A biomarker for diagnosing and treating oral squamous cell carcinoma and application thereof

Abstract

The application discloses a biomarker for diagnosing and treating oral squamous cell carcinoma and application thereof, and the biomarker is RP11-973H7.1. Through research on differential expression genes and cell experiment exploration on the influence of the differential expression gene RP11-973H7.1 on oral squamous cell carcinoma cells, it is found that inhibition of the expression of RP11-973H7.1 can significantly reduce the proliferation and migration ability of oral squamous cell carcinoma cells, and has important significance for promoting early diagnosis and individualized treatment of oral squamous cell carcinoma.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to a biomarker for diagnosing and treating oral squamous cell carcinoma and application thereof, in particular to a RP11-973H7.1 gene. BACKGROUND

[0002] Oral squamous cell carcinoma (OSCC / Squamous cell carcinoma of the head and neck, HNSCC) accounts for 8% of adult malignant tumors, and the number of new cases worldwide is close to 540,000 cases per year, of which the number of death cases is 271,000 cases, and the mortality rate is as high as 50%. According to the statistics of the National Central Cancer Registry of China (NCCR), in 2015, there were about 4.292 million new cases of invasive cancer in China, which is equivalent to 12,000 new cancer cases per day on average, and there were 2.814 million cancer death cases, which is equivalent to 7,500 people dying of cancer per day on average. Among them, there were 48,100 new cases of lip, oral and pharyngeal cancer per year, and 22,100 death cases per year. Almost 22% of global new cancer cases occur in China, and 27% of cancer death cases occur in China. A survey from 1985 to 1994 of 50 states in the United States by the National Cancer Database (NCDB) shows that oral and maxillofacial-head and neck tumors account for 6.6% of all malignant tumors, and among them, squamous cell carcinoma accounts for 55.8%, and the oral cavity and hypopharynx are high-risk sites (20.9%), and there is a trend of increasing year by year.

[0003] Oral squamous cell carcinoma (OSCC), simply oral squamous carcinoma, can occur in different parts of the oral cavity, mostly adjacent to important tissues and organs, such as the brain, upper respiratory tract, and important nerves and blood vessels of the neck, which severely restricts the range of surgery. Moreover, due to the rich blood vessels and lymphatic vessels of the head and neck and the frequent mechanical movement of the tongue, early cervical lymph node metastasis often occurs, which has a poor prognosis and seriously threatens people's life and health. With the development of comprehensive treatment of tumors, the treatment of oral squamous carcinoma has made some progress, but in recent years, the 5-year survival rate of patients has not improved significantly. At present, the treatment methods for oral squamous carcinoma in clinical practice include surgical treatment, preoperative or postoperative radiotherapy and chemotherapy, etc., but these treatment methods are far from meeting the needs of patient survival. There is a possibility of postoperative recurrence and metastasis in a part of patients even after surgery and adjuvant radiotherapy and chemotherapy, which seriously affects the overall survival time of patients. This is because the existing pathological diagnosis technology cannot accurately judge the biological characteristics of oral squamous carcinoma, and there is a lack of specific and efficient treatment drugs and programs for oral squamous carcinoma. With the rapid development of molecular biological technology, biomarkers related to the prognosis of patients with early oral squamous carcinoma have been discovered. A new biomarker for oral squamous carcinoma is screened in the present application, which is used for the diagnosis and treatment of oral squamous carcinoma. SUMMARY

[0004] In order to make up for the shortcomings of the prior art, the purpose of the present application is to provide a biomarker for diagnosing and treating oral squamous carcinoma and its application, thereby providing a molecular means for the diagnosis and treatment of oral squamous carcinoma.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] The first aspect of the present application provides a reagent.

[0007] Further, the reagent can specifically detect RP11-973H7.1 gene and / or its expression product.

[0008] Further, the reagent comprises:

[0009] a primer for specifically amplifying RP11-973H7.1;

[0010] a probe for specifically recognizing RP11-973H7.1.

