New use of biomarker af131217.1

Abstract

The application discloses a new use of a biomarker AF131217.1, and particularly discloses application of the biomarker AF131217.1 in diagnosis and treatment of oral squamous cell carcinoma. The application proves that the AF131217.1 can be used as a diagnostic marker and a therapeutic target of the oral squamous cell carcinoma. The oral squamous cell carcinoma can be diagnosed by detecting the expression level of the AF131217.1. The AF131217.1 is used as a drug target to assist clinical operation treatment by inhibiting the expression of the AF131217.1, so that the cure rate of patients is improved, and the life quality of the patients is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a new use of a biomarker AF131217.1, in particular, to application of the biomarker AF131217.1 in diagnosis and treatment of oral squamous cell carcinoma. BACKGROUND

[0002] Head and neck cancer is the sixth most common malignancy worldwide, causing approximately 350,000 deaths per year. Oral cancer is the most common type of head and neck tumor, and squamous cell carcinoma accounts for about 90% of all malignant tumors in the oral cavity, which has strong invasiveness, early recurrence and metastasis, etc. In the past few decades, although significant achievements have been made in its detection, prevention and treatment, oral squamous cell carcinoma still lacks reliable prognostic molecular markers, leading to its easy metastasis and recurrence, and poor prognosis. Therefore, studying the occurrence, development, invasion and metastasis mechanism of oral squamous cell carcinoma from the molecular level has become a hot spot for the treatment of oral squamous cell carcinoma.

[0003] The oral cavity is the starting point of the digestive tract and is connected with the respiratory tract, and has important physiological functions. Oral cancer is a general term for malignant tumors occurring in the oral and maxillofacial region, and can cause dysfunction of eating, chewing, speaking, swallowing and breathing, etc. of patients, and seriously affects the quality of life of patients. Oral squamous cell carcinoma is one of the most common malignant tumors in the oral and maxillofacial region, which is simply referred to as oral squamous cell carcinoma, and the annual survival rate is only about 60%. So far, the treatment of oral squamous cell carcinoma is still mainly surgical resection, supplemented by radiotherapy and chemical drugs, which emphasizes the important role of radical and thoroughness on the prognosis of patients. However, for patients with advanced tumors and recurrence, although the surgical method is constantly improved and the adjuvant therapy is constantly strengthened, the five-year survival rate has not increased significantly in recent years.

[0004] Biomarkers are indicators of the physiological state and changes of cells during the disease process, and have unique advantages in accurately and sensitively evaluating early and low-level damage, which can be used for early diagnosis, prognosis prediction and treatment of oral squamous cell carcinoma, and has a good application prospect in the clinical treatment of oral squamous cell carcinoma. Therefore, for oral squamous cell carcinoma, especially for early oral squamous cell carcinoma, it is of great significance to find specific cancer biomarkers with accurate diagnosis and prognosis prediction ability. SUMMARY

[0005] In order to make up for the shortcomings of the prior art, the purpose of the present application is to provide a biomarker related to the occurrence and development of oral squamous cell carcinoma, so as to provide a molecular means for diagnosis and treatment of oral squamous cell carcinoma.

[0006] The above-mentioned purpose of the present application is realized by the following technical scheme:

[0007] The first aspect of the present application provides use of a product for detecting the expression level of the AF131217.1 gene in the preparation of a diagnostic tool for oral squamous cell carcinoma.

[0008] Further, the tool comprises a chip, a reagent, a kit or a nucleic acid membrane strip.

[0009] The chip comprises a solid phase carrier and oligonucleotide probes immobilized on the solid phase carrier, wherein the solid phase carrier comprises inorganic carriers such as silicon carriers, glass carriers, ceramic carriers, etc., and organic carriers such as polypropylene films, nylon membranes, etc.

[0010] The kit comprises instructions or labels indicating that the kit is used for detecting oral squamous cell carcinoma, and preferably, the kit further comprises a label for labeling the RNA sample, and a substrate corresponding to the label. In addition, the kit can further comprise various reagents required for RNA extraction, PCR, hybridization, color development, etc., including but not limited to extraction solution, amplification solution, hybridization solution, enzyme, control solution, color development solution, washing solution, etc.

