CN106435002A

CN106435002A - OSCC (oral squamous cell carcinoma) biomarker and application thereof

Info

Publication number: CN106435002A
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: 2017-02-22
Anticipated expiration: 2036-12-12
Also published as: CN106435002B

Abstract

The invention discloses an OSCC (oral squamous cell carcinoma) biomarker and an application thereof. The biomarker is XIRP2, an experiment proves that an XIRP2 gene expresses up regulation in OSCC tissue, and the silent XIRP2 gene can reduce transcription and translation of the gene to inhibit excessive proliferation of OSCC cells. The invention provides the application of the gene or an expression product of the gene in diagnosis and treatment of OSCC.

Description

Oral squamous cell carcinoma biomarker and its application

Technical field

The invention belongs to biomedicine field, it is related to one kind in oral squamous cell carcinoma biomarker and its application, has The application being related in the notable XIRP2 raising of oral squamous cell carcinoma of body.

Background technology

Malignant tumor of mouth is the disease that a class seriously threatens human health.In malignant tumor of mouth, oral cavity squamous thin Born of the same parents' cancer (oral squamous cell carcinoma, OSCC) ratio accounts for 90%.As the generation of other tumors, OSCC Generation development be polygenes, polymolecular be in network structure, multi-step, multistage coefficient long-term complex process. Between each oncogene, molecule, mutual synergism promotes tumor cell formation and development.The at present mechanism of tumor and controlling Treat and all do not obtain important breakthrough, how early detection oncosises undermine radical cure is modern tumor worker's difficult problem and opportunity.

Tumor is a kind of disease of molecular level, and the research carrying out tumor from genes protein level is final approach.In recent years The genomics based on high flux molecule scanning means, proteomics and computer-aided design in integral level Provide new opportunity etc. the integration of technology and the application etc. of " Technology Chain " of being mutually related for the research of tumor, and exist Great successes are achieved in the research of breast carcinoma, pulmonary carcinoma, gastric cancer, colon cancer, ovarian cancer, melanoma etc..Wherein gene table Reaching spectrum chip is most typical representative, can detect and screen the difference of tumor tissues and normal structure by chip gene expression profile Allogene and oncogene, the change of antioncogene and variation relation, therefrom find the cause of disease, are early diagnosiss and radical cure offer line Rope and foundation.

Content of the invention

In order to make up the deficiencies in the prior art, it is an object of the invention to provide a kind of biological mark of oral squamous cell carcinoma Note thing, the sensitive and specific diagnosis realizing oral squamous cell carcinoma and treatment.

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

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

Further, described 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 product changing to diagnose oral squamous cell carcinoma of XIRP2 gene Product.

Wherein, blotting includes Northern, Southern, western blot method.Southern blotting is by from sample Originally the genomic DNA obtaining separates and fixes, and measures the XIRP2 base in sample by detecting DNA and the hybridization of XIRP2 gene Cause；Northern blotting be a kind of will be separated by the mRNA obtaining in sample, fixing, by detecting mRNA and XIRP2 gene The mRNA to detect this gene for the hybridization；Western blot method be a kind of by the Protein Separation in sample, fixation, by detection The expression degree to analyze gene for the immunoreation of antibody and XIRP2 albumen.

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

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

The invention provides a kind of product of diagnosis oral squamous cell carcinoma, described product can be by detecting in sample The expression of XIRP2 gene is diagnosing oral squamous cell carcinoma.

Further, described product includes chip, test kit or preparation.Wherein, described chip includes gene chip, protein Chip；Described gene chip includes solid phase carrier and the oligonucleotide probe being fixed on solid phase carrier, and described oligonucleotide is visited Pin includes the oligonucleotide probe for XIRP2 gene for detecting XIRP2 gene transcription level；Described protein chip bag The specific antibody including solid phase carrier and being fixed on the XIRP2 albumen of solid phase carrier；Described test kit includes gene test examination Agent box and protein immunization detection kit；Described gene detecting kit includes the examination for detecting XIRP2 gene transcription level Agent；Described protein immunization detection kit includes the specific antibody of XIRP2 albumen.

It is multiple including XIRP2 gene that gene chip of the present invention or gene detecting kit can be used for detection The expression of gene (for example, the multiple genes related to oral squamous cell carcinoma).Described protein chip or protein immunization inspection Test agent box can be used for multiple protein including XIRP2 albumen for the detection, and (for example related to oral squamous cell carcinoma is many Individual protein) expression.Multiple marks of oral squamous cell carcinoma are detected simultaneously, is greatly improved oral cavity squama The accuracy rate of shape cell carcinoma diagnosis.

