM drug-treated DMEM medium and 320 nM drug-treated DMEM medium to the wells according to the colony formation experimental group, 0.5 ml per well;

[0160] (6) Place the 12-well plate in an incubator at 37°C with 5% CO2 for 15-20 days. Replace the upper culture medium every three days. Stop cell culture when visible cell clones are present in the culture plate.

[0161] (7) Remove and discard the upper culture medium, add 1 ml of 0.01% crystal violet staining solution to each well, stain in the dark for 1 h, decolorize with PBS buffer until the clones are clearly visible, place the cell culture plate under an inverted microscope, observe and count the number of SCC4 and SCC4 / CTX cell clones.

[0162] See results Figure 17 As the drug concentration increased, the number of SCC4 cell clones decreased significantly, while the number of SCC4 / CTX cell clones did not decrease significantly under different drug concentrations and there was no significant difference. This indicates that cetuximab has a weak ability to inhibit the formation of SCC4 / CTX cell clones.

[0163] Example 17

[0164] Effects of cetuximab on tumor growth in SCC4 and SCC4 / CTX cell xenograft models

[0165] (1) Large-scale culture of SCC4 and SCC4 / CTX cells was performed using 0.25% trypsin solution for digestion. After digestion was terminated, the cell suspension was centrifuged at 1000 rpm for 5 min. The cells were then resuspended in serum-free DMEM medium and counted. The cell concentration was adjusted to 5 × 10⁶ cells / year. 7 pcs / ml;

[0166] (2) Each nude mouse (24 female BALB / c nude mice aged 4-6 weeks and weighing 14-16g were ordered and acclimatized in an SPF-grade animal housing for 1 week) was injected with 200μl of the corresponding group of cell suspension under its left axilla. The cell volume injected was 1×10⁻⁶. 7 indivual;

[0167] (3) Closely observe the tumor growth at the injection site of nude mice after inoculation. On the 7th day after inoculation, white nodules appeared at the injection site. They could be moved under the skin when touched. As the tumor tissue grew, the injection site gradually formed a hard tumor mass. The average volume of the tumor tissue reached 100 mm3 in about 14 days. BALB / c nude mice were randomly divided into four groups of 6 mice each. The weight of the animals was 16-18g when the drug was first administered.

[0168] (4) Measure and record the tumor volume every two days. The formula for calculating tumor volume (TV) is as follows:

[0169] Tumor volume = 0.5 × a × b^2

[0170] Where a is the length of the transplanted tumor and b is the width of the transplanted tumor.

[0171] See results Figure 18 In the SCC4 xenograft model, the cetuximab group (20 mg / kg) -1 The tumor volume of the cetuximab group was significantly reduced, showing a highly significant difference (P<0.001); in the SCC4 / CTX xenograft model, the cetuximab group (20 mg / kg) showed a significant reduction in tumor volume. -1 Under continuous pressure from cetuximab, no significant slowdown in tumor growth was observed, and the tumor growth curve was similar to that of the negative group. This indicates that the SCC4 / CTX cell xenograft model exhibits a cetuximab resistance phenotype, and that SCC4 / CTX cells possess cetuximab resistance in vivo.

[0172] Example 18

[0173] Detection of lnc RP11-499F3.2 expression levels in SCC4 and SCC4 / CTX cells after in vivo tumor drug administration;

[0174] After drug administration to SCC4 and SCC4 / CTX cell xenograft models in vivo, tumor tissues were dissected for qRT-PCR monitoring. The experimental results are shown below. Figure 19 Under cetuximab drug pressure (20 mg / kg) -1 Under these conditions, the expression level of lnc RP11-499F3.2 in SCC4 cell xenograft tissues was significantly decreased (P<0.05), while the expression in the SCC4 / CTX cell line did not change significantly, and the expression level of lnc RP11-499F3.2 in the SCC4 / CTX cell line was still significantly different from that in SCC4 cells (P<0.01). lnc RP11-499F3.2 expression is positively correlated with cetuximab resistance phenotype, and it may be a target molecule for reversing cetuximab resistance in head and neck squamous cell carcinoma.

[0175] Example 19

[0176] Dynamic growth of tumors in a cetuximab-sensitive PDX xenograft model

[0177] (1) Fresh tissue samples of head and neck squamous cell carcinoma were obtained directly after surgery. After the samples were removed from the body, they were quickly placed in sterile containers and transported to a sterile operating table.

[0178] (2) Fresh tumor tissue was quickly washed with saline containing antibiotics and immediately transferred to a 10cm cell culture dish containing DMEM medium containing antibiotics. Necrotic tumor tissue, intratumoral fibrous tissue and adipose tissue were cut off and washed again.