[0011] The nucleic acid membrane strip comprises a substrate and oligonucleotide probes immobilized on the substrate; the substrate can be any substrate suitable for immobilizing oligonucleotide probes, such as nylon membranes, nitrocellulose membranes, polypropylene membranes, glass slices, silica gel chips, microscale magnetic beads, etc.

[0012] The second aspect of the present application provides a tool for diagnosing oral squamous cell carcinoma.

[0013] Further, the tool comprises a product for detecting the expression level of the AF131217.1 gene.

[0014] Further, the product comprises:

[0015] a probe specifically recognizing the AF131217.1 gene; or

[0016] a primer specifically amplifying the AF131217.1 gene.

[0017] Further, the primer specifically amplifying the AF131217.1 gene has a sequence as shown in SEQ ID NO. 7-8.

[0018] Further, the primer specifically amplifying the AF131217.1 gene is designed according to the transcripts ENST00000433310.2, ENST00000430247.1 and ENST00000452028.1 of the AF131217.1 gene.

[0019] The third aspect of the present application provides use of AF131217.1 in the preparation of a pharmaceutical composition for treating oral squamous carcinoma.

[0020] Further, the pharmaceutical composition comprises an agent for inhibiting expression of AF131217.1.

[0021] Further, the agent is siRNA.

[0022] More preferably, the sequence of the siRNA is shown in SEQ ID NO. 17-18.

[0023] The fourth aspect of the present application provides a pharmaceutical composition.

[0024] Further, the pharmaceutical composition comprises the agent of the third aspect of the present application, and / or a pharmaceutically acceptable carrier and / or adjuvant.

[0025] Further, the pharmaceutically acceptable carrier and / or adjuvant comprises (but is not limited to) diluents, binders, surface active agents, wetting agents, adsorptive carriers, lubricants, fillers, disintegrants.

[0026] The diluents comprise lactose, sodium chloride, glucose, urea, starch, water, etc.; the binders comprise starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginic acid salt, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methyl cellulose, etc.; the surface active agents comprise polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, stearic acid monoglyceride, cetyl alcohol, etc.; the wetting agents comprise glycerol, starch, etc.; the adsorptive carriers comprise starch, lactose, bementite, silica gel, kaolin and soap clay, etc.; the lubricants comprise zinc stearate, glycerol monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauryl sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc.; the fillers comprise mannitol (granular or powdery), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupled sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate, etc.; the disintegrants comprise cross-linked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

[0027] The pharmaceutical composition in the present application can further comprise stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents and surfactants, etc.

[0028] The pharmaceutical composition of the present application can also be used in combination with other drugs for treating oral squamous cell carcinoma. The other therapeutic compounds can be administered simultaneously with the main active ingredient, even in the same composition. The other therapeutic compounds can also be administered separately from the main active ingredient, either in a separate composition or in a different dosage form. Part of the dose of the main ingredient can be administered simultaneously with the other therapeutic compounds, while the other doses can be administered separately. During the course of treatment, the dose of the pharmaceutical composition of the present application can be adjusted according to the severity of the symptoms, the frequency of recurrence and the physiological response to the treatment regimen.

[0029] The fifth aspect of the present application provides the use of AF131217.1 in screening a pharmaceutical composition for treating oral squamous cell carcinoma.

[0030] Further, the use includes a method for screening a pharmaceutical composition for treating oral squamous cell carcinoma.

[0031] Further, the method includes the following steps:

[0032] (1) treating a system expressing or containing AF131217.1 with a substance to be screened;

[0033] (2) detecting the expression of AF131217.1 in the system;

[0034] (3) if the substance to be screened can inhibit the expression of AF131217.1, it indicates that the substance to be screened is a drug for treating oral squamous cell carcinoma.

[0035] Further, the system includes (but is not limited to) a cell system, a subcellular system, a solution system, a tissue system, an organ system or an animal system.

[0036] Further, the steps further include performing further cell experiments and / or animal experiments on the obtained candidate drugs to further select a pharmaceutical composition for treating oral squamous cell carcinoma from the candidate drugs.

[0037] The terms used in the present application are explained as follows:

[0038] The term "diagnosing oral squamous cell carcinoma" in the present application includes judging whether a subject has already suffered from oral squamous cell carcinoma, judging whether a subject is at risk of suffering from oral squamous cell carcinoma, judging the responsiveness of a patient with oral squamous cell carcinoma to drug treatment, or judging the prognosis of a patient with oral squamous cell carcinoma.

