CN106435002B

CN106435002B - Oral squamous cell carcinoma biomarker and its application

Info

Publication number: CN106435002B
Application number: CN201611136247.1A
Authority: CN
Inventors: 宋宏涛; 马翠; 任静
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Priority date: 2016-12-12
Filing date: 2016-12-12
Publication date: 2019-07-12
Anticipated expiration: 2036-12-12
Also published as: CN106435002A

Abstract

The invention discloses oral squamous cell carcinoma biomarker and its applications, the biomarker is XIRP2, experiments have shown that, XIRP2 gene expresses up-regulation in oral squamous cell carcinoma, silencing XIRP2 gene can reduce the transcription and translation of the gene, inhibit the hyper-proliferative of oral squamous cell carcinoma cell.The present invention provides the application of the gene or its expression product in diagnosis and treatment oral squamous cell carcinoma.

Description

Oral squamous cell carcinoma biomarker and its application

Technical field

The invention belongs to biomedicine fields, are related to a kind of in oral squamous cell carcinoma biomarker and its application, tool The application of the XIRP2 for being related to significantly raising in oral squamous cell carcinoma of body.

Background technique

Malignant tumor of mouth is a kind of disease for seriously threatening human health.It is oral cavity squamous thin in malignant tumor of mouth Born of the same parents' cancer (oral squamous cell carcinoma, OSCC) ratio accounts for about 90%.As the generation of other tumours, OSCC Occurrence and development be a polygenes, polymolecular is in network structure, multi-step, multistage coefficient long-term complex process. Mutually synergistic effect promotes tumour cell formation and development between each oncogene, molecule.It the mechanism of tumour and controls at present Treatment does not obtain important breakthrough, and it is modern tumour worker problem and opportunity that how early detection neoplastic disease, which undermines radical cure,.

Tumour is a kind of disease of molecular level, and the research that tumour is carried out from genes protein level is final approach.In recent years Genomics, proteomics and CAD based in integral level by high-throughput molecule scanning means Etc. the integration of technologies and the application etc. of " Technology Chain " of being mutually related provide new opportunity for the research of tumour, and exist Great successes are achieved in the research of breast cancer, lung cancer, gastric cancer, colon cancer, oophoroma, melanoma etc..Wherein gene table It is most typical representative up to spectrum chip, can detecte and screen the difference of tumor tissues and normal tissue by chip gene expression profile Allogene and oncogene, the variation of tumor suppressor gene and variation relation, therefrom find the cause of disease, provide line to early diagnose and eradicating Rope and foundation.

Summary of the invention

In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a kind of biology marks of oral squamous cell carcinoma Remember object, the diagnosing and treating of sensitive and specific realization oral squamous cell carcinoma.

To achieve the goals above, the present invention adopts the following technical scheme:

The present invention provides application of the XIRP2 gene in the product of preparation diagnosis oral squamous cell carcinoma.

Further, the product include by chip, blotting, RT-PCR, real-time quantitative PCR, FISH method, CGH method or Array CGH method, bisulfite sequencing, COBRA method detect the variation of XIRP2 gene to diagnose the production of oral squamous cell carcinoma Product.

Wherein, blotting includes Northern, Southern, western blot method.Southern blotting is will be from sample Originally the genomic DNA obtained is separated and is fixed, and measures the XIRP2 base in sample by hybridizing for detection DNA and XIRP2 gene Cause；Northern blotting is a kind of will to pass through detection mRNA and XIRP2 gene by the mRNA separation, fixed obtained in sample Hybridization detect the mRNA of the gene；Western blot method is a kind of Protein Separation by sample, fixation, passes through detection The expression degree of gene is analyzed in the immune response of antibody and XIRP2 albumen.

Wherein, the product with RT-PCR diagnosis oral squamous cell carcinoma includes at least a pair of of specific amplified XIRP2 base The primer of cause；The product with real-time quantitative PCR diagnosis oral squamous cell carcinoma includes at least a pair of of specific amplified XIRP2 base The primer of cause.

Further, a pair of of specific amplified XIRP2 base that the product with real-time quantitative PCR diagnostic tube squamous carcinoma includes at least The primer sequence of cause is as shown in SEQ ID NO.3 and SEQ ID NO.4.

The present invention provides a kind of product for diagnosing oral squamous cell carcinoma, the product can be by detection sample The expression of XIRP2 gene diagnoses oral squamous cell carcinoma.

Further, the product includes chip, kit or preparation.Wherein, the chip includes genetic chip, protein Chip；The genetic chip includes solid phase carrier and the oligonucleotide probe for being fixed on solid phase carrier, and the oligonucleotides is visited Needle includes the oligonucleotide probe for XIRP2 gene for detecting XIRP2 gene transcription level；The protein-chip packet It includes solid phase carrier and is fixed on the specific antibody of the XIRP2 albumen of solid phase carrier；The kit includes genetic test examination Agent box and protein immunization detection kit；The gene detecting kit includes the examination for detecting XIRP2 gene transcription level Agent；The protein immunization detection kit includes the specific antibody of XIRP2 albumen.

Genetic chip or gene detecting kit of the present invention can be used for detecting multiple including XIRP2 gene The expression of gene (for example, multiple genes relevant to oral squamous cell carcinoma).The protein chip or protein immunization inspection Multiple protein that test agent box can be used for detecting including XIRP2 albumen are (such as relevant to oral squamous cell carcinoma more A protein) expression.Multiple markers of oral squamous cell carcinoma are detected simultaneously, are greatly improved oral cavity squama The accuracy rate of shape cell cancer diagnosis.

The present invention provides application of the XIRP2 gene in the pharmaceutical composition of preparation treatment oral squamous cell carcinoma.

Further, described pharmaceutical composition includes XIRP2 gene and/or the inhibitor of its expression product.The inhibitor Including inhibiting the substance of XIRP2 gene expression, inhibiting the substance of XIRP2 gene expression product stability, and/or inhibiting XIRP2 The active substance of gene expression product.

Further, the inhibitor is for the siRNA of XIRP2 gene, for the antibody of XIRP2 albumen；Preferably, institute Stating inhibitor is siRNA.

In a specific embodiment of the invention, the sequence such as SEQ ID NO.9 of the siRNA for XIRP2 gene With shown in SEQ ID NO.10.

The present invention also provides a kind of pharmaceutical composition for treating oral squamous cell carcinoma, the drug includes XIRP2 base Cause and/or its expression product inhibitor.The inhibitor includes the substance for inhibiting XIRP2 gene expression, inhibits XIRP2 gene table Up to the substance, and/or the inhibition active substance of XIRP2 gene expression product of product stability.