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

Further, described pharmaceutical composition includes the inhibitor of XIRP2 gene and/or its expression product.Described inhibitor Including the material of suppression XIRP2 gene expression, the material of suppression XIRP2 gene expression product stability and/or suppression XIRP2 The material of gene expression product activity.

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

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

Present invention also offers a kind of pharmaceutical composition treating oral squamous cell carcinoma, described pharmaceutical pack base containing XIRP2 Cause and/or its expression product inhibitor.Described inhibitor includes material, the suppression XIRP2 gene table suppressing XIRP2 gene expression Reach the material of product stability and/or the material of suppression XIRP2 gene expression product activity.

Present invention also offers a kind of method of suppression cell proliferation, methods described is by for XIRP2 gene SiRNA, shRNA, antisense oligonucleotide or Loss-of-function gene import in tumor cell in vitro.

The invention provides a kind of composition of medicine, described 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 chemoluminescence therapeutic agent such as NSC-241240 or a kind of platinum medicine of related category, taxane or A kind of taxanes of classification, or both.

Advantages of the present invention and beneficial effect：

Present invention firstly discovers that there is the related biomarker XIRP2 of development to oral squamous cell carcinoma, pass through The change of detection experimenter XIRP2, to realize the early diagnosiss of oral squamous cell carcinoma.

The invention provides the molecular target for the treatment of oral squamous cell carcinoma, treat disease by targeting molecular marker Disease has Sensitivity and Specificity.

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

Brief description

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

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

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

Fig. 4 display mtt assay detection impact to oral cavity epidermoid carcinoma cell proliferation activity for the XIRP2；

Fig. 5 shows the impact detecting XIRP2 gene pairss oral squamous cell carcinoma cell invasion using transwell cell.

Specific embodiment

Present invention firstly discovers that XIRP2 and the generation of oral squamous cell carcinoma develop related, and demonstrate XIRP2 in mouth High expression in the squamous cell carcinoma of chamber.XIRP2 can be as the independentpredictor of oral squamous cell carcinoma it is also possible to and other bases Because of mark use in conjunction.

In the present invention, term " biomarker " is its expression in tissue or cell and normal or healthy cell Or tissue expression compare any gene changing or albumen.

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

Biomarker as herein described includes gene and albumen.Such biomarker is included containing encoding human labelling The DNA of the complete or partial sequence of the complementary seriess of the nucleotide sequence of thing or this sequence.Biomarker nucleic acid also include containing The RNA of the complete or partial sequence of any nucleotide sequence of interest.Biomarker protein is by the biological mark of DNA of the present invention The albumen of note thing coding or corresponding to the present invention DNA biomarker.Biomarker protein comprises any biomarker Thing albumen or the complete or partial amino-acid series of polypeptide.The fragment of biomarker gene and albumen and variant are also included within this In the range of invention.So-called " fragment " refers to the parts of polynucleotide or aminoacid sequence thus one of the albumen of coding Point.For the fragment of biomarker nucleotide sequence polynucleotide generally comprise at least 10,15,20,50,75,100,150, 200th, 250,300,350,400,450,500,550,600,650,700,800,900,1,000,1,100,1,200,1,300 or The continuous nucleotide of Isosorbide-5-Nitrae 00, or at most it is present in the nucleotide in total length biomarker polynucleotide disclosed herein Number.The fragment of biomarker polynucleotide will generally encode at least 15,25,30,50,100,150,200 or 250 continuously Aminoacid, or it is present in the sum of the aminoacid in the total length biomarker protein of the present invention." variant " is intended to indicate that substantially Upper similar sequence.In general, the variant of the biomarker-specific thing of the present invention will have and be measured by alignment programs With described biomarker at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%th, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence iden.

The present invention can measure gene expression using any method known in the art.Those skilled in the art should manage Solution, the means measuring gene expression are not the importances of the present invention.Can detect in (i.e. albumen) level transcribing or translating The expression of biomarker.