[0039] The term "treatment" in the present invention refers to the medical management of a patient for the purpose of curing, improving, stabilizing, or preventing a disease, pathological condition, or disorder. The term encompasses active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, the term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment that relieves secondary effects caused by the specific treatment of the main disease, pathological condition, or disorder.

[0040] The term "biomarker" in the present invention is used interchangeably with "marker" and "molecular marker" to refer to a target molecule that is an indication of normal or abnormal progression in an individual or an indication of a disease or other state in an individual or that manifests these. In more detail, a "biomarker" is an anatomical, physiological, biochemical, or molecular parameter that is normal or abnormal, and if abnormal, is associated with the presence of a particular physiological state or progression, whether chronic or acute. Biomarkers can be detected and measured by a variety of methods including laboratory tests and medical imaging.

[0041] The term "probe" in the present invention refers to a molecule that binds to a particular sequence or subsequence or other part of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that binds to another polynucleotide by complementary base pairing. The probe has a base sequence that is complementary to a particular base sequence of a target gene, and "complementary" means hybridization, which can not be perfect complementarity. These polynucleotides generally have more than 80%, preferably more than 90%, more preferably more than 95%, and particularly preferably 100% homology with respect to the particular base sequence. These probes can be DNA or RNA, and in addition, can be polynucleotides in which the nucleotides in part or all of them are replaced with artificial nucleic acids such as PNA, LNA, ENA, GNA, TNA, etc.

[0042] The terms "down-regulation" and variations thereof in the present invention are used interchangeably to refer to a biomarker value or level in a biological sample that is less than the value or level (or range of values or levels) of the biomarker typically detected from a similar biological sample of a healthy or normal individual. The terms also refer to a biomarker value or level in a biological sample that is less than the value or level (or range of values or levels) of the biomarker that can be detected from a different step of a particular disease.

[0043] The term "sample" in the present invention refers to a composition obtained from a target patient, which comprises cells and / or other molecular entities to be characterized and / or identified, for example, according to physical, biochemical, chemical and / or physiological characteristics. For example, the phrase "clinical sample" or "disease sample" and variants thereof refers to any sample obtained from a target patient, from which cells and / or molecular entities, for example biomarkers to be characterized, are expected or known to be obtainable.

[0044] It has been disclosed that there are three transcripts of the AF131217.1 gene, and the sequences are shown in ENST00000433310.2, ENST00000430247.1 and ENST00000452028.1, respectively. The AF131217.1 gene is located on chromosome 21, and the Ensemble ID is ENSG00000232855.

[0045] The present invention has the advantages and beneficial effects that:

[0046] The present invention provides a molecular target AF131217.1 for diagnosing and treating oral squamous cell carcinoma. By targeting AF131217.1, the level of AF131217.1 is changed to treat oral squamous cell carcinoma with sensitivity and specificity.

[0047] The present invention provides a new means and approach for the research and development of drugs for treating oral squamous cell carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0048] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0049] Figure 1 is a differential expression diagram of the differentially expressed genes in oral squamous cell carcinoma tissue and paracancerous tissue;

[0050] Figure 2 is a result diagram of CCK-8 cell proliferation experiment for detecting the influence of differentially expressed genes on the proliferation activity of oral squamous cell carcinoma cells;

[0051] Figure 3 is a result diagram of cell migration experiment for detecting the influence of differentially expressed genes on the migration ability of oral squamous cell carcinoma cells. DETAILED DESCRIPTION

[0052] The present invention will be further described below in conjunction with specific examples, which are only used to explain the present invention and cannot be understood as a limitation of the present invention. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods in the following examples are not specified, and the detection is usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer.

[0053] Example 1 Screening of gene markers associated with oral squamous cell carcinoma

[0054] 1. Sample collection

[0055] Five cases of oral squamous cell carcinoma tissues and para-cancer tissues were collected, respectively, and were confirmed by pathological diagnosis. All patients did not receive any form of treatment before surgery. The surgical specimens were stored in liquid nitrogen. The collection of all the above specimens was approved by the ethics committee.