The present invention also provides a kind of method for inhibiting cell Proliferation, the method will be directed to XIRP2 gene SiRNA, shRNA, antisense oligonucleotides or Loss-of-function gene are imported into tumour cell in vitro.

The present invention provides a kind of composition of medicine, the composition of medicine includes above-mentioned pharmaceutical composition and contains antitumor The pharmaceutical composition of agent.

Further, the pharmaceutical composition of antitumor agent includes but is not limited to for example western appropriate former times monoclonal antibody of immunotherapeutic agent, Chemotherapeutant or a kind of platinum medicine of related category of chemoluminescence therapeutic agent such as Kapo Platinum, taxane are either A kind of taxanes or the two of classification.

The advantages of the present invention:

Present invention firstly discovers that biomarker-XIRP2 relevant to oral squamous cell carcinoma occurrence and development, passes through Detect the variation of subject XIRP2, the early diagnosis of Lai Shixian oral squamous cell carcinoma.

The present invention provides the molecular targets for the treatment of oral squamous cell carcinoma, treat disease by targeting molecular marker Disease has sensibility and specificity.

The present invention provides certain theoretical basis to the Mechanism Study of oral squamous cell carcinoma.

Detailed description of the invention

Fig. 1 shows the expression using QPCR detection XIRP2 gene in oral squamous cell carcinoma；

Fig. 2 shows the expression using QPCR detection XIRP2 gene in oral squamous cell carcinoma cell；

Fig. 3 shows the influence using QPCR detection siRNA to XIRP2 gene expression；

Fig. 4 shows influence of the mtt assay detection XIRP2 to oral cavity epidermoid carcinoma cell proliferation activity；

Fig. 5 shows the influence using the cell transwell detection XIRP2 gene pairs oral squamous cell carcinoma cell invasion.

Specific embodiment

Present invention firstly discovers that XIRP2 is related to the occurrence and development of oral squamous cell carcinoma, and demonstrate XIRP2 in mouth High expression in chamber squamous cell carcinoma.XIRP2 can be used as the independentpredictor of oral squamous cell carcinoma, can also be with other bases Because of marker use in conjunction.

Term " biomarker " is its expression and normal or healthy cell in tissue or cell in the present invention Or the expression of tissue compares any gene or albumen to change.

It would be recognized by those skilled in the art that practicability of the invention is not limited to marker gene of the invention The gene expression of any specific variants is quantified.As unrestricted example, marker gene can have SEQ ID NO.1 Or coded sequence or amino acid sequence that SEQ ID NO.2 is specified.In some embodiments, have with listed sequence extremely Few 85% the same or similar cDNA sequence or amino acid sequence, such as above-mentioned listed sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% the same or similar cDNA sequence or amino acid sequence.

Biomarker as described herein includes gene and albumen.Such biomarker includes marking containing encoding human Complete or partial sequence the DNA of the complementary series of the nucleic acid sequence of object or this sequence.Biomarker nucleic acid further include containing Complete or partial sequence the RNA of any nucleic acid sequence of interest.Biomarker protein is by DNA biology mark of the invention Remember object coding or corresponding to DNA biomarker of the invention albumen.Biomarker protein includes any biomarker The complete or partial amino-acid series of object albumen or polypeptide.The segment and variant of biomarker gene and albumen are also included within this In the range of invention.So-called " segment " refer to polynucleotides a part or amino acid sequence and thus encode one of albumen Point.For the segment of biomarker nucleotide sequence polynucleotides generally comprise at least 10,15,20,50,75,100,150, 200,250,300,350,400,450,500,550,600,650,700,800,900,1,000,1,100,1,200,1,300 or 00 continuous nucleotide of Isosorbide-5-Nitrae, or the nucleotide being at most present in overall length biomarker polynucleotides disclosed herein Number.The segment of biomarker polynucleotides will usually encode at least 15,25,30,50,100,150,200 or 250 continuously Amino acid, or the sum of amino acid being present in overall length biomarker protein of the invention." variant " is intended to indicate that substantially Upper similar sequence.In general, the variant of biomarker-specific object of the invention will have is measured by alignment programs With the biomarker at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.

The present invention can use any method known in the art measurement gene expression.Those skilled in the art should manage Solution, the means for measuring gene expression are not importances of the invention.It can be detected on transcribing or translating (i.e. albumen) level The expression of biomarker.

In some embodiments, the expression of biomarker is detected on transcriptional level.Hybridize skill using nucleic acid A variety of methods that art carries out specific DNA and RNA measurement are known to the skilled in the art.Certain methods are related to being separated by electrophoresis (for example, the Southern trace for detecting DNA and Northern trace for detecting RNA), but can also be unfavorable With the measurement (for example, passing through Dot blot) for carrying out DNA and RNA in the case where electrophoretic separation.Genomic DNA is (for example, come from People) Southern trace can be used for screening restriction fragment length polymorphism (RFLP), influence polypeptide of the present invention to detect The presence of inherited disorder.Can detecte the RNA of form of ownership, including but not limited to mRNA (mRNA), microRNA (miRNA), RRNA (rRNA) and transfer RNA (tRNA).

Pharmaceutical composition in the present invention further includes pharmaceutically acceptable carrier, and this kind of carrier includes (but and unlimited In): diluent, excipient such as lactose, sodium chloride, glucose, urea, starch, water etc., filler such as starch, sucrose etc.；Bonding Agent such as simple syrup, glucose solution, starch solution, cellulose derivative, alginates, gelatin and polyvinylpyrrolidone；It is wet Agent such as glycerol；Disintegrating agent such as dried starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate；It absorbs Promotor quaternary ammonium compound, lauryl sodium sulfate etc.；Surfactant such as polyoxyethylene sorbitan aliphatic ester, 12 Sodium alkyl sulfate, glyceryl monostearate, hexadecanol etc.；Humectant such as glycerol, starch etc.；Absorption carrier for example starch, lactose, Bentonite, silica gel, kaolin and soap clay etc.；Lubricant such as talcum powder, calcium stearate and magnesium, polyethylene glycol, boric acid powder etc..

Pharmaceutical composition of the invention can be used different additives and be prepared, such as buffer, stabilizer, antibacterial Agent, isotonic agent, chelating agent, pH controlling agent and surfactant.