In some embodiments, the expression of biomarker is detected on transcriptional level.Using nucleic acid hybridization skill Multiple methods that art carries out concrete DNA and RNA measurement are well known by persons skilled in the art.Certain methods are related to be separated by electrophoresis (for example, for detecting the Southern trace of DNA and the Northern trace for detecting RNA), but can also be unfavorable Carry out the measurement (for example, by Dot blot) of DNA and RNA with electrophoresis in the case of detached.Genomic DNA (for example, is derived from People) Southern trace can be used for screening restriction fragment length polymorphism (RFLP), affect polypeptide of the present invention to detect The presence of inherited disorder.Can detect the RNA of form of ownership, including but not limited to messenger RNA (mRNA), microRNA (miRNA), Ribosomal RNA (rRNA) and transfer RNA (tRNA).

Pharmaceutical composition in the present invention also includes pharmaceutically acceptable carrier, and this kind of carrier includes (but not limiting In)：Diluent, excipient such as Lactose, sodium chloride, glucose, carbamide, starch, water etc., filler such as starch, sucrose etc.；Bonding Agent such as simple syrup, glucose solution, starch solution, cellulose derivative, alginate, gelatin and Polyvinylpyrrolidone；Moistening Agent such as glycerol；Disintegrating agent such as dried starch, sodium alginate, laminarin powder, agar powder, Calcium Carbonate and sodium bicarbonate；Absorb Accelerator quaternary ammonium compound, sodium lauryl sulphate etc.；Surfactant for example polyoxyethylene sorbitan fatty acid ester, 12 Alkyl sodium sulfate, glycerol 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 Pulvis Talci, calcium stearate and magnesium, Polyethylene Glycol, boric acid powder etc..

The pharmaceutical composition of the present invention can be prepared using different additives, for example buffer agent, stabilizer, antibacterial Agent, isotonic agent, chelating agen, pH controlling agent and surfactant.

Buffer agent can 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, potassium salt, calcium salt and magnesium salt).Isotonic agent includes potassium chloride, sodium chloride, sugar and glycerol.Chelating Agent includes sodium ethylene diamine tetracetate and citric acid.Antibacterial includes but is not limited to valid density (for example<Benzylalcohol 1%w/v), benzene Phenol, metacresol, methaform, methyl parahydroxybenzoate and/or propyl p-hydroxybenzoate.Stabilizer includes serum human egg In vain, l-amino acid, sugar and cellulose derivative.L-amino acid can also include appointing in glycine, cysteine and glutamic acid Meaning one.Saccharide includes monosaccharide, such as glucose, mannose, galactose, Fructose etc.；Sugar alcohol, such as Mannitol, inositol, wood Sugar alcohol etc.；Disaccharide, such as sucrose, maltose, Lactose etc.；Polysaccharide, such as glucosan, hydroxypropyl starch, sulfuration chrondroitin, thoroughly Bright matter acid waits and their derivant.Cellulose derivative includes methylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxyl Propyl cellulose, hydroxypropyl methylcellulose and Carboxymethyl cellulose sodium.Surfactant includes ion or non-ionic surface active Agent, such as polyethylene glycol oxide Arrcostab, sorbitan monoacyl ester, fatty glyceride.

The medicine of the present invention may also include pharmaceutically acceptable coating material and includes but is not limited to, fast decoupled coating Material, stain, 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, hydroxypropyl methylcellulose acetass, hydroxypropyl methylcellulose 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, by suck spray delivery, locally give Medicine, rectally, nasal administration, cheek administration, vagina administration or the storage medicine device administration by implanting.Preferred oral administration or injection Administration.Pharmaceutical composition of the present invention can contain any commonly employed nontoxic pharmaceutical acceptable carrier, adjuvant or excipient.In some situations Under, medicinal acid, alkali or buffer agent can be used to adjust the pH of preparation to improve stablizing of prepared compound or its form of administration Property.Terms used herein parenteral route includes in subcutaneous, Intradermal, intravenouss, intramuscular, intraarticular, intra-arterial, intrasynovial, breastbone, Bring up in interior, 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 in the form of any peroral dosage form oral administration, including but not limited to capsule, piece Agent, Emulsion and water slurry, dispersant and solution.For oral tablet, common carrier includes Lactose and corn starch.Typically also Add lubricant such as magnesium stearate.In order to be administered with capsules per os, applicable diluent includes Lactose and anhydrous Semen Maydiss Starch.When Orally administered water slurry and/or emulsion, active component can be suspended or dissolved in oil phase, and and emulsifying agent And/or suspending agent merges.If necessary, some sweeting agents and/or correctivess and/or coloring agent can be added.When suitably, can The dosage unit preparations bag microcapsule of oral administration will be used for.For example, by polymer, wax etc. by particulate matter coating or bag Bury, also can prepare described preparation and extend or maintained release.It is endogenic that the pharmaceutical composition of the present invention can be used for supplement The disappearance of XIRP2 albumen or deficiency, by improve XIRP2 albumen expression or strengthen XIRP2 albumen function, thus treat because The oral squamous cell carcinoma that the minimizing of XIRP2 albumen leads to.