[0056] 2. Extraction of tissue RNA

[0057] About 50 mg of cancer tissue and para-cancer tissue samples stored in liquid nitrogen were taken out, and the tissue samples were ground in a pre-cooled mortar. When there were no large particles of tissue, they were transferred to a 1.5 mL EP tube. RNA was extracted and separated according to the instructions in the kit. The specific extraction steps are as follows:

[0058] (1) Add 1 mL Trizol and stand at room temperature for 5 min;

[0059] (2) Add 0.2 mL chloroform, shake the centrifuge tube vigorously, mix well, and stand at room temperature for 5-10 min;

[0060] (3) Centrifuge at 12000 rpm and 4°C for 15 min. Then gently transfer the upper aqueous phase to another new EP tube (be careful not to suck the protein material between the two aqueous phases). Add an equal volume of -20°C pre-cooled isopropanol, mix well by inverting, and place on ice for 10 min;

[0061] (4) Centrifuge at 12000 rpm and 4°C for 15 min. Carefully discard the supernatant, add 1 mL of 75% DEPC ethanol for washing the precipitate (stored at 4°C), mix well, and centrifuge at 4°C and 12000 rpm for 5 min;

[0062] (5) Discard the ethanol supernatant and stand at room temperature for 5 min. Dissolve the precipitate with DEPC water;

[0063] (6) After quantification, use or store in a -80°C freezer for later use.

[0064] 3. Quantification and purity analysis of total RNA

[0065] The extracted RNA was subjected to agarose gel electrophoresis. The concentration and purity of the extracted RNA were determined using Nanodrop 2000. The integrity of the RNA was detected by agarose gel electrophoresis, and the RIN value was measured by Agilent 2100. The total amount of RNA required for single library construction was 5 μg, and the concentration was ≥200 ng / μL.

[0066] 4. Construction of cDNA library

[0067] Ribosomal RNA in total RNA was removed using Ribo-Zero kit of Epicentre; for intact RNA sequence, random breaking was performed using metal ions to randomly break the RNA into small fragments of about 200 bp; cDNA library construction was performed using Illumina Truseq™ RNA sample Prep Kit.

[0068] 5. Sequencing

[0069] Sequencing was performed using Illumina X-Ten sequencing platform, 2*150 bp.

[0070] 6. High-throughput transcriptome sequencing data analysis

[0071] LncRNA that is not easy to detect is deleted, and DESeq2 in R-3.3.3 tool is used for differential expression analysis of reads number, and the differential expression LncRNA screening standard is FDR<0.05, abs(log2FC)>2.

[0072] 7. Experimental results

[0073] The results show that the expression levels of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 in oral squamous cell carcinoma tissues are significantly up-regulated compared with the para-cancer tissues.

[0074] Example 2 QPCR verification of differential expression of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1

[0075] 1. Tissue collection

[0076] The collection method described in Example 1 was used to collect 60 tissue samples of oral squamous cell carcinoma and their corresponding para-cancer tissue samples, and large sample differential expression genes of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 were verified.

[0077] 2. Extraction of tissue RNA

[0078] The extraction steps are the same as in Example 1.

[0079] 3. QPCR experiment

[0080] (1) Reverse transcription reaction

[0081] LncRNA reverse transcription was carried out by using FastQμant cDNA first strand synthesis kit (item number: KR106), first remove genomic DNA reaction, add 5x gDNA Bμffer 2.0 μL, total RNA 1 μg, add RNase Free ddH2O to make the total volume to 10 μL in the test tube, heat in the water bath 42℃ for 3 min.

[0082] Add 10x Fast RT Bμffer 2.0 μL, RT Enzyme Mix 1.0 μL, FQ-RT Primer Mix 2.0 μL, RNase Free ddH2O 5.0 μL, mix and add to the above test tube, mix together for a total of 20 μL, heat in the water bath 42℃ for 15 min, 95℃ for 3 min.