Buffer may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and corresponding salt (their alkali metal or alkali Property rare earth metal salt, such as sodium salt, sylvite, calcium salt and magnesium salts).Isotonic agent includes potassium chloride, sodium chloride, sugar and glycerol.Chelating Agent includes sodium ethylene diamine tetracetate and citric acid.Bacteriostatic agent includes but is not limited to the benzylalcohol of effective concentration (such as < 1%w/v), benzene Phenol, metacresol, methaform, methyl p-hydroxybenzoate and/or propylparaben.Stabilizer includes human serum egg White, l-amino acid, sugar and cellulose derivative.L-amino acid can also include appointing in glycine, cysteine and glutamic acid Meaning one.Carbohydrate includes monosaccharide, such as glucose, mannose, galactolipin, fructose etc.；Sugar alcohol, such as mannitol, inositol, wood Sugar alcohol etc.；Disaccharides, such as sucrose, maltose, lactose etc.；Polysaccharide, for example, glucan, hydroxypropul starch, vulcanization chondroitin, thoroughly Bright matter acid etc. and their derivative.Cellulose derivative includes methylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxyl Propyl cellulose, hypromellose and sodium cellulose glycolate.Surfactant includes ion or non-ionic surface active Agent, such as polyethylene glycol oxide Arrcostab, sorbitan monoacyl ester, fatty glyceride.

Drug of the invention may also include pharmaceutically acceptable coating material, fast decoupled coating Material, coloring agent, enteric polymer, plasticizer, water-soluble polymer, insoluble polymer, dyestuff, pigment, other collapse Powder.Common fast decoupled coating material includes OPADRY；Enteric polymer includes methylacrylic acid polymer, phosphorus hydroxypropyl first Base cellulose phthalate, hypromellose acetic acid esters, hypromellose succinate, hydroxyl first ethyl cellulose Element, cellulose acetophthalate；Plasticizer includes polyethylene glycol (PEG), propylene glycol etc..

Pharmaceutical composition Orally-administrable of the present invention, parenteral administration are given by sucking spray delivery, part Medicine, rectally, nasal administration, cheek administration, vagina administration are administered by the storage medicine device of implantation.It is preferred that oral administration or injection Administration.Pharmaceutical composition of the present invention contains any commonly employed nontoxic pharmaceutical acceptable carrier, auxiliary material or excipient.In certain situations Under, medicinal acid, alkali or buffer can be used to adjust the pH of preparation to improve the stabilization of prepared compound or its form of administration Property.Terms used herein parenteral route include subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, in breastbone, In bringing up, in damage location and intracranial injection or infusion techniques.As long as destination organization can be reached, pharmaceutical composition of the present invention Receptor can be given by any approach.

Pharmaceutical composition of the present invention can be administered orally in the form of any peroral dosage form, including but not limited to capsule, piece Agent, emulsion and water slurry, dispersing agent and solution.For oral tablet, common carrier includes lactose and cornstarch.It is general to go back Lubricant such as magnesium stearate is added.In order to be administered with capsules per os, applicable diluent includes lactose and anhydrous corn Starch.When water slurry and/or lotion is administered orally, active component can be suspended or dissolved in oily phase, and and emulsifier And/or suspending agent merges.If necessary, some sweeteners and/or corrigent and/or colorant can be added.Where appropriate, can The dosage unit preparations packet micro-capsule that will be used to be administered orally.For example, by the way that particulate matter is coated or is wrapped in polymer, wax etc. It buries, the preparation can also be prepared and extended or maintained release.Pharmaceutical composition of the invention can be used for supplementing endogenic The missing or deficiency of XIRP2 albumen, by improve XIRP2 albumen expression or enhance XIRP2 albumen function, thus treat because Oral squamous cell carcinoma caused by XIRP2 albumen is reduced.

Drug of the invention can also be with the drug combination of other treatment oral squamous cell carcinoma, and other therapeutic compound can To be administered simultaneously with main active constituent, or even it is administered simultaneously in same composition.Can also with individual composition or The dosage form different from main active constituent individually gives other therapeutic compounds.The Fractional of main component can be with It is administered simultaneously with other therapeutic compounds, and other dosage can be administered alone.It over the course for the treatment of, can be according to symptom The physiologic response of severity, the frequency of recurrence and therapeutic scheme adjusts the dosage of pharmaceutical composition of the present invention.

Pharmaceutical composition of the present invention can also be administered in the form of Liposomal delivery systems, such as small monolayer vesicle, big list Layer vesica and multi-layer vesicles.Liposome can be formed there are many phosphatide, such as cholesterine, stearic amine or phosphatidyl choline.

Pharmaceutical composition of the present invention can be configured to ointment, cream, suspension with the pharmaceutical composition of local administration Agent, lotion, powder, solution, paste, gelling agent, spray, aerosol or finish.

The carrier of the present invention for carrying gene is various carriers known in the art, such as commercially available carrier including plasmid, Clay, bacteriophage, virus etc..

Term " probe ", which refers to, in the present invention to divide in conjunction with the particular sequence of another molecule or subsequence or other parts Son.Unless otherwise indicated, " probe " is often referred to match and another polynucleotides (often referred to as " target multicore by complementary base Thuja acid ") combine polynucleotide probes.According to the preciseness of hybridization conditions, probe energy and with the probe lack sufficient sequence it is mutual The target polynucleotide of benefit property combines.Probe can make direct or indirect label, and range includes primer.Crossing system, including, but It is not limited to: solution phase, solid phase, mixed phase or in situ hybridization measuring method.

As probe, fluorescent marker, radio-labeled, biotin labeling etc. can be used, cancer detection is carried out with polynucleotides The label probe of label.Labeling method of polynucleotides itself is well known.Can check by the following method in sample whether There are subject nucleic acids: fixed subject nucleic acid or its amplified matter are hybridized with label probe, are washed, and then measurement with The label of solid phase binding.Alternatively, cancer detection polynucleotides can be also fixed, subject nucleic acid is hybrid with it, then application mark The detections such as note probe are incorporated into the subject nucleic acid in solid phase.In this case, the cancer detection multicore glycosides being incorporated into solid phase Acid is also referred to as probe.It the use of the method for polynucleotide probes measurement subject nucleic acid in this field is also well known.Can as follows into Row this method: connect polynucleotide probes and subject nucleic acid (preferably within ± 4 DEG C) at or near Tm Touching is washed, the label probe or the template nucleic acid in conjunction with solid phase probe for then measuring hybridization for hybridizing.

