CN112481377A - Application of RP11-575F12.2 in preparation of preparations for diagnosing and treating oral squamous cell carcinoma - Google Patents

Abstract

The invention discloses application of RP11-575F12.2 in preparation of a preparation for diagnosing and treating oral squamous cell carcinoma, and through detecting oral squamous cell carcinoma tissues and tissue samples beside the oral squamous cell carcinoma, a biomarker RP11-575F12.2 is found to be remarkably highly expressed in the oral squamous cell carcinoma tissues, and further cell experiments verify that the proliferation and invasion of the oral squamous cell carcinoma cells can be inhibited by regulating the expression level of RP11-575F12.2, so that a new detection and target site is provided for the diagnosis and treatment of the oral squamous cell carcinoma, and a theoretical basis is provided for disclosing the pathogenesis of the oral squamous cell carcinoma.

Description

Application of RP11-575F12.2 in preparation of preparations for diagnosing and treating oral squamous cell carcinoma

Technical Field

The invention belongs to the field of biological medicines, relates to a biomarker, and particularly relates to application of the biomarker RP11-575F12.2 in preparation of a preparation for diagnosing and treating oral squamous cell carcinoma.

Background

Oral Squamous Cell Carcinoma (OSCC), abbreviated as Oral squamous cell carcinoma, is a malignant tumor with the highest incidence rate of Oral and maxillofacial regions, has the characteristics of high local infiltration, high cervical lymph node metastasis rate, easy relapse and the like in Oral malignant diseases with the proportion of about 90 percent, and has poorer prognosis. In recent years, the survival rate of oral squamous cell carcinoma patients after operation for 5 years is about 60 percent all the time, and the survival rate is not obviously improved along with the improvement of diagnosis and treatment technology. Clinically, when a patient is diagnosed with symptoms such as pain, ulcer, bleeding and lumps, large-area infiltration growth and even local and remote metastasis often occur, and surgical treatment adopted at this stage generally causes the facial appearance and oral cavity function of the patient to be damaged, seriously affects the food intake, speech, social interaction and the like of the patient, and brings great influence to the patient and the family.

If the patient with oral squamous cell carcinoma can be diagnosed in time at the early stage of onset, the 5-year survival rate after operation can reach 80% -90%, and the influence on the patient is small due to small tumor infiltration area and small surgical excision range at the early stage of onset. Therefore, the early discovery, early diagnosis and early treatment are important means for improving the prognosis of oral squamous cell carcinoma. At present, oral squamous carcinoma is mainly diagnosed by tissue biopsy, but due to the characteristic of heterogeneity of tumor lesions, part of tissues cut by biopsy cannot completely reflect the nature of lesions, even the detection of tumors is influenced, thus resulting in missed diagnosis. Therefore, the development of noninvasive early screening and diagnosis by using a modern biological method is very important for improving the current situations of high incidence rate and poor prognosis of oral squamous cell carcinoma.

In recent years, extensive research efforts have been made for early noninvasive diagnosis of oral cancer. However, each method has certain disadvantages, such as: the fluorescein detection utilizes the diffusion degree of the fluorescent liquid in the tissues to judge the benign and malignant degree of the lesion, the fluorescent liquid needs to be immediately observed in a dark room after being injected into the lesion area, the detection condition can not be completely realized clinically, and the increase of hemoglobin at the hyperemia part can enhance the absorption of light, cause fluorescence deficiency and easily cause false positive; the vital tissue staining method stains the tissue to show the cancerous tissue site, but the dye also has affinity for epithelial dysplastic tissue, mitotically active cells, and rough or keratinized tissue, and the false positive rate can reach 42.2%. Therefore, it is of great significance to find a stable, reliable and easily-obtained biomarker molecule for early diagnosis and later treatment of oral squamous cell carcinoma.

Disclosure of Invention

Aiming at the defects in the prior art, the invention researches the genes which are differentially expressed in the oral squamous cell carcinoma tissue and the tissues beside the oral squamous cell carcinoma, and researches the influence of the differentially expressed genes on the oral squamous cell carcinoma cells through further cell experiments, thereby providing detection and target sites for the diagnosis and treatment of the oral squamous cell carcinoma and simultaneously providing a theoretical basis for disclosing the pathogenesis of the oral squamous cell carcinoma.