[0083] (2) Design and preparation of primers

[0084] According to the coding sequence of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 genes and GAPDH gene in Genebank, QPCR amplification primers were designed, and when designing the primers of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1, the common sequence of different transcription product sequences was selected for design, and the specific primer sequences were as follows:

[0085] RP11-575F12.2 gene:

[0086] The forward primer was 5'-TTAGAAGAAGAACCATTG-3'(SEQ ID NO.1)

[0087] The reverse primer was 5'-TCCTATTCAAGACCATAA-3'(SEQ ID NO.2)

[0088] RP11-54A9.1 gene:

[0089] The forward primer was 5'-GACTGACACTGCTTGATT-3'(SEQ ID NO.3)

[0090] The reverse primer was 5'-TCCTTCACTGTAAGAGTTATC-3'(SEQ ID NO.4)

[0091] RP11-973H7.1 gene:

[0092] The forward primer was 5'-TTGCCATTCTAATGTAAG-3'(SEQ ID NO.5)

[0093] Reverse primer was 5'-TGTTGCCTATTCATAAGAC-3'(SEQ ID NO. 8)

[0094] AF131217.1 gene:

[0095] Forward primer was 5'-ATTCTCGTTACTCCTTGT-3'(SEQ ID NO. 7)

[0096] Reverse primer was 5'-TGTTGCCTATTCATAAGAC-3'(SEQ ID NO. 8)

[0097] GAPDH gene:

[0098] Forward primer was 5'-CTCTGGTAAAGTGGATATTGT-3'(SEQ ID NO. 9)

[0099] Reverse primer was 5'-GGTGGAATCATATTGGAACA-3'(SEQ ID NO. 10)

[0100] (3) Real-time quantitative PCR reaction

[0101] Amplification was performed with SuperReal PreMix Plus (SYBR Green) (item number: FP205).

[0102] A 20μL reaction system was used: 2x SuperReal PreMix Plus 10μL, forward and reverse primers (10μM) 0.6μL each, 5x ROX Reference Dye △ 2μL, DNA template 2μL, sterilized distilled water 4.8μL. Three parallel tubes were set for each sample, and all amplification reactions were repeated more than three times to ensure the reliability of the experimental results.

[0103] The amplification program was as follows:

[0104] RP11-575F12.2 gene: 95℃ 15min, (95℃ 10s, 50℃ 30s, 72℃ 32s) x 45 cycles;

[0105] RP11-54A9.1 gene: 95℃ 15min, (95℃ 10s, 54℃ 30s, 72℃ 32s) x 40 cycles;

[0106] RP11-973H7.1 gene: 95℃ 15min, (95℃ 10s, 56℃ 30s, 72℃ 32s) x 39 cycles;

[0107] AF131217.1 gene: 95 ℃ 15 min, (95 ℃ 10 s, 52 ℃ 30 s, 72 ℃ 32 s) x 45 cycles.

[0108] 4. Statistical analysis

[0109] Statistical software SPSS20.0 was used for statistical analysis, and the measurement data were expressed as mean ± standard deviation (mean ± SD). Paired T test was used for comparison between two groups, single factor analysis of variance was used for three or more groups, and LSD-t test was used for multiple comparisons. All experiments were repeated three times, and P<0.05 was considered statistically significant.

[0110] 5. Experimental results

[0111] The results of QPCR are shown in Figure 1 , and the results show that the expression of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 in oral squamous cell carcinoma tissue is significantly up-regulated compared with the adjacent tissue, and the difference is statistically significant (P<0.05), indicating that RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 can be used as molecular markers for diagnosis and treatment of oral squamous cell carcinoma.

[0112] Example 3 Detection of silencing of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 and functional verification

[0113] 1. Cell culture

[0114] The human oral squamous cell carcinoma SCC-15 cells stored in liquid nitrogen were taken out and inoculated in DMEM medium, and the cells were cultured in a 37℃, 5% CO2 constant temperature incubator. After 24h, the cells showed adherent growth, which indicated that the recovery was successful. The medium was changed every 1-2d, and the cells were digested with trypsin and prepared into cell suspension for experiment.

[0115] 2. Cell transfection

[0116] The cells were inoculated into a six-well cell culture plate at 2x10 5 / well, and cultured in a 37℃, 5% CO2 incubator. The cells in the logarithmic growth phase (about 80%) were discarded, washed with PBS for 2 times, and 2mL DMEM was added for starvation culture in the incubator for 1h. Lipofectamine transfection reagent 2000 (Invitrogen) was used for transfection, and the specific operation was carried out according to the instruction manual.

[0117] The experiment was divided into three groups: blank control group (SCC-15), negative control group (siRNA-NC) and experimental group (siRNA group), among which the siRNA in the negative control group had no sequence homology with RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1.