The polynucleotides used as probe be preferably sized to 18 or more nucleotide, more preferably 20 or The overall length or less of more nucleotide and coding region.As primer in use, the polynucleotides are preferably sized to 18 Or more nucleotide and 50 or more Oligonucleotide.

Term " chip " is also referred to as " array ", refers to the solid support of the nucleic acid comprising connection or peptide probes.Array is usual A variety of different nucleic acid or peptide probes comprising being connected to substrate surface according to different known locations.These arrays, also referred to as " microarray " can use mechanical synthesis methods or light guidance synthetic method usually to generate these arrays, and the light guidance is closed The combination of photolithography method and solid phase synthesis process is incorporated at method.Array may include flat surface, or can be pearl Son, gel, polymer surfaces, the fiber of such as optical fiber, glass or nucleic acid or peptide in any other suitable substrate.It can be with Certain mode carrys out array of packages, to allow to carry out the manipulation of diagnosis of global function device or other means.

In the present invention, term " antibody " refers to the natural or synthetic antibody of selective binding target antigen.The term packet Include polyclonal and monoclonal antibody.Other than complete immunoglobulin molecules, the segment of those immunoglobulin molecules or poly- It is " anti-that the mankind or humanization form for closing the immunoglobulin molecules of object and selective binding target antigen are also included within term In the range of body ", as long as they show desired biological activity." monoclonal antibody " refers to from a group substantially homogeneity Antibody obtain antibody, i.e., composition group each antibody it is identical and/or combine same epitope, in addition to produce monoclonal antibody During outside issuable possible variant, such variant is generally with indivisible presence.Such monoclonal antibody is typically wrapped The antibody comprising the polypeptide sequence in conjunction with target is included, wherein target combination polypeptide sequence is by including in the more peptide sequence of comforming The process including single target combination polypeptide sequence is selected to obtain.

Monoclonal antibody further includes " chimeric " antibody, wherein a part of heavy chain and/or light chain be derived from particular species Or the corresponding sequence belonged in the antibody of specific antibodies classification or subclass is identical or homologous, and the remainder of chain with derived from another One species belong to that corresponding sequence in the antibody of another antibody isotype or subclass is identical or homologous and the piece of such antibody Section, as long as they show desired biological activity.

Polyclonal antibody includes that antibody obtained by XIRP2 protein is immunized to the animal (for example, mouse) for generating human antibody. After being prepared for chimeric antibody or humanized antibody, the amino acid in variable region (for example, FR) and/or constant region can be used Other amino acid substitutions etc..

The replacement of amino acid is, for example, less than 15, less than 10,8 or less, 7 or less, 6 or less, 5 or less, 4 A following, 3 or less or 2 amino acid, preferably 1~5 amino acid, more preferable 1 or 2 amino acid below replacement, is replaced Changing antibody should functionally be equal with antibody is not replaced.It is expected that replacement is conservative amino acid substitution, this is charge, side chain, pole Replacement between the kin amino acid such as property, aromatic series.Kin amino acid can for example be classified as alkaline ammonia Base acid (arginine, lysine, histidine), acidic amino acid (aspartic acid, glutamic acid), uncharged polar amino acid (sweet ammonia Acid, asparagine, glutamine, serine, threonine, cysteine, tyrosine), apolar amino acid it is (leucine, different bright Propylhomoserin, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched-chain amino acid it is (leucine, valine, different Leucine), aromatic amino acid (phenylalanine, tyrosine, tryptophan, histidine) etc..

Term " treatment " refers to for the purpose of healing, improvement, stabilization or prevention disease, pathological state or illness in the present invention The medical supervision that patient is carried out.The term includes active treatment, i.e., specially for the purpose of improving disease, pathological state or illness Treatment, and further include etiological treatment, i.e. the treatment for the purpose of removing related disease, pathological state or the cause of disease of illness. In addition, the term further includes palliative treatment, i.e., controlling for disease, pathological state or illness is cured designed for alleviating symptom It treats；Prophylactic treatment, i.e., utmostly to reduce or partially or completely inhibit the development of related disease, pathological state or illness For the purpose for the treatment of；And supportive treatment, i.e., for supplementing another kind to improve related disease, pathological state or illness as mesh Specific therapy treatment.

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

Embodiment 1 screens gene marker relevant to oral squamous cell carcinoma

1, sample collection

6 surrounding normal mucosal tissues and oral squamous cell carcinoma are respectively collected, is confirmed through pathological diagnosis, is owned Patient is preoperative not to receive any type for the treatment of.The sample cut of performing the operation freezes in liquid nitrogen, the equal informed consent of patient, above-mentioned institute There is the acquirement of sample to pass through the agreement of the committee, organizational ethics.

2, the preparation (being operated using the tissue RNA extracts kit of QIAGEN) of RNA sample

Taking-up freezes the tissue samples in liquid nitrogen, and tissue samples are put into the mortar being pre-chilled and are ground, according to Specification in kit extracts separation RNA.It is specific as follows:

1) Trizol is added, is placed at room temperature for 5min；

2) chloroform 0.2ml is added to be mixed well with forced oscillation centrifuge tube, places 5-10min at room temperature；

3) 12000rpm is centrifuged 15min, and upper strata aqueous phase is moved on in another new centrifuge tube and (is careful not to be drawn onto two layers of water Protein substance between phase), the isopropanol of -20 DEG C of isometric pre-coolings is added, is sufficiently mixed by inversion, is placed in 10min on ice；

4) 12000rpm high speed is added 75% in the ratio of 1ml/ml Trizol from supernatant is carefully discarded after 15min DEPC ethanol washing precipitating (4 DEG C of preservations), washing precipitate, oscillation mixes, and 4 DEG C, 12000rpm is centrifuged 5min；

5) ethanol liquid is discarded, places 5min at room temperature, DEPC water dissolution precipitating is added；

6) it with Nanodrop2000 ultraviolet specrophotometer measurement RNA purity and concentration, freezes in -70 DEG C of refrigerators.

3, reverse transcription and label

With Low RNA Input Linear Amplification Kit by mRNA reverse transcription at cDNA, while using Cy3 Labelling experiment group and control group respectively.

4, hybridize

Genetic chip uses people's full-length genome chip of expression spectrum of Aglient company, and every chip includes 45015 few cores Thuja acid, wherein having 43376 people's gene probes and 1639 experiment control probes.It is carried out by the step of chip operation instructions, Temperature rolls through 17h 10r/min and hybridizes, 37 DEG C are developed a film at 65 DEG C.