[0011] Further, the primer for specifically amplifying RP11-973H7.1 is shown in SEQ ID NO. 5-6.

[0012] Further, the primer sequence is designed according to the transcript ENST00000563722.1 of RP11-973H7.1 gene.

[0013] Further, the detection of RP11-973H7.1 according to the present application is performed by using various nucleic acid techniques known to those skilled in the art, including (but not limited to): nucleic acid sequencing, nucleic acid hybridization and nucleic acid amplification techniques.

[0014] The second aspect of the present application provides a kit.

[0015] Further, the kit comprises the reagent according to the first aspect of the present application.

[0016] Further, the kit further comprises an instruction manual or label, a positive control, a negative control, a buffer, an adjuvant or a solvent.

[0017] Further, the instruction manual or label indicates that the kit is used for detecting oral squamous cell carcinoma.

[0018] 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 extracting RNA, 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.

[0019] The third aspect of the present application provides a chip.

[0020] Further, the chip comprises the reagent according to the first aspect of the present application.

[0021] Further, the chip comprises a probe hybridized with the nucleic acid sequence of RP11-973H7.1 gene.

[0022] Further, the chip further comprises a solid support and an oligonucleotide probe immobilized on the solid support.

[0023] Further, the oligonucleotide probe specifically corresponds to part or all of the sequence of RP11-973H7.1 gene.

[0024] Further, the oligonucleotide probe is used for detecting the transcription level of RP11-973H7.1 gene.

[0025] Further, the solid support comprises an inorganic support and an organic support, the inorganic support comprising (but not limited to): silicon support, glass support, ceramic support, etc.; the organic support comprising (but not limited to): polypropylene film, nylon film, etc.

[0026] "Probe" refers to a molecule that binds to a particular sequence or subsequence or other portion of another molecule. In the present invention, the term "probe" generally refers to a polynucleotide probe that binds to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing, unless otherwise indicated. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks perfect sequence complementarity to the probe. The probe can be directly or indirectly labeled, and ranges from primers. Hybridization formats include, but are not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays.

[0027] Exemplary probes in the present invention include PCR primers and gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection assay probes, probes linked to molecular barcodes, and probes immobilized on beads.

[0028] Further, these probes have a base sequence complementary to a specific base sequence of a target gene. Here, "complementary" means hybridization, and can not be perfect complementarity. These polynucleotides generally have 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% homology with respect to the specific base sequence. These probes can be DNA or RNA, and in addition, can be polynucleotides in which the nucleotides in part or all thereof are replaced with artificial nucleic acids such as PNA, LNA, ENA, GNA, TNA, etc.

[0029] The fourth aspect of the present invention provides a composition.

[0030] Further, the composition includes an agent that inhibits the expression of RP11-973H7.1.

[0031] Further, the agent that inhibits the expression of RP11-973H7.1 refers to any substance that can reduce the stability of the RP11-973H7.1 gene and / or its expression product, down-regulate the expression of RP11-973H7.1, reduce the effective action time of RP11-973H7.1, or inhibit the transcription of the RP11-973H7.1 gene, and these substances can be used in the present invention as substances useful for down-regulating the expression of the RP11-973H7.1 gene.

[0032] Further, the agent is a double-stranded molecule siRNA;

[0033] Preferably, the sequence of the siRNA is shown in SEQ ID NO. 15-16.

[0034] Further, the sequence of the siRNA is designed based on the transcript ENST00000563722.1 of the RP11-973H7.1 gene. Further, the sequence of the siRNA is designed based on the transcript ENST00000563722.1 of the RP11-973H7.1 gene.

[0035] The siRNAs of the application can include partially purified RNA, reasonably pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that render the siRNA resistant to nuclease digestion.

[0036] One or both strands of the siRNAs of the application can also comprise a 3' overhang. By "3' overhang" is meant at least one unpaired nucleotide extending from the 3' end of the RNA strand. Thus, in one embodiment, the siRNAs of the application comprise at least one 3' overhang of from 1 to about 6 nucleotides (including ribonucleotides or deoxyribonucleotides), preferably from 1 to about 5 nucleotides, more preferably from 1 to about 4 nucleotides, and particularly preferably from about 1 to about 4 nucleotides.