The above object of the present invention is achieved by the following technical solutions:

the invention provides application of a detection reagent of a biomarker in preparing a product for diagnosing oral squamous cell carcinoma.

Further, the biomarker is RP11-575F 12.2.

Further, the detection reagent comprises a reagent for detecting the RP11-575F12.2 gene and/or an expression product thereof;

preferably, the agent is selected from: a primer for specifically amplifying the RP11-575F12.2 gene and a probe for specifically recognizing the RP11-575F12.2 gene;

more preferably, the primer sequence of the specific amplification RP11-575F12.2 gene is shown in SEQ ID NO. 1-2.

Furthermore, the primer sequence is designed according to transcripts ENST00000541419.1 and ENST00000535022.1 of RP11-575F12.2 gene.

In a second aspect of the invention, there is provided a product for diagnosing oral squamous carcinoma.

Further, the product comprises a reagent for detecting the expression level of the RP11-575F12.2 gene.

Further, the product comprises a kit, a chip or a test strip.

Further, the reagent comprises a reagent for detecting the expression level of RP11-575F12.2 by reverse transcription PCR, real-time quantitative PCR, in-situ hybridization and a gene chip;

preferably, the reagent for detecting the expression level of the RP11-575F12.2 by reverse transcription PCR and/or real-time quantitative PCR at least comprises a pair of primers for specifically amplifying the RP11-575F 12.2;

preferably, the reagent for detecting the expression level of RP11-575F12.2 by in situ hybridization and/or gene chip comprises a probe hybridized with a nucleic acid sequence of RP11-575F 12.2.

The third aspect of the invention provides an application of RP11-575F12.2 in preparing a pharmaceutical composition for treating oral squamous carcinoma.

Further, the pharmaceutical composition includes an agent that inhibits expression of RP11-575F 12.2;

preferably, the agent inhibiting the expression of RP11-575F12.2 is a double-stranded molecule;

more preferably, the double stranded molecule comprises siRNA;

most preferably, the sequence of the siRNA is shown in SEQ ID NO. 11-12.

Furthermore, the sequence of the siRNA is designed according to transcripts ENST00000541419.1 and ENST00000535022.1 of RP11-575F12.2 gene.

In a fourth aspect of the invention, a pharmaceutical composition is provided.

Further, the pharmaceutical composition includes an agent that inhibits expression of RP11-575F 12.2;

preferably, the agent inhibiting the expression of RP11-575F12.2 is a double-stranded molecule;

more preferably, the double stranded molecule comprises siRNA;

most preferably, the sequence of the siRNA is shown in SEQ ID NO. 11-12.

Furthermore, the sequence of the siRNA is designed according to transcripts ENST00000541419.1 and ENST00000535022.1 of RP11-575F12.2 gene.

Furthermore, the pharmaceutical composition can also comprise an effective amount of drugs for treating oral squamous cell carcinoma and pharmaceutically acceptable carriers and/or auxiliary materials.

Further, the pharmaceutical composition and the medicament for treating oral squamous cell carcinoma can be prepared into separate preparations for combined application, and the two can also be prepared into one preparation for application in the form of a composition.

Further, the carrier and/or adjuvant includes pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the designed dosage form. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols (e.g. polyethylene glycol, water, sucrose, ethanol, glycerol), various preservatives, lubricants, dispersants, flavoring agents, wetting agents, sweeteners, fragrances, emulsifiers, suspending agents, preservatives, antioxidants, colorants, stabilizers, salts, buffers and the like, to which suitable pharmaceutically acceptable carriers and/or adjuvants are also added, as well as those which are described in detail in Remington's Pharmaceutical Sciences (19th ed.,1995) for aiding the stability of the formulation or for enhancing the activity or its bioavailability or for producing an acceptable mouthfeel or odor in the case of oral administration, and those which can be used in such Pharmaceutical compositions can be in the form of their original compounds per se, or optionally in the form of a pharmaceutically acceptable salt thereof. The pharmaceutical composition thus formulated may be administered by any suitable means known to those skilled in the art, as desired, by administering a safe and effective amount of the drug of the present invention to a human.