5, data processing

Chip Agilent scanner scanning after hybridization, resolution ratio are 5 μm, and scanner is automatically with 100% and 10%PMT Each scanning 1 time, 2 times result Agilent software merges automatically.Scan image data is using at Feature Extraction Reason analysis, obtained initial data application Bioconductor program bag carry out follow-up data processing.Last Ratio value is experiment Group and control group.Differential gene screening criteria: ratio >=4 are up-regulation gene, and ratio≤0.25 is down-regulated gene.

6, result

Compared with normal mucosa tissue, expression quantity of the XIRP2 gene in oral squamous cell carcinoma is significantly raised.

The differential expression of 2 QPCR sequence verification XIRP2 gene of embodiment

1, large sample QPCR verifying is carried out to XIRP2 gene differential expression.It is selected according to the sample collection mode in embodiment 1 Select normal mucosa tissue and oral squamous cell carcinoma each 80.

2, RNA extraction step is as described in Example 1.

3, reverse transcription:

1) reaction system:

1 reverse transcription reaction system of table

2) reverse transcription reaction condition

It is carried out according to reverse transcription reaction condition in RNA PCR Kit (AMV) Ver.3.0.

42 DEG C of 60min, 99 DEG C of 2min, 5 DEG C of 5min.

3) polymerase chain reaction

1) design of primers

QPCR amplimer is designed according to the coded sequence of XIRP2 gene and GAPDH gene in Genebank, by Bo Maide Biotech firm's synthesis.Specific primer sequence is as follows:

XIRP2 gene:

Forward primer is 5 '-GCAGCCTTATCTACAGTC-3 ' (SEQ ID NO.3)；

Reverse primer is 5 '-TCTTCCTTCCTTCCTTCT-3 ' (SEQ ID NO.4).

The primer sequence of house-keeping gene GAPDH are as follows:

Forward primer: 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQ ID NO.5)

Reverse primer: 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6)

2) 25 μ l PCR reaction systems are prepared according to table 1:

2 PCR reaction system of table

3) PCR reaction condition: 94 DEG C of 4min, (94 DEG C of 20s, 60 DEG C of 30s, 72 DEG C of 30s) × 30 circulations.With SYBR Green carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, is analyzed by melt curve analysis as fluorescent marker Determine that purpose band, Δ Δ CT method carry out relative quantification with electrophoresis, each sample carries out 3 repetitions and tests.

5, statistical method

Using GAPDH as internal reference, the reality of oral squamous cell carcinoma and normal mucosa histofluorescence quantitative RT-PCR is calculated It tests as a result, using SPSS18.0 statistical software come for statistical analysis, difference between the two is examined using t, with the tool of P < 0.05 There is statistical difference.

6, result

As a result as shown in Figure 1, compared with surrounding normal mucosal tissue, XIRP2 gene is in oral squamous cell carcinoma Expression up-regulation, difference has statistical significance (P < 0.05), consistent with RNA-sep result.

Differential expression of the 3 XIRP2 gene of embodiment in oral squamous cell carcinoma cell line

1, cell culture

Oral squamous cell carcinoma cell line Tca8113, HN13, normal mucosa epithelial cell line HIOEC are handed over purchased from Shanghai Logical attached 9th the People's Hospital, university.The culture medium of HIOEC is K-SFM；The culture medium of Tca8113, HN13 are DMEM；To contain The culture medium of 10% fetal calf serum and 1%P/S are in 37 DEG C, 5%CO₂, relative humidity be 90% incubator in cultivate.2-3 days It changes liquid 1 time, trypsase conventional digestion passage of the use 0.25% containing EDTA.

2, the extraction of cell total rna

1) culture is terminated when cell is merged up to 80-90%, 0.25% trypsin digestion is collected cell and managed in 1.5m1EP In, lm1Trizol is added in every pipe and slowly shakes smudge cells, places 10min on ice.

2) deproteinized removes DNA: 0.2ml chloroform is added in each 1.5m1EP pipe, rocks 15s, is placed at room temperature for 10min.4 DEG C, 12000rpm is centrifuged 15min.

Remaining operation step is the same as RNA extraction process in tissue.

3, reverse transcription

Specific steps are the same as embodiment 2.

4, statistical method

Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS18.0 statistical software come for statistical analysis, difference between the two is examined using t, it is believed that is had as P < 0.05 It is statistically significant.

5, result

As a result as shown in Fig. 2, compared with normal mucosa epithelial cell, XIRP2 gene is in oral squamous cell carcinoma cell It expresses in Tca8113, HN13 and raises, difference has statistical significance (P < 0.05), consistent with RNA-sep result.

The silencing of 4 XIRP2 gene of embodiment

1, cell culture

Human mouth epidermoid carcinoma cell strain Tca8113 is existed with the culture medium DMEM containing 10% fetal calf serum and 1%P/S 37 DEG C, 5%CO₂, relative humidity be 90% incubator in cultivate.It changes within 2-3 days liquid 1 time, pancreas egg of the use 0.25% containing EDTA White enzyme conventional digestion passage.

2, siRNA is designed

For the siRNA sequence of XIRP2 gene:

Negative control siRNA sequence (siRNA-NC):

Positive-sense strand is 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.7),

Antisense strand is 5 '-ACGUGACACGUUCGGAGAA-3 ' (SEQ ID NO.8)；

SiRNA1-XIRP2:

Positive-sense strand is 5 '-UGUUGAUAUUGGUAUUGAGCA-3 ' (SEQ ID NO.9),

Antisense strand is 5 '-CUCAAUACCAAUAUCAACAUC-3 ' (SEQ ID NO.10)；

SiRNA2-XIRP2:

Positive-sense strand is 5 '-AUAACAUCCUCUUUCUGACAA-3 ' (SEQ ID NO.11),

Antisense strand is 5 '-GUCAGAAAGAGGAUGUUAUAG-3 ' (SEQ ID NO.12)；

Cell is pressed 4 × 10⁴/ hole is inoculated into six porocyte culture plates, in 37 DEG C, 5%CO₂Cell culture in incubator For 24 hours, in DMEM culture medium without double antibody, containing 10%FBS, transfection (is purchased from according to lipofectamine 2000 Invitrogen company) specification transfection.

Experiment is divided into three groups: control group (Tca8113), negative control group (siRNA-NC) and experimental group (siRNA1- XIRP2, siRNA2-XIRP2), wherein the sequence of negative control group siRNA and XIRP2 gene is without homology, concentration 20nM/ Hole, while being transfected respectively.

3, QPCR detects the transcriptional level of XIRP2 gene

The extraction of 3.1 cell total rnas

Specific steps are the same as embodiment 3.