[0037] In embodiments in which both strands of the siRNA molecule comprise a 3' overhang, the length of the overhang can be the same or different for each strand. In the most preferred embodiment, a 3' overhang of 2 nucleotides is present on both strands of the siRNA. To enhance the stability of the siRNAs of the application, the 3' overhang can also be stabilized against degradation. In one embodiment, the overhang is stabilized using purine nucleotides, such as adenosine or guanosine nucleotides.

[0038] Alternatively, substitution of pyrimidine nucleotides with modified analogs, such as 2' deoxythymidine in place of uridine nucleotides in the 3' overhang, is tolerated without affecting the efficiency of RNAi degradation. In particular, the absence of the 2' hydroxyl group in 2' deoxythymidine significantly enhances nuclease resistance of the 3' overhang in tissue culture media.

[0039] The siRNAs of the application can be obtained using any of a number of techniques known to those skilled in the art. For example, the siRNAs can be chemically synthesized or recombinantly produced using methods known in the art. Preferably, the siRNAs of the application are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer.

[0040] Further, the composition also includes other drugs compatible with the agent, as well as pharmaceutically acceptable carriers and / or adjuvants.

[0041] The pharmaceutically acceptable carrier can be one or more and includes, but is not limited to, binders, sweeteners, disintegrants, diluents, flavoring agents, preservatives, lubricants, time delay agents and / or coating agents. Suitable binders include acacia, gelatin, cornstarch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrants include cornstarch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavoring agents include peppermint, oil of wintergreen, cherry, citrus, or raspberry flavoring. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate. The pharmaceutical compositions of the present application can also include stabilizers, bacteriostats, buffers, isotonic agents, chelating agents, pH control agents and surfactants. Suitable coating agents include polymers or copolymers of acrylic and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten.

[0042] A fifth aspect of the present application provides a method for screening a candidate drug for preventing or treating oral squamous cell carcinoma.

[0043] Further, the method comprises the following steps:

[0044] treating a system expressing or containing RP11-973H7.1 with a substance to be screened;

[0045] detecting the expression level of RP11-973H7.1 in the system;

[0046] If the substance to be screened can inhibit the expression of RP11-973H7.1, it indicates that the candidate substance is a candidate drug for preventing or treating oral squamous cell carcinoma.

[0047] Further, the method further comprises: further testing the candidate drug obtained in the above step for its effect of inhibiting oral squamous cell carcinoma, and if the tested substance has a significant inhibitory effect on oral squamous cell carcinoma, it indicates that the substance is a candidate substance for preventing or treating oral squamous cell carcinoma.

[0048] 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 (such as an animal model, preferably a non-human mammalian animal model, such as a mouse, a rabbit, a sheep, a monkey, etc.).

[0049] Further, when the candidate drug screened by the screening method of the present application is administered to a human or other mammal, including but not limited to mice, rats, guinea pigs, rabbits, cats, dogs, sheep, pigs, cows, monkeys, baboons, chimpanzees, the candidate drug can be administered directly, or can be formulated into various dosage forms using known pharmaceutical preparation methods. For example, the drug can be administered orally as a sugar-coated tablet, a capsule, an elixir, and a microcapsule, as needed, or can be administered non-orally in the form of a sterile solution or suspension prepared by formulation with water or any other pharmaceutically acceptable liquid, for injection. For example, the candidate drug can be mixed with a pharmaceutically acceptable carrier or medium, including but not limited to sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., in a unit dosage form required for generally accepted drug administration. Depending on the content of the active ingredient in these formulations, a suitable amount of administration within a specified range can be obtained.