The appropriate dose of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the method of preparation, the mode of administration, the age, body weight, sex, disease state, diet, time of administration, route of administration, excretion rate and reaction sensitivity of the patient, and a skilled physician can easily determine the prescription and the dose of administration effective for the desired treatment or prevention.

The fifth aspect of the invention provides application of RP11-575F12.2 in screening candidate drugs for treating oral squamous cell carcinoma.

Further, the use includes a method of screening a candidate drug for preventing or treating oral squamous cell carcinoma.

In a sixth aspect, the present invention provides a method for screening a candidate drug for preventing or treating oral squamous cell carcinoma.

Further, the method comprises the steps of:

(1) treating a system expressing or containing the RP11-575F12.2 gene with a test substance;

(2) detecting the expression of RP11-575F12.2 gene in the system;

(3) selecting the test substance capable of reducing the expression level of the RP11-575F12.2 gene as a candidate drug.

Further, the system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

Further, the test substances include, but are not limited to: interfering molecules, nucleic acid inhibitors, small molecule compounds and the like designed aiming at the RP11-575F12.2 gene or upstream or downstream genes thereof.

Further, the selected test agent described in step (3) is one that significantly reduces the expression level of RP11-575F12.2 as compared to the expression level detected in the absence of the test agent.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, some terms are explained as follows.

As used herein, "biomarker" and "marker" may be used in combination to refer to an indication of normal or abnormal progression in an individual or an indication of a disease or other condition in an individual or a target molecule that expresses these. In more detail, "biomarker" and "marker" are normal or abnormal and, if abnormal, anatomical, physiological, biochemical or molecular parameters associated with the presence of a particular physiological state or progression, either chronic or acute. Biomarkers can be detected and measured by a variety of methods including laboratory testing and medical imaging.

The term "effective amount" as used herein, refers to an amount that has a therapeutic effect or is required to produce a therapeutic effect in a subject being treated. For example, a therapeutically or pharmaceutically effective amount of a drug refers to the amount of drug required to produce the desired therapeutic effect, which can be reflected in the results of clinical trials, model animal studies, and/or in vitro studies. The pharmaceutically effective amount will depend on several factors including, but not limited to, the characteristics of the subject (e.g., height, weight, sex, age, and medical history), and the severity of the disease.

The term "pharmaceutically acceptable carrier and/or adjuvant" as used herein refers to a carrier and/or adjuvant that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

The term "excipient", as used herein, refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

In the embodiment of the invention, the invention discovers for the first time that the expression of the RP11-575F12.2 gene in the oral squamous cell carcinoma tissue is obviously higher than that of the normal oral mucosal tissue through intensive research, and verifies that the RP11-575F12.2 gene also shows high expression in the oral squamous cell carcinoma cell by using related cell experiments, and the reduction of the expression level of the RP11-575F12.2 gene can inhibit the proliferation and invasion of the oral squamous cell carcinoma cell, thereby prompting that the RP11-575F12.2 gene can be used as a target for diagnosing and treating the oral squamous cell carcinoma in clinic.

It has been disclosed that two transcripts exist in the RP11-575F12.2 gene, the sequences are respectively shown as ENST00000541419.1 and ENST00000535022.1, the RP11-575F12.2 gene is located on chromosome 12, and Ensemble ID is ENSG 00000256001.

The invention has the following advantages and beneficial effects:

(1) the invention firstly proves that RP11-575F12.2 can be used as a biomarker for diagnosing and treating oral squamous cell carcinoma, and the expression level of RP11-575F12.2 in the oral squamous cell carcinoma tissue is obviously higher than that in the tissue beside the carcinoma.