[0118] Among them, the siRNA sequence for RP11-575F12.2 is as follows:

[0119] The positive strand is 5'-UUGUAAAAGAAUUCUUUGCUC-3' (SEQ ID NO. 11)

[0120] The antisense strand is 5'-GCAAAGAAUUCUUUUACAAAG-3' (SEQ ID NO.12)

[0121] The siRNA sequences targeting RP11-54A9.1 are as follows:

[0122] The positive strand is 5'-ACAAUCUGUGCAAUUACAGUU-3' (SEQ ID NO. 13)

[0123] The antisense strand is 5'-CUGUAAUUGCACAGAUUGUUC-3' (SEQ ID NO. 14)

[0124] The siRNA sequences targeting RP11-973H7.1 are as follows:

[0125] The positive strand is 5'-UUCAAAAGUGAAACUACAGAC-3' (SEQ ID NO. 15)

[0126] The antisense strand is 5'-CUGUAGUUUCACUUUUGAACC-3' (SEQ ID NO. 16)

[0127] The siRNA sequences targeting AF131217.1 are as follows:

[0128] The positive strand is 5'-AUCUUAGACUGGGUAAAUCAU-3' (SEQ ID NO. 17)

[0129] The antisense strand is 5'-GAUUUACCCAGUCUAAGAUAU-3' (SEQ ID NO. 18)

[0130] 3. QPCR detection of transcription levels of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 genes

[0131] After 48h of transfection, total RNA was extracted from each group of cells using the Trizol method, and reverse transcription and real-time quantitative PCR detection were performed according to the method in Example 2.

[0132] 4. Cell proliferation experiment

[0133] The cells in the negative control group and the experimental group 24h after transfection were digested and centrifuged by a conventional method, and the supernatant was discarded. 1 mL of complete medium was added to resuspend the cells, which were mixed by blowing. 3000 cells per well were inoculated into a 96-well plate, and the complete medium was supplemented to 100 μL. 100 mL of DEPC water was added to the outermost circle of the well plate, and the 96-well plate was placed in a constant temperature incubator for culture. After 48h of culture, 100 μL of medium containing 10% CCK-8 was added, and the plate was further cultured in the incubator for 1h. The absorbance at 450 nm was measured on a microplate reader, and the experimental data were counted and recorded.

[0134] 5. Cell migration experiment

[0135] The Transwell chamber was placed in a 24-well plate, 200 μL of DMEM solution was added to the upper chamber, and the chamber was hydrated in the incubator for 1h. According to the method of plating 2x10 4 cells per chamber, the liquid in the upper chamber was supplemented to 200 μL, mixed by blowing, and 700 μL of complete medium was added to the lower chamber. The plate was further cultured in the incubator for 36h. The chamber was removed, and the medium in the upper and lower chambers was discarded. The residual medium and cells in the upper chamber were gently wiped off with a cotton swab, the chamber was washed with PBS, and the PBS was discarded after 5min of shaking. 500 μL of 4% paraformaldehyde was added to the lower chamber, and the chamber was fixed at room temperature for 30min. The fixing solution was discarded, and the chamber was washed with PBS for 3 times, 5min of shaking, and PBS was discarded. The chamber was placed in a fume hood and air-dried for 30min. 500 μL of prepared 0.1% crystal violet solution was added to the lower chamber, and bubbles were excluded. The chamber was left still for 30min. The crystal violet solution was discarded, and the chamber was washed with PBS for 3 times, 5min of shaking, and PBS was discarded. The excess liquid in the upper chamber was gently wiped off with a dry cotton swab, and the chamber was placed under a microscope for cell counting and recording.

[0136] 6. Statistical analysis

[0137] Statistical software SPSS20.0 was used for statistical analysis. The measurement data were expressed as mean ± standard deviation (mean ± SD). Paired T test was used for comparison between two groups, one-way ANOVA was used for comparison among three or more groups, and LSD-t test was used for multiple comparisons. All experiments were repeated three times, and P<0.05 was considered statistically significant.

[0138] 7. Experimental results

[0139] The transfection results showed that, taking the expression level of the blank control group RP11-575F12.2 as reference, set to 1, compared with the expression amount of the blank control group RP11-575F12.2 (relative expression amount was 1) and the expression amount of the transfection siRNA-NC negative control group RP11-575F12.2 (relative expression amount was 0.948±0.026), the expression amount of RP11-575F12.2 in the transfection siRNA experimental group (relative expression amount was 0.541±0.085) was significantly down-regulated, and the difference was statistically significant (experimental group vs blank control group, P<0.05; experimental group vs negative control group, P<0.05), and there was no significant difference between the siRNA-NC group and the blank control group (P>0.05).