3.2 reverse transcription steps are the same as embodiment 2.

3.3 QPCR amplification steps are the same as embodiment 2.

4, statistical method

Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD, Using SPSS18.0 statistical software come for statistical analysis, the difference between XIRP2 gene expression panel and control group is interfered to adopt It is examined with t, it is believed that there is statistical significance as P < 0.05.

5, result

As a result such as Fig. 3 is shown, compared to TCA8113, transfection zero load siRNA-NC, siRNA2-XIRP2 group, siRNA1- XIRP2 group can significantly reduce the expression of XIRP2 gene, and difference has statistical significance (P < 0.05).

5 ELISA of embodiment detects the protein expression of XIRP2 in Tca8113 cell

Using double-antibody sandwich enzyme-labeled immunity (Enzyme-Linked Immunosorbent Assay, ELISA) analytic approach Measure XIRP2 protein level in Tca8113 cell conditioned medium.The 6th day after RNA interference, three groups of Tca8113 cells are collected respectively Supernatant, according to the concentration of XIRP2 in ELISA kit operating process quantitative detection tumour cell supernatant.

1, configuration concentration is that the standard items of 70000pg/ml then carry out 2 times of doubling dilutions after 10 times of dilutions, share 7 it is dilute Degree of releasing.

2, it is loaded: setting blank well, gauge orifice, sample to be tested hole respectively.Blank well adds 50 μ l of sample diluting liquid, remaining hole difference Add the standard items and each 50 μ l of sample to be tested of various concentration gradient.Mixing is shaked gently, ELISA Plate is plus lid, 37 DEG C of reaction 2h.

3, liquid is discarded, is dried.Every hole adds 200 μ l VEGF-C conjugates.37 DEG C, after 120min, liquid in hole is discarded, It dries, PBS board-washing 3 times.

4, sequentially every hole adds 200 μ l of substrate solution, and 37 DEG C are protected from light colour developing 30min.

5, sequentially every hole adds 50 μ l of stop bath, terminates reaction.

6, the optical density (OD value) in each hole is sequentially measured in 450nm wavelength with enzyme-linked instrument.All standard items and sample to be tested OD value be both needed to subtract the OD value in zero hole to obtain corrected value.

7, the actual concentrations of sample are calculated.

8, result is as shown in table 3 below, the XIRP2 gene of siRNA silencing Tca8113 cell, the protein content of XIRP2 also phase It should reduce, illustrate that silencing XIRP2 gene can inhibit the expression of XIRP2 gene.

3 ELISA of table detects the expression of the XIRP2 albumen in different group cells

6 mtt assay of embodiment detects Tca8113 cell-proliferation activity

It is heavy using MTT (Methyl thiazolyl tetrazolium, methyl thiazolyl tetrazolium) method detection XIRP2 gene Influence after silent to Tca8113 cell-proliferation activity.

1, Tca8113 cell is pressed 1 × 10 by cell culture³/ hole is inoculated in 96 orifice plates, and every 1,37 DEG C of 100 μ of hole, 5%CO₂It incubates Culture is incubated in case.

2, cell transfecting step is the same as embodiment 3.

3, MTT is detected

1) when transfecting 1~7 day, each hole culture medium is discarded, MTT (5mg/ml) 20 μ l is added.Continue routine culture 4h。

2) mixed liquor is sucked, 200 μ l of DMSO is added in every hole, and concussion 10min makes to crystallize abundant dissolution.In enzyme linked immunological instrument The upper absorbance value surveyed at 490nm, records result.

4, using the time as horizontal axis, absorbance value (OD) is that the longitudinal axis draws cell growth curve.

5, result:

As a result as shown in figure 4, compared with the control, the cell Proliferation of transfection siRNA1-XIRP2 group significantly reduces.

7 Transwell cells in vitro Matrigel of embodiment

Different groups of other Tca8113 cells are collected within the 6th day after RNA interference, is resuspended in culture solution, makes final concentration of cells It is 2 × 10⁶/ ml draws 100 μ l cell suspensions and is added in the cell Transwell.It is observed using the cell Transwell method Influence of the XIRP2 gene silencing to Tca8113 cellular invasiveness.

1, Matrigel (4 μ g/ μ l) is put into 4 DEG C of thawings, prepared ice chest (ice bath environment).Matrigel is diluted with DMEM It is used after 8 times.In the cell Transwell, the outer surface of filter membrane (8 μm of apertures) applies 8 μ g people's fibronectin, sets wind in super-clean bench It is dry.

2, it spreads in the inner surface of the 6 hole cell Transwell filter membranes with the Matrigel glue in 1/ hole 100 μ, 37 DEG C, 5%CO₂ It is incubated for 1h in incubator, it is spare to form a matrix barrier layer.

3, the DMEM culture solution 2.5m1 containing 20%FBS is added in the every hole of 6 orifice plates.

4, the cell for collecting logarithmic growth phase, is resuspended in culture solution, final concentration of 2 × 10⁶/ml。

5, cell suspension is added in the cell Transwell, cell is dipped in the conditioned medium of 6 orifice plates by every 100 μ 1 of hole In, 37 DEG C, 5%CO₂It is incubated for for 24 hours in incubator.

6, the cell Transwell is taken out, filter membrane fixes 1 minute with methanol.

7, HE is dyed: hematoxylin dyes 3min, washing；10~30s of eosin stains, washing.And it is wiped and is not passed through with cotton swab The cell of film.

8, microscopically observation, photograph and invasion cell number is counted, every film counts in up and down the saturating of 5 different visuals field Cell number is crossed, average value is calculated.Every group sets 3 filter membranes in parallel.

9, data processing

Statistical analysis is carried out to data with SPSS18.0 software.Measurement data is indicated with mean ± standard deviation.Multiple samples This mean compares using one-way analysis of variance, and P < 0.05 is that difference is statistically significant.

10, result

As a result as shown in figure 5, Tca8113, siRNA1-XIRP2, siRNA-NC group cell are trained in the cell transwell After supporting for 24 hours, the cell number in room face is substantially reduced under siRNA1-XIRP2 group polycarbonate membrane.