(2) According to the invention, the detection finds that the RP11-575F12.2 has high specificity expression in oral squamous cell carcinoma, the detection accuracy is high, and a more accurate and rapid detection method is provided for clinic.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a graph showing that differentially expressed genes are differentially expressed in oral squamous carcinoma tissues and paracarcinoma tissues;

FIG. 2 is a diagram showing the results of CCK-8 cell proliferation assay for detecting the effect of differentially expressed genes on the proliferative activity of oral squamous carcinoma cells;

FIG. 3 is a graph showing the results of cell migration experiments to determine the effect of differentially expressed genes on the migration ability of oral squamous cell carcinoma cells.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Example 1 screening of Gene markers associated with oral squamous cell carcinoma

1. Sample collection

5 cases of oral squamous carcinoma tissues and tissues beside the carcinoma were collected respectively, and all patients were confirmed by pathological diagnosis not to receive any form of treatment before operation, and the samples cut by the operation were frozen in liquid nitrogen. All the specimens were obtained with the consent of the tissue ethics committee.

2. Extraction of tissue RNA

Taking out about 50mg of cancer tissue and para-cancer tissue samples frozen in liquid nitrogen, putting the tissue samples into a precooled mortar for grinding, transferring the tissue samples into a 1.5mL EP tube when no large-particle tissue exists, and extracting and separating RNA according to the instruction in the kit. The specific extraction steps are as follows:

(1) adding 1mL of Trizol, and standing at room temperature for 5 min;

(2) adding 0.2mL of chloroform, forcibly oscillating the centrifuge tube, fully and uniformly mixing, and standing at room temperature for 5-10 min;

(3) centrifuging at 12000rpm and 4 deg.C for 15min, transferring the upper water phase into another new EP tube (taking care not to absorb protein substances between the two water phases), adding equal volume of isopropanol precooled at-20 deg.C, reversing thoroughly, mixing, and placing on ice for 10 min;

(4) centrifuging at 12000rpm and 4 deg.C for 15min, carefully removing supernatant, adding 1mL 75% DEPC ethanol, washing precipitate (storing at 4 deg.C), shaking, mixing, and centrifuging at 12000rpm for 5 min;

(5) removing ethanol supernatant, standing at room temperature for 5min, adding DEPC water to dissolve precipitate;

(6) quantitatively using or freezing in a refrigerator at-80 deg.C for use.

3. Quantitative and purity analysis of Total RNA

The extracted RNA was subjected to agarose gel electrophoresis, the concentration and purity of the extracted RNA were determined using Nanodrop2000, the integrity of the RNA was checked by agarose gel electrophoresis, and the RIN value was determined by Agilent 2100. The total amount of RNA required for single library construction is 5 mug, and the concentration is more than or equal to 200 ng/mug.

4. Construction of cDNA library

Ribosomal RNA was removed from total RNA using the Ribo-Zero kit from Epicentre; randomly breaking the complete RNA sequence by using metal ions, and randomly breaking the RNA into small fragments of about 200 bp; the construction of cDNA library was performed using Illumina Truseq RNA sample Prep Kit.

5. Sequencing

2X 150bp sequencing was performed using the Illumina X-Ten sequencing platform.

6. High throughput transcriptome sequencing data analysis

Deletion of undetectable LncRNA differential expression analysis of reads numbers using DESeq2 in R-3.3.3 tool differential expression LncRNA screening criteria: FDR <0.05, abs (log2FC) > 2.

7. Results of the experiment

The results show that the expression levels of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 are significantly up-regulated in oral squamous carcinoma tissues compared to paracarcinoma tissues.

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

1. Tissue collection

Tissue samples of 60 oral squamous cell carcinomas and corresponding paracancerous tissue samples were collected by the collection method described in example 1, and large-sample differential expression genes were verified for RP11-575F12.2, RP11-54A9.1, RP11-973H7.1, and AF 131217.1.

2. Extraction of tissue RNA

The extraction procedure was the same as in example 1.

3. QPCR experiment

(1) Reverse transcription reaction

Using the FastQ μ ant cDNA first strand synthesis kit (cat # KR106) to perform LncRNA reverse transcription, genomic DNA reaction was first removed, 5 XgDNA B μ ffer 2.0 μ L, total RNA 1 μ g, RNase Free ddH were added to the test tube₂O to make the total volume to 10 μ L, heating in water bath at 42 deg.C for 3 min.