[0050] The sixth aspect of the present application provides the use of any one of:

[0051] a. the agent of the first aspect of the present application in the manufacture of a product for diagnosing oral squamous cell carcinoma;

[0052] b. the kit of the second aspect of the present application in the manufacture of a product for diagnosing oral squamous cell carcinoma;

[0053] c. the chip of the third aspect of the present application in the manufacture of a product for diagnosing oral squamous cell carcinoma;

[0054] d. the composition of the fourth aspect of the present application in the manufacture of a medicament for treating oral squamous cell carcinoma;

[0055] e. the method of the fifth aspect of the present application in the screening of a candidate drug for preventing or treating oral squamous cell carcinoma.

[0056] The drug of the present application can also be used in combination with other drugs for treating oral squamous cell carcinoma. Other therapeutic compounds can be administered simultaneously with the main active ingredient, even in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a different dosage form from the main active ingredient. 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 drug of the present application can be adjusted according to the severity of the symptoms, the frequency of recurrence, and the physiological response of the treatment regimen.

[0057] At present, it has been disclosed that the RP11-973H7.1 gene exists one transcript, and the sequence is shown as ENST00000563722.1, the RP11-973H7.1 gene is located on chromosome 18, and the Ensemble ID is ENSG00000260302.

[0058] Due to the adoption of the above scheme, the advantages and beneficial effects of the present application are as follows:

[0059] (1) The present application provides a LncRNA RP11-973H7.1 which is significantly highly expressed in oral squamous cell carcinoma, and the RP11-973H7.1 is related to the proliferation and migration of oral squamous cell carcinoma cells.

[0060] (2) The discovery of the relationship between the RP11-973H7.1 and oral squamous cell carcinoma has important significance for promoting the early diagnosis and personalized treatment of oral squamous cell carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0061] Hereinafter, the embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0062] Figure 1 is a differential expression diagram of differential expression genes in oral squamous cell carcinoma tissues and para-cancer tissues;

[0063] Figure 2 is a result diagram of CCK-8 cell proliferation experiment for detecting the influence of differential expression genes on the proliferation activity of oral squamous cell carcinoma cells;

[0064] Figure 3 is a result diagram of cell migration experiment for detecting the influence of differential expression genes on the migration ability of oral squamous cell carcinoma cells. DETAILED DESCRIPTION

[0065] The present application will be further described below in combination with specific examples, which are only used to explain the present application and cannot be understood as a limitation of the present application. 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 application, and the scope of the present application 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 suggested by the manufacturer.

[0066] Example 1: Screening of gene markers related to oral squamous cell carcinoma

[0067] 1. Sample collection

[0068] 5 cases of oral squamous cell carcinoma tissues and para-cancer tissues were collected respectively, which were confirmed by pathology. All patients did not receive any form of treatment before surgery. The samples were stored in liquid nitrogen. The collection of all the above samples was approved by the ethics committee.

[0069] 2. Extraction of tissue RNA

[0070] About 50 mg of the cancer tissue and para-cancer tissue samples stored in liquid nitrogen were taken out and 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:

[0071] (1) Add 1 mL Trizol and stand at room temperature for 5 min;

[0072] (2) Add 0.2 mL chloroform, shake the centrifuge tube vigorously, mix well, and stand at room temperature for 5-10 min;

[0073] (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 and mix well by inverting. Place on ice for 10 min;

[0074] (4) Centrifuge at 12000 rpm and 4°C for 15 min. Carefully discard the supernatant and add 1 mL of 75% DEPC ethanol wash the precipitate (stored at 4°C). Shake well and mix, centrifuge at 4°C and 12000 rpm for 5 min;

[0075] (5) Discard the ethanol supernatant and stand at room temperature for 5 min. Add DEPC water to dissolve the precipitate;

[0076] (6) Quantify and use or store in a -80°C freezer for later use.

[0077] 3. Quantification and purity analysis of total RNA

[0078] 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.

[0079] 4. Construction of cDNA library

[0080] Ribosomal RNA in total RNA was removed using Ribo-Zero kit of Epicentre; for the intact RNA sequence, random breaking was performed using metal ions to randomly break the RNA into small fragments of about 200 bp; Illumina TruseqTM RNA sample Prep Kit was used for construction of cDNA library.