The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.

daSEQUENCE LISTING

<110>Beijing Yang Shen biology information technology Co., Ltd

<120>oral squamous cell carcinoma biomarker and its application

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 2817

<212> DNA

<213>source of people

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atgttcccaa tgcagaaggg ctccctcaac ctcctgaggc agaaatggga atcttgtgat 60

tatcagagaa gtgagtgtca tcccagggac agccattgta caattttcca gcctcaggaa 120

agcaaattgc ttgcgcctga aggagaggta gtatcagcac ctcaatcttt ggatcccaca 180

agtctgccct acagtacagg ggaagagatg tggagttcga agccggaaga gaaggattct 240

gtggacaaga gtaacaacac cagggaatat ggtcggccag aagtgctgaa ggaggattcc 300

ctgagcagtc ggcgcaggat tgaacgcttt tccattgccc ttgatgagct gaggagtgtg 360

tttgaggctc ctaagagtgg aaacaaacca gctgagtacg gtggaaagga agtggaaatt 420

gagcgaagtt tgtgctcgcc agcttttaag agtcaccctg ggagccagct ggaggattct 480

gtgaaagatt cagacaagaa aggcaaggaa acatcttttg acaagatgtc acctgaaagt 540

ggtcacagcc gcatctttga agcgactgct ggccctaata agcctgagag tggatttgca 600

gaagacagtg ctgctcgggg cgagggtgtg tcagacctcc acgaagtggt ctccctgaag 660

gagcggatgg cgaggtacca ggcagctgtt tccaggggtg actgccgcag cttctctgct 720

aatatgatgg aagaatcaga aatgtgcgca gtgcctggtg gtttggccaa ggtgaagaaa 780

caatttgagg acgaaattac ttcttcccgt aatacctttg ctcaatacca atatcaacat 840

cagaacagat ctgagcagga ggcaattcat agcagccagg ttggcacttc aagaagcagc 900

caggaaatgg caagaaatga acaagaaggg tccaaagtac agaaaattga tgttcatgga 960

acagaaatgg tctctcatct tgaaaagcac accgaggaag taaaccaagc atctcagttt 1020

catcaatatg ttcaagaaac tgtcattgat acacctgagg atgaagagat tccaaaggtt 1080

tcgactaagt tgttaaaaga gcagtttgaa aagtctgccc aggaaaagat cctttattct 1140

gacaaagaga tgacaacccc agccaagcag attaagaagc tgctgctcca agacaaggaa 1200

atatgtatac tttgtcaaaa gacagtttat ccaatggagt gcctagtggc agacaagcag 1260

aattttcata agtcctgctt ccgatgccac cattgcaaca gtaaactaag tttgggaaat 1320

tatgcatcac ttcatggaca aatatactgt aaacctcact ttaaacaact tttcaaatcc 1380

aaaggaaatt atgatgaagg ttttggacat aagcagcata aagatagatg gaactgcaaa 1440

aaccaaagca gatcagtgga ctttattcct aatgaagaac caaatatgtg taaaaatatt 1500

gcagaaaaca cccttgtacc tggagatcgt aatgaacatt tagatgctgg taacagtgaa 1560

gggcaaagga atgatttgag aaaattaggg gaaaggggaa aattaaaagt catttggcct 1620

ccttccaagg agatccctaa gaaaacctta ccctttgagg aagagctcaa aatgagtaaa 1680

cctaagtggc cacctgaaat gacaaccctg ctatcccctg aatttaaaag tgaatctctg 1740

ctagaagatg ttagaactcc agaaaataaa ggacaaagac aagatcactt tccatttttg 1800

cagccttatc tacagtccac ccatgtttgt cagaaagagg atgttatagg aatcaaagaa 1860

atgaaaatgc ctgaaggaag aaaagatgaa aagaaggaag gaaggaagaa tgtgcaagat 1920

aggccgagtg aagctgaaga cacaaagagt aacaggaaaa gtgctatgga tcttaatgac 1980

aacaataatg tgattgtgca gagtgctgaa aaggagaaaa atgaaaaaac taaccaaact 2040

aatggtgcag aagttttaca ggttactaac actgatgatg agatgatgcc agaaaatcat 2100

aaagaaaatt tgaataagaa taataataac aattatgtag cagtctcata tctgaataat 2160

tgcaggcaga agacatctat tttagaattt cttgatctat tacccttgtc gagtgaagca 2220

aatgacactg caaatgaata tgaaattgag aagttagaaa atacatctag aatctcagag 2280

ttacttggta tatttgaatc tgaaaagact tattcgagga atgtactagc aatggctctg 2340

aagaaacaga ctgacagagc agctgctggc agtcctgtgc agcctgctcc aaaaccaagc 2400

ctcagcagag gccttatggt aaagggggga agttcaatca tctctcctga tacaaatctc 2460

ttaaacatta aaggaagcca ttcaaagagc aaaaatttac actttttctt ttctaacacc 2520

gtgaaaatca ctgcattttc caagaaaaat gagaacattt tcaattgtga tttaatagat 2580

tctgtagatc aaattaaaaa tatgccatgc ttggatttaa gggaatttgg aaaggatgtt 2640

aaaccttggc atgttgaaac aacagaagct gcccgcaata atgaaaacac aggttttgat 2700

gctctgagcc atgaatgtac agctaagcct ttgtttccca gagtggaggt gcagtcagaa 2760

caactcacgg tggaagagca gattaaaaga aacaggtgct acagtgacac tgagtaa 2817

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Glu Val Val Ser Ala Pro Gln Ser Leu Asp Pro Thr Ser Leu Pro Tyr

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Ser Thr Gly Glu Glu Met Trp Ser Ser Lys Pro Glu Glu Lys Asp Ser

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Val Asp Lys Ser Asn Asn Thr Arg Glu Tyr Gly Arg Pro Glu Val Leu

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Ala Leu Asp Glu Leu Arg Ser Val Phe Glu Ala Pro Lys Ser Gly Asn

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Lys Pro Ala Glu Tyr Gly Gly Lys Glu Val Glu Ile Glu Arg Ser Leu

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Cys Ser Pro Ala Phe Lys Ser His Pro Gly Ser Gln Leu Glu Asp Ser

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Val Lys Asp Ser Asp Lys Lys Gly Lys Glu Thr Ser Phe Asp Lys Met

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Ser Pro Glu Ser Gly His Ser Arg Ile Phe Glu Ala Thr Ala Gly Pro

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Asn Lys Pro Glu Ser Gly Phe Ala Glu Asp Ser Ala Ala Arg Gly Glu

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Gly Val Ser Asp Leu His Glu Val Val Ser Leu Lys Glu Arg Met Ala

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Arg Tyr Gln Ala Ala Val Ser Arg Gly Asp Cys Arg Ser Phe Ser Ala

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Asn Met Met Glu Glu Ser Glu Met Cys Ala Val Pro Gly Gly Leu Ala