10 XFast RT B. mu.ffer 2.0. mu.L, RT Enzyme Mix 1.0μL，FQ-RT Primer Mix 2.0μL，RNase Free ddH₂O5.0 μ L, mixing, adding into the above test tube, mixing to give 20 μ L, heating in water bath at 42 deg.C for 15min, and heating at 95 deg.C for 3 min.

(2) Design and preparation of primers

QPCR amplification primers are designed according to coding sequences of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1, AF131217.1 genes and GAPDH genes in Genebank, and when primer design of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 is carried out, common sequences of different transcription product sequences are selected for design, and specific primer sequences are as follows:

RP11-575F12.2 gene:

the forward primer is 5'-TTAGAAGAAGAACCATTG-3' (SEQ ID NO.1)

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

RP11-54A9.1 gene:

the forward primer is 5'-GACTGACACTGCTTGATT-3' (SEQ ID NO.3)

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

RP11-973H7.1 gene:

the forward primer is 5'-TTGCCATTCTAATGTAAG-3' (SEQ ID NO.5)

The reverse primer is 5'-ACTCCTATTATCTGTATGT-3' (SEQ ID NO.6)

AF131217.1 gene:

the forward primer is 5'-ATTCTCGTTACTCCTTGT-3' (SEQ ID NO.7)

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

GAPDH gene:

the forward primer is 5'-CTCTGGTAAAGTGGATATTGT-3' (SEQ ID NO.9)

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

(3) Real-time quantitative PCR reaction

Amplification was performed using SuperReal PreMix Plus (SYBR Green) (cat # FP 205).

A20. mu.L reaction was used: 2 XSuperReal Premix Plus 10. mu.L, positive and negativePrimer pair (10. mu.M) 0.6. mu.L each, 5 XROX Reference Dye^△2. mu.L, DNA template 2. mu.L, sterilized distilled water 4.8. mu.L. Each sample was provided with 3 parallel channels and all amplification reactions were repeated three more times to ensure the reliability of the experimental results.

The amplification procedures were:

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

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

RP11-973H7.1 gene: 15min at 95 ℃ (10 s at 95 ℃, 30s at 56 ℃, 32s at 72 ℃) multiplied by 39 cycles;

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

4. Statistical analysis

Statistical analysis was performed using statistical software SPSS20.0, and the data were expressed as mean. + -. standard deviation (mean. + -. SD). The two groups of comparisons adopt a paired T test, the three groups and above adopt a one-factor variance analysis, and the multiple comparisons adopt an LSD-T test. All experiments were repeated three times, with differences of P <0.05 being statistically significant.

5. Results of the experiment

The QPCR result is shown in figure 1, and the result shows that compared with the tissues beside the cancer, the expression levels of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 in the oral squamous cell carcinoma tissues are obviously up-regulated, and the difference has statistical significance (P <0.05), thereby indicating that RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 can be used as molecular markers for diagnosing and treating the oral squamous cell carcinoma.

Example 3 silencing detection and functional validation of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1

1. Cell culture

Taking out human oral squamous cell carcinoma SCC-15 cell stored in liquid nitrogen, recovering, inoculating in DMEM medium, culturing at 37 deg.C and 5% CO₂Cells were cultured in a thermostated incubator. After 24h, the cells grow adherently, namely the cells are recovered successfully, the liquid is changed for 1 time every 1-2d, the cells are digested by trypsin and prepared into cell suspension for experiment。

2. Cell transfection

The cells were arranged at 2X 10⁵One well was inoculated into six well cell culture plates at 37 ℃ with 5% CO₂Culturing in an incubator. Cells in the logarithmic phase of proliferation (about 80%) were washed with PBS 2 times after discarding the medium, starvation-cultured in an incubator for 1 hour with 2mL of DMEM, and transfected with Lipofectin 2000 (Invitrogen), according to the instructions.

The experiment was divided into three groups: a blank control group (SCC-15), a negative control group (siRNA-NC) and an experimental group (siRNA group), wherein the siRNA of the negative control group has no homology with the sequences of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF 131217.1.