[0179] (3) Cut the remaining active tumor tissue into tumor tissue blocks of 2×2×2mm3. Take 2-3 BALB / c nude mice, disinfect the skin of the forelimb dorsal and ventral sides with 75% ethanol, anesthetize the mice with isoflurane, cut a 2mm skin incision in the axilla of the forelimb of the mouse, fill the tumor tissue block into an 18-gauge cannula puncture needle, puncture into the subcutaneous tissue of the shoulder and back of the mouse forelimb, forming a 10mm long subcutaneous transitional sinus tract, push the core of the cannula puncture needle to inoculate the tumor tissue block into the subcutaneous tissue of the shoulder and back, inoculate two sites per mouse, inoculate 2-3 mice per sample (determined according to the amount of tumor sample), the process from sample ex vivo to tumor tissue transplantation should not exceed 4 hours, the head and neck squamous cell carcinoma PDX xenograft model is named G1 generation;

[0180] (4) When the average tumor volume of the PDX xenograft models in each experimental group grows to 150 mm 3 And the number of PDX model mice of the same generation meets the grouping requirements (no less than 8 mice), and in vivo cetuximab administration begins to verify the sensitivity of PDX parent model mice to cetuximab.

[0181] (5) PDX model mice in each experimental group were randomly divided into two groups, with 4 mice in each group. The groups were set as G1 negative control (physiological saline) and G2 cetuximab (20 mg·kg-1), and the dosing period was 21 days.

[0182] See results Figure 20 In nude mice, PDX groups whose tumor volume decreased by more than 50% after drug treatment were defined as drug-sensitive groups, those whose tumor volume increased by more than 35% after drug treatment were defined as drug-tolerant groups, and the remaining groups were defined as drug-stable groups. Two drug-sensitive PDX models were obtained. In this invention, a moderately sensitive PDX model (Group C2, oral squamous cell carcinoma patient) was selected as the parental model for establishing a cetuximab-resistant head and neck squamous cell carcinoma PDX xenograft model for in vivo drug administration experiments.

[0183] Example 20

[0184] Dynamic growth of cetuximab-resistant PDX xenografts of head and neck squamous cell carcinoma

[0185] After passage of the selected drug-sensitive PDX xenografts of head and neck squamous cell carcinoma, the tumors were allowed to grow to 150 mm in size. 3 Cetuximab was administered intravenously to induce a drug-resistant phenotype in a drug-sensitive PDX xenograft model. After three rounds of intravenous administration of cetuximab and tumor tissue passage, the C2 group PDX model showed a significant drug-resistant phenotype, with tumor volume still showing significant growth under cetuximab drug pressure. Results are shown below. Figure 21 In the cetuximab-sensitive oral squamous cell carcinoma PDX model (OSCC), the tumor volume decreased significantly after cetuximab administration compared to the negative control group (saline group). However, in the oral squamous cell carcinoma PDX model (OSCC-CR) induced by repeated in vivo administration of cetuximab, the tumor growth volume in the negative control group was significantly higher than that in the drug-sensitive model (OSCC) (P<0.01). Under the pressure of cetuximab, the negative control group showed significant tumor growth, with the tumor volume increasing by more than 35%, exhibiting a significant drug resistance phenotype, which meets the definition of the drug-resistant group. Thus, the cetuximab-resistant PDX xenograft model of head and neck squamous cell carcinoma was successfully established.

[0186] Example 21

[0187] Detection of lnc RP11-499F3.2 expression levels in cetuximab-sensitive and cetuximab-tolerant HNSCC-PDX model tumor tissues

[0188] After in vivo administration of cetuximab-sensitive and tolerable HNSCC-PDX models, tumor tissue was dissected and monitored by qRT-PCR. The experimental results are shown below. Figure 22The expression level of lnc RP11-499F3.2 in the cetuximab-resistant OSCC-CR PDX model was significantly different from that in the drug-sensitive model (P<0.01). Simultaneously, under cetuximab stress, the relative expression of lnc RP11-499F3.2 in the tumor tissue of the drug-sensitive model decreased significantly (P<0.05), while the expression in the OSCC-CR model tumor tissue did not change significantly; its lnc RP11-499F3.2 expression level remained significantly higher than that of the drug-sensitive model (P<0.01). These results are consistent with the expression of lnc RP11-499F3.2 in SCC4 / CTX (Example 18), further demonstrating through clinical tumor tissue that the expression level of lnc RP11-499F3.2 is positively correlated with cetuximab resistance, and verifying the successful establishment of the cetuximab-resistant head and neck squamous cell carcinoma PDX model in this invention.

[0189] Example 22

[0190] Dynamic tumor growth monitoring after cetuximab-resistant HNSCC PDX model treated with lnc RP11-499F3.2 targeted therapy.

[0191] To verify in vivo the role of lnc RP11-499F3.2 in the formation of a cetuximab-resistant PDX xenograft model of head and neck squamous cell carcinoma, this invention designs an LNA targeting lnc RP11-499F3.2 for intratumoral injection.

[0192] (1) Order 20 female BALB / c nude mice aged 4-6 weeks and weighing 14-16g. Adapt them in an SPF-grade animal house for 1 week. Resuscitate and inoculate them with tissue blocks of the OSCC-CR cetuximab-resistant oral squamous cell carcinoma model. Closely observe the growth of the xenograft tumor at the inoculation site of the nude mice.