[0081] 5. Sequencing

[0082] Using Illumina X-Ten sequencing platform, 2*150 bp sequencing was performed.

[0083] 6. High-throughput transcriptome sequencing data analysis

[0084] LncRNAs that are not easy to detect are deleted, and DESeq2 in R-3.3.3 tools is used for differential expression analysis of reads number, and the screening criteria for differentially expressed LncRNAs are FDR <0.05 and abs(log2FC) >2.

[0085] 7. Experimental results

[0086] 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.

[0087] Example 2 QPCR verification of differential expression of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1

[0088] 1. Tissue collection

[0089] Using the collection method described in Example 1, 60 tissue samples of oral squamous cell carcinoma and their corresponding para-cancer tissue samples were collected, and RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 were verified as large sample differential expression genes.

[0090] 2. Extraction of tissue RNA

[0091] The extraction steps are the same as in Example 1.

[0092] 3. QPCR experiment

[0093] (1) Reverse transcription reaction

[0094] LncRNA reverse transcription was carried out by using FastQμant cDNA first strand synthesis kit (item number: KR106). First, the genomic DNA reaction was removed. In the test tube, 5x gDNA Bμffer 2.0 μL, total RNA 1 μg, RNase Free ddH2O was added to make the total volume 10 μL, and the water bath was heated at 42°C for 3 min. 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 were mixed, and then added to the above test tube and mixed together to make a total volume of 20 μL. The water bath was heated at 42°C for 15 min and 95°C for 3 min.

[0095] (2) Design and preparation of primers

[0096] QPCR amplification primers were designed according to the coding sequences of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 genes and GAPDH gene in Genebank. When designing the primers of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1, the common sequences of different transcription product sequences were selected for design. The specific primer sequences are as follows:

[0097] RP11-575F12.2 gene:

[0098] The forward primer is 5'-TTAGAAGAAGAACCATTG-3'(SEQ ID NO. 1)

[0099] The reverse primer is 5'-TCCTATTCAAGACCATAA-3'(SEQ ID NO. 2)

[0100] RP11-54A9.1 gene:

[0101] The forward primer is 5'-GACTGACACTGCTTGATT-3'(SEQ ID NO. 3)

[0102] The reverse primer is 5'-TCCTTCACTGTAAGAGTTATC-3'(SEQ ID NO. 4)

[0103] RP11-973H7.1 gene:

[0104] The forward primer is 5'-TTGCCATTCTAATGTAAG-3'(SEQ ID NO. 5)

[0105] The reverse primer is 5'-TGTTGCCTATTCATAAGAC-3' (SEQ ID NO. 8)

[0106] AF131217.1 gene:

[0107] The forward primer is 5'-ATTCTCGTTACTCCTTGT-3' (SEQ ID NO. 7)

[0108] The reverse primer is 5'-TGTTGCCTATTCATAAGAC-3' (SEQ ID NO. 8)

[0109] GAPDH gene:

[0110] The forward primer is 5'-CTCTGGTAAAGTGGATATTGT-3' (SEQ ID NO. 9)

[0111] The reverse primer is 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO. 10)

[0112] (3) Real-time quantitative PCR reaction

[0113] The amplification was performed using SuperReal PreMix Plus (SYBR Green) (item number: FP205).

[0114] A 20 μL reaction system was used: 2x SuperReal PreMix Plus 10 μL, 0.6 μL of forward and reverse primers (10 μM) each, 5x ROX Reference Dye △ 2 μL, 2 μL of DNA template, and 4.8 μL of sterilized distilled water. 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.