Wherein, the siRNA sequence aiming at RP11-575F12.2 is as follows:

the sense strand is 5'-UUGUAAAAGAAUUCUUUGCUC-3' (SEQ ID NO.11)

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

The siRNA sequence for RP11-54A9.1 is as follows:

the sense strand is 5'-ACAAUCUGUGCAAUUACAGUU-3' (SEQ ID NO.13)

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

The siRNA sequence for RP11-973H7.1 is as follows:

the sense strand is 5'-UUCAAAAGUGAAACUACAGAC-3' (SEQ ID NO.15)

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

The siRNA sequence for AF131217.1 is as follows:

the sense strand is 5'-AUCUUAGACUGGGUAAAUCAU-3' (SEQ ID NO.17)

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

3. QPCR detection of the transcript level of RP11-575F12.2, RP11-54A9.1, RP11-973H7.1 and AF131217.1 genes

After 48h of transfection and culture of each group of cells, total RNA of the cells was extracted by Trizol method, reverse transcription and real-time quantitative PCR detection were performed according to the method of example 2.

4. Cell proliferation assay

Digesting and centrifuging a negative control group transfected for 24 hours and cells of an experimental group by a conventional method, removing supernatant, adding 1mL of complete culture medium to suspend the cells, blowing, beating and uniformly mixing, inoculating 3000 cells per hole to a 96-well plate, and supplementing the complete culture medium to 100 mu L; 100mL of DEPC water was added to the outermost turn of the plate, and the 96-well plate was incubated in a incubator. After culturing for 48h, adding 100 mu L of culture medium containing 10% CCK-8, continuously culturing in an incubator for 1h, measuring absorbance at 450nm by using an enzyme-labeling instrument, and counting and recording experimental data.

5. Cell migration assay

Placing the Transwell chamber in a 24-hole plate, adding 200 mu L DMEM solution into the upper chamber, placing the upper chamber in an incubator, and hydrating for 1 h; according to 2X 10 per cell⁴Plating the cells, supplementing the liquid in the upper chamber to 200 mu L, blowing, beating and uniformly mixing, adding 700 mu L of complete culture medium in the lower chamber, and continuously culturing for 36h in an incubator; taking out the small chamber, discarding the culture medium in the upper chamber and the lower chamber, wiping off the residual culture medium and cells in the upper chamber with a cotton swab, washing the small chamber with PBS, shaking for 5min, and discarding the PBS; adding 500 μ L of 4% paraformaldehyde into the lower chamber, fixing at room temperature for 30min, removing the fixing solution, washing with PBS for 3 times, shaking for 5min, and removing PBS; placing the small chamber in a fume hood, and air-drying for 30 min; adding 500 μ L of prepared 0.1% crystal violet solution into lower chamber, removing air bubbles, and standing for 30 min; discard crystal violet solution, wash 3 times with PBS, shake for 5min, discard PBS, gently wipe off excess liquid in upper chamber with dry cotton swab, place chamber under microscope, count cell number and record.

6. Statistical analysis

Statistical analysis was performed using statistical software SPSS20.0, and the data were expressed as mean. + -. standard deviation (mean. + -. SD). The two groups of comparisons adopt a paired T test, the three groups and above adopt a one-factor variance analysis, and the multiple comparisons adopt an LSD-T test. All experiments were repeated three times, with differences of P <0.05 being statistically significant.

7. Results of the experiment

The transfection result shows that the expression level of the blank control group RP11-575F12.2 is set as 1 as a reference, compared with the expression level (relative expression level is 1) of the blank control group RP11-575F12.2 and the expression level (relative expression level is 0.948 +/-0.026) of the transfection siRNA-NC negative control group RP11-575F12.2, the expression level (relative expression level is 0.541 +/-0.085) of the transfection siRNA experimental group RP11-575F12.2 is obviously reduced, and the difference has statistical significance (the experimental group vs blank control group, P < 0.05; the experimental group vs negative control group, P <0.05) and no obvious difference (P >0.05) between the siRNA-NC group and the blank control group.

The transfection results show that the expression level of the blank control group RP11-54A9.1 is set as 1 as a reference, compared with the expression level (relative expression level is 1) of the blank control group RP11-54A9.1 and the expression level (relative expression level is 0.948 +/-0.026) of the transfection siRNA-NC negative control group RP11-54A9.1, the expression level (relative expression level is 0.355 +/-0.027) of the transfection siRNA experimental group RP11-54A9.1 is significantly reduced, and the difference has statistical significance (the experimental group vs blank control group, P < 0.05; the experimental group vs negative control group, P <0.05) and no significant difference (P >0.05) between the siRNA-NC group and the blank control group.