[0193] (2) When the average tumor volume reached 150 mm3, BALB / c nude mice were randomly divided into four groups of 5 mice each. The groups were set as follows: G1 negative control group (0.2 ml / 20 g), G2 cetuximab group (20 mg·kg-1), G3 LNA group (5 mg·kg-1), and G4 cetuximab + LNA group (cetuximab: 20 mg·kg-1; LNA: 5 mg·kg-1).

[0194] (3) The administration cycle was 21 days. After the administration cycle ended, the PDX model mice were observed for one week, and the changes in tumor volume of the PDX model were measured and recorded.

[0195] (4) After the experiment, the mice were euthanized by dislocation, the tumor was removed, the tumor volume was calculated and photographed, and the tumor tissue blocks of each PDX model group were retained for liquid nitrogen quick-freezing and cryopreservation.

[0196] See results Figure 23 After the completion of the treatment cycles in the C2 group of the cetuximab-resistant oral squamous cell carcinoma PDX model (OSCC-CR), the tumor volumes in the saline group, cetuximab group, LNA group, and cetuximab + LNA group were (1959.08±79.09) mm. 3 (832.08±92.08)mm 3 (419.03±73.38)mm 3 and (97.05±35.04)mm 3 (See Figure 24 The OSCC-CR model showed a tumor volume increase of over 35% under cetuximab stress, exhibiting a significant drug resistance phenotype, demonstrating cetuximab resistance. LNA administration alone significantly reduced tumor growth volume compared to the control group (P<0.001), indicating that LNA-targeted downregulation of lnc RP11-499F3.2 can inhibit tumor growth in the cetuximab-resistant model. After the completion of the cetuximab + LNA treatment cycle, the tumor volume showed negative growth, meaning that under cetuximab stress, simultaneous downregulation of intratumoral lnc RP11-499F3.2 expression in the OSCC-CR model could reverse the original drug resistance phenotype to a significant therapeutic effect. This suggests that LNA-targeted downregulation of lnc RP11-499F3.2 can resensitize cetuximab-resistant head and neck squamous cell carcinoma.

[0197] Example 23

[0198] HE staining results of major organs in a cetuximab-resistant HNSCC PDX model after targeted therapy with lnc RP11-499F3.2

[0199] After cetuximab-resistant HNSCC PDX models underwent targeted therapy with lnc RP11-499F3.2, heart, liver, spleen, lung, and kidney tissues were collected from the model mice, fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to observe the histopathological condition. Tissues from each experimental group of head and neck squamous cell carcinoma OSCC-CR PDX models treated with lnc RP11-499F3.2 were collected, stained with HE, and the histopathological condition of each tissue was observed.

[0200] See results Figure 25No obvious pathological symptoms were observed in the heart, liver, spleen, and kidneys in any group. However, lung infiltration was observed in the G3 cetuximab-treated group, indicating that tumor metastasis occurred in this group. This suggests that lung metastasis occurred to some extent during the establishment of the cetuximab-resistant PDX xenograft model of head and neck squamous cell carcinoma.

[0201] Example 24

[0202] Effects of targeted interference lnc RP11-499F3.2 on the proliferation of parental and drug-resistant colon cancer cells HT29 (CTX-R), esophageal squamous cell carcinoma cells TE13 (CTX-R), and non-small cell lung cancer cells A549 (CTX-R).

[0203] HT-29, TE13 and A549 resistant cell lines were established using a cetuximab concentration gradient method, as detailed in Example 13. The effects of lnc RP11-499F3.2-decanucleotide on the proliferation of HT29, TE13 and A549 sensitive and resistant cells were detected using the MTT assay, as detailed in Example 9.

[0204] The results are shown in Table 4. Compared with the control group, the lnc RP11-499F3.2 locked nucleoside designed in this invention significantly reduced the proliferation of HT-29, TE13 and A549 resistant cells when used alone. The proliferation was also significantly reduced when combined with cetuximab. This indicates that targeting and specifically reducing the expression of lnc RP11-499F3.2 can resensitize cetuximab-resistant colon cancer, esophageal squamous cell carcinoma and non-small cell lung cancer.

[0205] Table 4. Effects of targeted interference lnc RP11-499F3.2 on drug-resistant cells.

[0206]

[0207] The experiment was independently repeated 3 times. The results were expressed as mean ± SD and statistical t-tests were performed. *P < 0.05 was considered statistically significant, and **P < 0.01 was considered highly statistically significant.

Claims

1. Application of a reagent for detecting long non-coding RNA RP11-499F3.2 in the preparation of oral squamous cell carcinoma detection products, wherein the long non-coding RNA sequence is shown in SEQ ID NO.1 in the sequence listing.

2. The application according to claim 1, characterized in that, The reagent used to detect long-chain non-coding RP11-499F3.2 is a real-time quantitative PCR detection reagent.

3. The application according to claim 2, characterized in that, The real-time quantitative PCR detection reagent includes detection primers, the primer sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3 in the sequence listing.

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