[0140] The transfection results showed that, taking the expression level of the blank control group RP11-54A9.1 as reference, set to 1, compared with the expression amount of the blank control group RP11-54A9.1 (relative expression amount was 1) and the expression amount of the transfection siRNA-NC negative control group RP11-54A9.1 (relative expression amount was 0.948±0.026), the expression amount of RP11-54A9.1 in the transfection siRNA experimental group (relative expression amount was 0.355±0.027) was significantly down-regulated, and the difference was statistically significant (experimental group vs blank control group, P<0.05; experimental group vs negative control group, P<0.05), and there was no significant difference between the siRNA-NC group and the blank control group (P>0.05).

[0141] The transfection results showed that, taking the expression level of the blank control group RP11-973H7.1 as reference, set to 1, compared with the expression amount of the blank control group RP11-973H7.1 (relative expression amount was 1) and the expression amount of the transfection siRNA-NC negative control group RP11-973H7.1 (relative expression amount was 0.948±0.026), the expression amount of RP11-973H7.1 in the transfection siRNA experimental group (relative expression amount was 0.743±0.096) was significantly down-regulated, and the difference was statistically significant (experimental group vs blank control group, P<0.05; experimental group vs negative control group, P<0.05), and there was no significant difference between the siRNA-NC group and the blank control group (P>0.05).

[0142] The results of transfection showed that the expression level of AF131217.1 in the blank control group was set as 1, and compared with the expression level of AF131217.1 in the blank control group (the relative expression was 1) and the expression level of AF131217.1 in the transfection siRNA-NC negative control group (the relative expression was 0.948±0.026), the expression level of AF131217.1 in the transfection siRNA experimental group (the relative expression was 0.131±0.053) was significantly down-regulated, and the difference was statistically significant (experimental group vs. blank control group, P<0.05; experimental group vs. negative control group, P<0.05), and there was no significant difference between the siRNA-NC group and the blank control group (P>0.05).

[0143] The CCK-8 cell proliferation activity results are shown in Table 5. Figure 2 The results showed that the OD450 of the transfection siRNA experimental group (0.634±0.064) was significantly lower than that of the transfection siRNA-NC control group (1.236±0.051), and P<0.05, indicating that RP11-575F12.2 in this study played an important role in the proliferation of oral squamous cell carcinoma cells, and changing the expression level of RP11-575F12.2 could change the proliferation ability of oral squamous cell carcinoma cells.

[0144] The CCK-8 cell proliferation activity results are shown in Table 5. Figure 2 The results showed that the OD450 of the transfection siRNA experimental group (0.506±0.038) was significantly lower than that of the transfection siRNA-NC control group (1.236±0.051), and P<0.05, indicating that RP11-54A9.1 in this study played an important role in the proliferation of oral squamous cell carcinoma cells, and changing the expression level of RP11-54A9.1 could change the proliferation ability of oral squamous cell carcinoma cells.

[0145] The CCK-8 cell proliferation activity results are shown in Table 5. Figure 2 The results showed that the OD450 of the transfection siRNA experimental group (0.820±0.110) was significantly lower than that of the transfection siRNA-NC control group (1.236±0.051), and P<0.05, indicating that RP11-973H7.1 in this study played an important role in the proliferation of oral squamous cell carcinoma cells, and changing the expression level of RP11-973H7.1 could change the proliferation ability of oral squamous cell carcinoma cells.

[0146] The CCK-8 cell proliferation activity results are shown in Table 5. Figure 2, the result shows that the OD450 of the experimental group transfected with siRNA (0.336±0.051) is significantly lower than that of the control group transfected with siRNA-NC (1.236±0.051), and P<0.05, which indicates that the AF131217.1 in this study plays an important role in the proliferation of oral squamous cell carcinoma cells, and changing the expression level of AF131217.1 can change the proliferation ability of oral squamous cell carcinoma cells.

[0147] The results of the cell migration experiment are shown in Figure 3 , the result shows that the number of migrated cells of the experimental group transfected with siRNA (69±6.245) is significantly lower than that of the control group transfected with siRNA-NC (111±5.568), and P<0.05, which indicates that the RP11-575F12.2 in this study plays an important role in the migration of oral squamous cell carcinoma cells, and changing the expression level of RP11-575F12.2 can change the migration ability of oral squamous cell carcinoma cells.