245 250 255

Lys Val Lys Lys Gln Phe Glu Asp Glu Ile Thr Ser Ser Arg Asn Thr

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Phe Ala Gln Tyr Gln Tyr Gln His Gln Asn Arg Ser Glu Gln Glu Ala

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Ile His Ser Ser Gln Val Gly Thr Ser Arg Ser Ser Gln Glu Met Ala

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Arg Asn Glu Gln Glu Gly Ser Lys Val Gln Lys Ile Asp Val His Gly

305 310 315 320

Thr Glu Met Val Ser His Leu Glu Lys His Thr Glu Glu Val Asn Gln

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Ala Ser Gln Phe His Gln Tyr Val Gln Glu Thr Val Ile Asp Thr Pro

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Glu Asp Glu Glu Ile Pro Lys Val Ser Thr Lys Leu Leu Lys Glu Gln

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Phe Glu Lys Ser Ala Gln Glu Lys Ile Leu Tyr Ser Asp Lys Glu Met

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Thr Thr Pro Ala Lys Gln Ile Lys Lys Leu Leu Leu Gln Asp Lys Glu

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Ile Cys Ile Leu Cys Gln Lys Thr Val Tyr Pro Met Glu Cys Leu Val

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Ala Asp Lys Gln Asn Phe His Lys Ser Cys Phe Arg Cys His His Cys

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Asn Ser Lys Leu Ser Leu Gly Asn Tyr Ala Ser Leu His Gly Gln Ile

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Tyr Cys Lys Pro His Phe Lys Gln Leu Phe Lys Ser Lys Gly Asn Tyr

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Asp Glu Gly Phe Gly His Lys Gln His Lys Asp Arg Trp Asn Cys Lys

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Asn Gln Ser Arg Ser Val Asp Phe Ile Pro Asn Glu Glu Pro Asn Met

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Cys Lys Asn Ile Ala Glu Asn Thr Leu Val Pro Gly Asp Arg Asn Glu

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His Leu Asp Ala Gly Asn Ser Glu Gly Gln Arg Asn Asp Leu Arg Lys

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Leu Gly Glu Arg Gly Lys Leu Lys Val Ile Trp Pro Pro Ser Lys Glu

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Ile Pro Lys Lys Thr Leu Pro Phe Glu Glu Glu Leu Lys Met Ser Lys

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Pro Lys Trp Pro Pro Glu Met Thr Thr Leu Leu Ser Pro Glu Phe Lys

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Ser Glu Ser Leu Leu Glu Asp Val Arg Thr Pro Glu Asn Lys Gly Gln

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Arg Gln Asp His Phe Pro Phe Leu Gln Pro Tyr Leu Gln Ser Thr His

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Val Cys Gln Lys Glu Asp Val Ile Gly Ile Lys Glu Met Lys Met Pro

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Glu Gly Arg Lys Asp Glu Lys Lys Glu Gly Arg Lys Asn Val Gln Asp

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Arg Pro Ser Glu Ala Glu Asp Thr Lys Ser Asn Arg Lys Ser Ala Met

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Asp Leu Asn Asp Asn Asn Asn Val Ile Val Gln Ser Ala Glu Lys Glu

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Lys Asn Glu Lys Thr Asn Gln Thr Asn Gly Ala Glu Val Leu Gln Val

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Thr Asn Thr Asp Asp Glu Met Met Pro Glu Asn His Lys Glu Asn Leu

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Asn Lys Asn Asn Asn Asn Asn Tyr Val Ala Val Ser Tyr Leu Asn Asn

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Cys Arg Gln Lys Thr Ser Ile Leu Glu Phe Leu Asp Leu Leu Pro Leu

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Ser Ser Glu Ala Asn Asp Thr Ala Asn Glu Tyr Glu Ile Glu Lys Leu

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Glu Asn Thr Ser Arg Ile Ser Glu Leu Leu Gly Ile Phe Glu Ser Glu

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Lys Thr Tyr Ser Arg Asn Val Leu Ala Met Ala Leu Lys Lys Gln Thr

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Asp Arg Ala Ala Ala Gly Ser Pro Val Gln Pro Ala Pro Lys Pro Ser

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Leu Ser Arg Gly Leu Met Val Lys Gly Gly Ser Ser Ile Ile Ser Pro

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Asp Thr Asn Leu Leu Asn Ile Lys Gly Ser His Ser Lys Ser Lys Asn

820 825 830

Leu His Phe Phe Phe Ser Asn Thr Val Lys Ile Thr Ala Phe Ser Lys

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Lys Asn Glu Asn Ile Phe Asn Cys Asp Leu Ile Asp Ser Val Asp Gln

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Ile Lys Asn Met Pro Cys Leu Asp Leu Arg Glu Phe Gly Lys Asp Val

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Lys Pro Trp His Val Glu Thr Thr Glu Ala Ala Arg Asn Asn Glu Asn

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Thr Gly Phe Asp Ala Leu Ser His Glu Cys Thr Ala Lys Pro Leu Phe

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Pro Arg Val Glu Val Gln Ser Glu Gln Leu Thr Val Glu Glu Gln Ile

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Lys Arg Asn Arg Cys Tyr Ser Asp Thr Glu

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gcagccttat ctacagtc 18

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tcttccttcc ttccttct 18

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ggtggaatca tattggaaca 20

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uguugauauu gguauugagc a 21

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cucaauacca auaucaacau c 21

<210> 11

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auaacauccu cuuucugaca a 21

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Claims

Application of the 1.XIRP2 gene in the product of preparation diagnosis oral squamous cell carcinoma.
2. application according to claim 1, which is characterized in that the product include by chip, blotting, RT-PCR, The change of real-time quantitative PCR, FISH method, CGH method or array CGH method, bisulfite sequencing, COBRA method detection XIRP2 gene Change to diagnose the product of oral squamous cell carcinoma.
3. application according to claim 2, which is characterized in that described to diagnose oral squamous cell carcinoma with real-time quantitative PCR Product include at least the primer of a pair of of specific amplification XIRP2 gene, the primer such as SEQ ID NO.3 and SEQ ID Shown in NO.4.
4. application according to claim 1, which is characterized in that the product includes chip, kit or preparation.
Application of the 5.XIRP2 gene in the pharmaceutical composition of preparation treatment oral squamous cell carcinoma.
6. application according to claim 5, which is characterized in that described pharmaceutical composition includes XIRP2 gene and/or its table Up to the inhibitor of product.