The transfection results show that the expression level of the blank control group RP11-973H7.1 is set as 1 as a reference, compared with the expression level (relative expression level is 1) of the blank control group RP11-973H7.1 and the expression level (relative expression level is 0.948 +/-0.026) of the transfection siRNA-NC negative control group RP11-973H7.1, the expression level (relative expression level is 0.743 +/-0.096) of the RP11-973H7.1 of the transfection siRNA experimental group is significantly reduced, and the difference has statistical significance (the experimental group vs blank control group, P < 0.05; the experimental group vs negative control group, P <0.05), while the siRNA-NC group and the blank control group have no significant difference (P > 0.05).

The transfection results show that, with the expression level of AF131217.1 in the blank control group as a reference set as 1, compared with the expression level (relative expression level of 1) of AF131217.1 in the blank control group and the expression level (relative expression level of 0.948 +/-0.026) of AF131217.1 in the transfected siRNA-NC negative control group, the expression level (relative expression level of 0.131 +/-0.053) of AF131217.1 in the transfected siRNA experimental group is significantly reduced, and the difference has statistical significance (experimental group vs blank control group, P < 0.05; experimental group vs negative control group, P <0.05), while there is no significant difference (P >0.05) between the siRNA-NC group and the blank control group.

The result of the proliferation activity of CCK-8 cells is shown in figure 2, and the result shows that the OD450(0.634 +/-0.064) of the experimental group transfected with siRNA is obviously lower than that of the control group (1.236 +/-0.051) transfected with siRNA-NC, and P is less than 0.05, which indicates that RP11-575F12.2 plays an important role in the proliferation of oral squamous cell carcinoma cells, and the proliferation capacity of the oral squamous cell carcinoma cells can be changed by changing the expression level of RP11-575F 12.2.

The result of CCK-8 cell proliferation activity is shown in figure 2, and the result shows that the OD450(0.506 +/-0.038) of the experimental group transfected with siRNA is obviously lower than that of the control group (1.236 +/-0.051) transfected with siRNA-NC, and P is less than 0.05, which indicates that RP11-54A9.1 plays an important role in the proliferation of oral squamous cell carcinoma cells, and the expression level of RP11-54A9.1 can change the proliferation capacity of the oral squamous cell carcinoma cells.

The result of CCK-8 cell proliferation activity is shown in figure 2, and the result shows that the OD450(0.820 +/-0.110) of the experimental group transfected with siRNA is obviously lower than that of the control group (1.236 +/-0.051) transfected with siRNA-NC, and P is less than 0.05, which shows that RP11-973H7.1 plays an important role in the proliferation of oral squamous cell carcinoma cells, and that the proliferation capacity of the oral squamous cell carcinoma cells can be changed by changing the expression level of RP11-973H 7.1.

The result of the proliferation activity of CCK-8 cells is shown in figure 2, and the result shows that the OD450(0.336 +/-0.051) of the experimental group transfected with siRNA is obviously lower than that of the control group (1.236 +/-0.051) transfected with siRNA-NC, and P is less than 0.05, which indicates that the AF131217.1 in the research plays an important role in the proliferation of oral squamous cell carcinoma cells, and the change of the expression level of AF131217.1 can change the proliferation capacity of the oral squamous cell carcinoma cells.

The results of cell migration experiments are shown in FIG. 3, and the results show that the number of migrating cells (69 + -6.245) in the experimental group transfected with siRNA is obviously lower than that in the control group transfected with siRNA-NC (111 + -5.568), and P is less than 0.05, which indicates that RP11-575F12.2 plays an important role in the migration of oral squamous cell carcinoma cells, and that the migration capability of the oral squamous cell carcinoma cells can be changed by changing the expression level of RP11-575F 12.2.