[0148] The results of the cell migration experiment are shown in Figure 3 , the result shows that the number of migrated cells of the experimental group transfected with siRNA (55±14.107) is significantly lower than that of the control group transfected with siRNA-NC (111±5.568), and P<0.05, which indicates that the RP11-54A9.1 in this study plays an important role in the migration of oral squamous cell carcinoma cells, and changing the expression level of RP11-54A9.1 can change the migration ability of oral squamous cell carcinoma cells.

[0149] The results of the cell migration experiment are shown in Figure 3 , the result shows that the number of migrated cells of the experimental group transfected with siRNA (84.333±4.163) is significantly lower than that of the control group transfected with siRNA-NC (111±5.568), and P<0.05, which indicates that the RP11-973H7.1 in this study plays an important role in the migration of oral squamous cell carcinoma cells, and changing the expression level of RP11-973H7.1 can change the migration ability of oral squamous cell carcinoma cells.

[0150] The results of the cell migration experiment are shown in Figure 3 , the result shows that the number of migrated cells of the experimental group transfected with siRNA (35.667±8.622) is significantly lower than that of the control group transfected with siRNA-NC (111±5.568), and P<0.05, which indicates that the AF131217.1 in this study plays an important role in the migration of oral squamous cell carcinoma cells, and changing the expression level of AF131217.1 can change the migration ability of oral squamous cell carcinoma cells.

[0151] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the protection scope of the claims of the present application. SEQUENCE LISTING <110> Central South University Xiangya No. 2 Hospital Qingdao Youshen Biomedical Co., Ltd. Central South University Xiangya Stomatological Hospital <120> New use of biomarker AF131217.1 <141> 2020-11-27 <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 1 ttagaagaag aaccattg 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 2 tcctattcaa gaccataa 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 3 gactgacact gcttgatt 18 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 4 tccttcactg taagagttat c 21 <210> 5​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <211> 18 <212> DNA <213> Artificial Sequence <400> 5 ttgccattct aatgtaag 18 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <400> 6 actcctatta tctgtatgt 19 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <400> 7 attctcgtta ctccttgt 18 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <400> 8 tgttgcctat tcataagac 19 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <400> 9 ctctggtaaa gtggatattg t 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 ggtggaatca tattggaaca 20 <210> 11 <211> 21 <212> RNA <213> Artificial Sequence <400> 11 uuguaaaaga auucuuugcu c 21 <210> 12 <211> 21 <212> RNA <213> Artificial Sequence <400> 12 gcaaagaauu cuuuuacaaa g 21 <210> 13 <211> 21 <212> RNA <213> Artificial Sequence <400> 13 acaaucugug caauuacagu u 21 <210> 14 <211> 21 <212> RNA <213> Artificial Sequence <400> 14 cuguaauugc acagauuguu c 21 <210> 15 <211> 21 <212> RNA <213> Artificial Sequence <400> 15 uucaaaagug aaacuacaga c 21 <210> 16 <211> 21 <212> RNA <213> Artificial Sequence <400> 16 cuguaguuuc acuuuugaac c 21 <210> 17 <211> 21 <212> RNA <213> Artificial Sequence <400> 17 aucuuagacu ggguaaauca u 21 <210> 18 <211> 21 <212> RNA <213> Artificial Sequence <400> 18 gauuuaccca gucuaagaua u 21

Claims

1. Application of products for detecting AF131217.1 gene expression levels in the preparation of diagnostic tools for oral squamous cell carcinoma.

2. The use according to claim 1, characterized in that The tool is a chip, a preparation, a kit or a nucleic acid membrane strip.

3. The use according to claim 1, characterized in that The product is a primer for specifically amplifying the AF131217.1 gene.

4. The use according to claim 3, characterized in that The primer sequences for specifically amplifying the AF131217.1 gene are shown in SEQ ID NOs. 7-8.

5. Use of an agent for inhibiting the expression of AF131217.1 in the preparation of a pharmaceutical composition for treating oral squamous cell carcinoma, characterized in that: The reagent is siRNA, and the sequence of the siRNA is shown in SEQ ID NO. 17-18.

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