The medicine of the present invention also can be with the drug combination of other treatment oral squamous cell carcinoma, and other therapeutic compound can To be administered simultaneously with main active component, or even it is administered simultaneously in same compositionss.Can also with single compositionss or The dosage form different from main active component individually gives other therapeutic compounds.The Fractional of main component is permissible It is administered simultaneously with other therapeutic compounds, and other dosage can be administered alone.Over the course for the treatment of, can be according to symptom The physiologic response of the order 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 the administration of Liposomal delivery systems form, such as little unilamellar vesicle, single greatly Layer vesicle and multilamellar vesicle.Liposome can have multiple phospholipid to be formed, such as cholesterol, stearic amine or phosphatidylcholine.

The pharmaceutical composition that pharmaceutical composition of the present invention can locally be administered, can be configured to ointment, ointment, suspension Agent, lotion, powder, solution, paste, gel, spray, aerosol or oil preparation.

The carrier carrying gene of the present invention is various carrier known in the art, such as commercially available carrier, include plasmid, Cosmid, phage, virus etc..

In the present invention term " probe " refer to be combined with the particular sequence of another molecule or subsequence or other parts point Son.Unless otherwise noted, " probe " is often referred to match and another polynucleotide (often referred to as " target multinuclear by complementary base Thuja acid ") polynucleotide probes that combine.According to the preciseness of hybridization conditions, probe energy and with this probe lack sufficient sequence mutual The target polynucleotide of benefit property combines.Probe can make direct or indirect labelling, and its scope includes primer.Crossing system, including, but It is not limited to：Solution, solid phase, mixed phase or in situ hybridization algoscopy.

As probe, it is possible to use fluorescent labeling, radio-labeled, biotin labeling etc. are carried out with polynucleotide to cancer detection The label probe of labelling.The labeling method of polynucleotide is known in itself.Whether can check in sample by the following method There is subject nucleic acid：Fixing subject nucleic acid or its amplified matter, are hybridized with label probe, washing, and and then measure with The labelling of solid phase binding.Alternatively, also can fix cancer detection polynucleotide, so that 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, many nucleoside are used in the cancer detection being incorporated in solid phase Acid is also referred to as probe.The method measuring subject nucleic acid using polynucleotide probes is also known in this area.Can enter as follows Row the method：Polynucleotide probes and subject nucleic acid near Tm or its (preferably within ± 4 DEG C) is made to connect in buffer Touch for hybridizing, washing, then measure the label probe of hybridization or the template nucleic acid being combined with solid phase probe.

The polynucleotide using as probe be preferably sized to 18 or more nucleotide, more preferably 20 or More nucleotide, and the total length or less of coding region.When using as primer, this polynucleotide is preferably sized to 18 Or more nucleotide, and 50 or more Oligonucleotide.

Term " chip " is also referred to as " array ", refers to comprise the solid support of the nucleic acid or peptide probes connecting.Array is usual Comprise multiple different nucleic acid or the peptide probes connecting to substrate surface according to different known location.These arrays, also referred to as " microarray ", generally can produce these arrays using mechanical synthesis methods or light guiding synthetic method, described light guiding is closed One-tenth method incorporates the combination of photoetching method and solid phase synthesis process.Array can comprise flat surface, or can be pearl Son, gel, polymer surfaces, the fiber of such as optical fiber, glass or any other suitably suprabasil nucleic acid or peptide.Can be with Certain mode carrys out array of packages, thus allowing to carry out the diagnosis of global function device or the manipulation of alternate manner.

In the present invention, term " antibody " refers to the naturally occurring or synthetic antibody of selective binding target antigen.This term bag Include polyclone and monoclonal antibody.In addition to complete immunoglobulin molecules, the fragment of those immunoglobulin molecules or poly- The mankind of the immunoglobulin molecules of compound and selective binding target antigen or humanization form are also included within term and " resist In the range of body ", as long as they show desired biologic activity." monoclonal antibody " refers to from a group substantially homogeneity The antibody that obtains of antibody, that is, each antibody constituting colony is identical and/or combine same epitope, except producing monoclonal antibody During issuable may become external, such variant is typically with indivisible presence.Such monoclonal antibody is typically wrapped Include the antibody comprising the peptide sequence with reference to target, wherein target Binding peptide sequence is by including comforming in many peptide sequences Single target Binding peptide sequence is selected to obtain in interior process.