The results of cell migration experiments are shown in FIG. 3, and the results show that the number of migrating cells (55 + -14.107) in the experimental group transfected with siRNA is obviously lower than that in the control group transfected with siRNA-NC (111 + -5.568), and P is less than 0.05, which indicates that RP11-54A9.1 plays an important role in the migration of oral squamous cell carcinoma cells, and that the migration capability of oral squamous cell carcinoma cells can be changed by changing the expression level of RP11-54A 9.1.

The results of cell migration experiments are shown in FIG. 3, and the results show that the number of migrating cells (84.333 + -4.163) in the experimental group transfected with siRNA is obviously lower than that in the control group (111 + -5.568) transfected with siRNA-NC, and P is less than 0.05, which indicates that RP11-973H7.1 plays an important role in the migration of oral squamous cell carcinoma cells, and that the migration capability of oral squamous cell carcinoma cells can be changed by changing the expression level of RP11-973H 7.1.

The results of cell migration experiments are shown in FIG. 3, and the results show that the number of migrating cells (35.667 + -8.622) in the experimental group transfected with siRNA is obviously lower than that in the control group transfected with siRNA-NC (111 + -5.568), and P is less than 0.05, which indicates that AF131217.1 plays an important role in migration of oral squamous cell carcinoma cells, and that the migration ability of oral squamous cell carcinoma cells can be changed by changing the expression level of AF 131217.1.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> Hunan-ya oral Hospital, Zhongnan university in Hunan

Application of <120> RP11-575F12.2 in preparation of preparation for diagnosing and treating oral squamous cell carcinoma

<141> 2020-11-27

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Claims (10)

1. The application of the detection reagent of the biomarker in the preparation of products for diagnosing oral squamous cell carcinoma is characterized in that the biomarker is RP11-575F 12.2.

2. The use of claim 1, wherein the detection reagents comprise reagents for detecting the RP11-575F12.2 gene and/or its expression products;

preferably, the agent is selected from: a primer for specifically amplifying the RP11-575F12.2 gene and a probe for specifically recognizing the RP11-575F12.2 gene;

more preferably, the primer sequence of the specific amplification RP11-575F12.2 gene is shown in SEQ ID NO. 1-2.

3. A product for diagnosing oral squamous carcinoma, which comprises a reagent for detecting the expression level of RP11-575F12.2 gene.

4. The product of claim 3, wherein the product comprises a kit, chip, or strip.

5. The product of claim 3, wherein the reagents comprise reagents for detecting the expression level of RP11-575F12.2 by reverse transcription PCR, real-time quantitative PCR, in situ hybridization, gene chip;

preferably, the reagent for detecting the expression level of the RP11-575F12.2 by reverse transcription PCR and/or real-time quantitative PCR at least comprises a pair of primers for specifically amplifying the RP11-575F 12.2;

preferably, the reagent for detecting the expression level of RP11-575F12.2 by in situ hybridization and/or gene chip comprises a probe hybridized with a nucleic acid sequence of RP11-575F 12.2.

Application of RP11-575F12.2 in preparing a pharmaceutical composition for treating oral squamous carcinoma.

7. The use of claim 6, wherein the pharmaceutical composition comprises an agent that inhibits the expression of RP11-575F 12.2;

preferably, the agent inhibiting the expression of RP11-575F12.2 is a double-stranded molecule;

more preferably, the double stranded molecule comprises siRNA;

most preferably, the sequence of the siRNA is shown in SEQ ID NO. 11-12.

8. A pharmaceutical composition comprising an agent that inhibits the expression of RP11-575F 12.2;

preferably, the agent inhibiting the expression of RP11-575F12.2 is a double-stranded molecule;

more preferably, the double stranded molecule comprises siRNA;

most preferably, the sequence of the siRNA is shown in SEQ ID NO. 11-12.

Application of RP11-575F12.2 in screening of candidate drugs for treating oral squamous cell carcinoma.

10. A method for screening a candidate drug for preventing or treating oral squamous cell carcinoma, comprising the steps of:

(1) treating a system expressing or containing the RP11-575F12.2 gene with a test substance;

(2) detecting the expression of RP11-575F12.2 gene in the system;

(3) selecting the test substance capable of reducing the expression level of the RP11-575F12.2 gene as a candidate drug.

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