[0115] The amplification programs were as follows:

[0116] RP11-575F12.2 gene: 95℃ 15min, (95℃ 10s, 50℃ 30s, 72℃ 32s) x 45 cycles;

[0117] RP11-54A9.1 gene: 95℃ 15min, (95℃ 10s, 54℃ 30s, 72℃ 32s) x 40 cycles;

[0118] RP11-973H7.1 gene: 95℃ 15min, (95℃ 10s, 56℃ 30s, 72℃ 32s) x 39 cycles;

[0119] AF131217.1 gene: 95 ℃ 15 min, (95 ℃ 10 s, 52 ℃ 30 s, 72 ℃ 32 s) x 45 cycles.

[0120] 4. Statistical analysis

[0121] Statistical software SPSS20.0 was used for statistical analysis, and 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.

[0122] 5. Experimental results

[0123] 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 paracancerous 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.

[0124] Example 3 Detection of silencing of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 and functional verification

[0125] 1. Cell culture

[0126] 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.

[0127] 2. Cell transfection

[0128] 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.

[0129] 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.

[0130] Among them, the siRNA sequence for RP11-575F12.2 is as follows:

[0131] The positive strand is 5'-UUGUAAAAGAAUUCUUUGCUC-3' (SEQ ID NO. 11)

[0132] The antisense strand is 5'-GCAAAGAAUUCUUUUACAAAG-3' (SEQ ID NO.12)

[0133] The siRNA sequences targeting RP11-54A9.1 are as follows:

[0134] The positive strand is 5'-ACAAUCUGUGCAAUUACAGUU-3' (SEQ ID NO. 13)

[0135] The antisense strand is 5'-CUGUAAUUGCACAGAUUGUUC-3' (SEQ ID NO. 14)

[0136] The siRNA sequences targeting RP11-973H7.1 are as follows:

[0137] The positive strand is 5'-UUCAAAAGUGAAACUACAGAC-3' (SEQ ID NO. 15)

[0138] The antisense strand is 5'-CUGUAGUUUCACUUUUGAACC-3' (SEQ ID NO. 16)

[0139] The siRNA sequences targeting AF131217.1 are as follows:

[0140] The positive strand is 5'-AUCUUAGACUGGGUAAAUCAU-3' (SEQ ID NO. 17)

[0141] The antisense strand is 5'-GAUUUACCCAGUCUAAGAUAU-3' (SEQ ID NO. 18)

[0142] 3. QPCR detection of transcription levels of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 genes

[0143] After 48h of transfection, total RNA was extracted from each group of cells using Trizol method, and reverse transcription and real-time quantitative PCR detection were performed according to the method in Example 2.

[0144] 4. Cell proliferation experiment

[0145] 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 culture 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.

[0146] 5. Cell migration experiment

[0147] 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 2×10 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.

[0148] 6. Statistical analysis

[0149] Statistical software SPSS20.0 was used for statistical analysis. The measurement data were represented by mean ± standard deviation (mean ± SD). Paired T test was used for comparison between two groups, single factor analysis of variance 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.

[0150] 7. Experimental results

[0151] 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).

[0152] 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).

[0153] 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).

[0154] 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).

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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> Xiangya Stomatological Hospital of Central South University Qingdao Youshen Biomedical Co., Ltd. <120> A biomarker for diagnosing and treating oral squamous cell carcinoma and application thereof <141> 2020-11-27 <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 ttagaagaag aaccattg 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 tcctattcaa gaccataa 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <400> 3 gactgacact gcttgatt 18 <210> 4 <211> 21 <212> DNA <213> 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> twenty one <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> twenty one <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. Use of a reagent for specific detection of the RP11-973H7.1 gene in the preparation of a product for diagnosing oral squamous cell carcinoma.

2. The use according to claim 1, characterized in that The reagent is a primer for specifically amplifying RP11-973H7.

1.

3. The use according to claim 2, characterized in that The primers for specific amplification of RP11-973H7.1 are shown in SEQ ID NOs. 5-6.

4. Use of an agent for inhibiting the expression of RP11-973H7.1 in the preparation of a drug for treating oral squamous cell carcinoma, characterized in that: The reagent is a double-stranded siRNA, and the sequence of the siRNA is shown in SEQ ID NO. 15-16.

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