Monoclonal antibody also includes " being fitted together to " antibody, wherein a part for heavy chain and/or light chain with derived from particular species Or belong to that corresponding sequence in specific antibodies classification or the antibody of subclass is identical or homology, and the remainder of chain with derived from another One species or belong to that corresponding sequence in another antibody isotype or the antibody of subclass is identical or homology, and the piece of this antibody-like Section, as long as they show desired biologic activity.

Polyclonal antibody comprises to the antibody obtained by animal (for example, mice) immunity XIRP2 protein producing human antibody. After being prepared for chimeric antibody or humanized antibody, the aminoacid in variable region (for example, FR) and/or constant region can be used Other amino acid substitutions etc..

The replacement of aminoacid be e.g. less than 15, less than 10, less than 8, less than 7, less than 6, less than 5,4 Individual following, less than 3 or less than 2 aminoacid, preferably 1～5 aminoacid, the replacement of more preferably 1 or 2 aminoacid, replace Change antibody to be functionally equal to not replacing antibody.Expecting to replace is conservative amino acid substitution, and this is electric charge, side chain, pole Replacement between the kin aminoacid such as property, aromatic series.Kin aminoacid for example can be categorized as alkaline ammonia Base acid (arginine, lysine, histidine), acidic amino acid (aspartic acid, glutamic acid), uncharged polar amino acid (sweet ammonia Acid, agedoite, L-Glutamine, serine, threonine, cysteine, tyrosine), apolar amino acid (leucine, different bright Propylhomoserin, alanine, L-Valine, proline, Phenylalanine, tryptophan, Methionine), branched-chain amino acid (leucine, L-Valine, different Leucine), aromatic amino acid (Phenylalanine, tyrosine, tryptophan, histidine) etc..

In the present invention, term " treatment " refers to cure, to improve, stably or for the purpose of prevention disease, pathological state or disease The medical supervision that patient is carried out.This term includes active treatment, that is, specially for the purpose of improving disease, pathological state or disease Treatment, and also include etiological treatment, i.e. treatment for the purpose of the cause of disease removing relevant disease, pathological state or disease. Additionally, this term also includes palliative treatment, that is, it is designed for controlling of relief of symptoms rather than cure diseases, pathological state or disease Treat；Prophylactic treatment, that is, at utmost to reduce or partially or completely to suppress the development of relevant disease, pathological state or disease For the purpose for the treatment of；And supportive treatment, that is, be used for supplementing another kind of to improve relevant disease, pathological state or disease as mesh Specific therapy treatment.

The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this Invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally 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 condition proposed by manufacturer.

The embodiment 1 screening gene marker related to oral squamous cell carcinoma

1st, sample collection

Respectively collect 6 surrounding normal mucosal tissues and oral squamous cell carcinoma, all confirm through pathological diagnosis, own Do not accept any type for the treatment of before operation in patients.The sample cutting of performing the operation is frozen in liquid nitrogen, the equal informed consent of patient, above-mentioned institute The acquirement having specimen is all by the agreement of committee of organizational ethics.

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

Take out frozen tissue samples in liquid nitrogen, be ground in mortar tissue samples being put into pre-cooling, according to Description in test kit is extracted and is separated RNA.Specific as follows：

1) add Trizol, room temperature places 5min；

2) add chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, under room temperature, place 5-10min；

3) 12000rpm centrifugation 15min, upper strata aqueous phase is moved on in another new centrifuge tube and (is careful not to be drawn onto two-layer water Protein substance between phase), add the isopropanol of isopyknic -20 DEG C of pre-coolings, fully reverse mixing, it is placed in 10min on ice；

4) 12000rpm high speed carefully discards supernatant after 15min, adds 75% in the ratio of 1ml/ml Trizol DEPC washing with alcohol precipitation (4 DEG C of preservations), washing precipitate, vibration mixes, 4 DEG C, and 12000rpm is centrifuged 5min；

5) discard ethanol liquid, under room temperature, place 5min, add DEPC water dissolution precipitation；

6) use Nanodrop2000 ultraviolet spectrophotometer measurement RNA purity and concentration, frozen in -70 DEG C of refrigerators.

3rd, reverse transcription and labelling

With Low RNA Input Linear Amplification Kit, mRNA reverse transcription is become cDNA, use Cy3 simultaneously Labelling experiment group and matched group respectively.

4th, hybridize

Gene chip adopts people's full-length genome chip of expression spectrum of Aglient company, and every chip includes 45015 few cores Thuja acid, wherein has 43376 people's gene probes and 1639 experiment control probes.Carry out by the step of chip operation instructions, Temperature, at 65 DEG C, rolls hybridization through 17h 10r/min, develops a film for 37 DEG C.

5th, data processing

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

6th, result

Compared with normal mucosa tissue, expression in oral squamous cell carcinoma for the XIRP2 gene significantly raises.

The differential expression of embodiment 2 QPCR sequence verification XIRP2 gene

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

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

3rd, reverse transcription：

1) reaction system：

Table 1 reverse transcription reaction system

2) reverse transcription reaction condition

Carry 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

According to the coded sequence design QPCR amplimer of XIRP2 gene and GAPDH gene in Genebank, by Bo Maide Biotech firm synthesizes.Concrete 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 is：

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：

Table 2 PCR reaction system

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, as fluorescent marker, in the reaction of Light Cycler quantitative real time PCR Instrument enterprising performing PCR, is analyzed by melt curve analysis Determine purpose band with electrophoresis, Δ Δ CT method carries out relative quantification, each sample carries out 3 times repeating to test.

5th, statistical method

With GAPDH as internal reference, calculate the reality of oral squamous cell carcinoma and normal mucosa histofluorescence quantitative RT-PCR Test result, statistical analysiss are carried out using SPSS18.0 statistical software, difference between the two adopts t to check, with P<0.05 tool There is significant difference.

6th, result

Result is as shown in figure 1, compared with surrounding normal mucosal tissue, XIRP2 gene is in oral squamous cell carcinoma Up-regulated, difference has statistical significance (P<0.05), consistent with RNA-sep result.

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

1st, cell culture

Oral squamous cell carcinoma cell line Tca8113, HN13, normal mucosa epithelial cell line HIOEC is purchased from Shanghai and hands over Attached 9th the People's Hospital of logical university.The culture medium of HIOEC is K-SFM；The culture medium of Tca8113, HN13 is DMEM；To contain The culture medium of 10% hyclone and 1%P/S is in 37 DEG C, 5%CO₂, relative humidity be 90% incubator in cultivate.2-3 days Change liquid 1 time, passed on using the 0.25% trypsin conventional digestion containing EDTA.

2nd, the extraction of cell total rna

1) terminate culture when 80-90% merges when cell reaches, 0.25% trypsinization is collected cell and managed in 1.5m1EP In, often add lm1Trizol slowly to shake smudge cellses in pipe, place 10min on ice.

2) Deproteinization, removes DNA：Each 1.5m1EP pipe adds 0.2ml chloroform, rocks 15s, and room temperature places 10min.4 DEG C, 12000rpm is centrifuged 15min.

Remaining operation step is with RNA extraction process in organizing.

3rd, reverse transcription

Concrete steps are with embodiment 2.

4th, statistical method

Experiment all to complete according to being repeated 3 times, and result data is all to be represented in the way of mean+SD, Statistical analysiss are carried out using SPSS18.0 statistical software, difference between the two adopts t to check it is believed that working as P<Have when 0.05 Statistically significant.

5th, result

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

The silence of embodiment 4 XIRP2 gene

1st, cell culture

Human mouth epidermoid carcinoma cell strain Tca8113, is existed with culture medium DMEM containing 10% hyclone and 1%P/S 37 DEG C, 5%CO₂, relative humidity be 90% incubator in cultivate.Change within 2-3 days liquid 1 time, using the 0.25% pancreas egg containing EDTA White enzyme conventional digestion passes on.

2nd, siRNA design

SiRNA sequence for 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 in six porocyte culture plates, in 37 DEG C, 5%CO₂Cell culture in incubator 24h, in DMEM culture medium that is no dual anti-, containing 10%FBS, transfection (is purchased from according to lipofectamine 2000 Invitrogen company) description transfection.

Experiment is divided into three groups：Matched group (Tca8113), negative control group (siRNA-NC) and experimental group (siRNA1- XIRP2, siRNA2-XIRP2), the wherein sequence no homology of negative control group siRNA and XIRP2 gene, concentration is 20nM/ Hole, is transfected simultaneously respectively.

3rd, QPCR detects the transcriptional level of XIRP2 gene

The extraction of 3.1 cell total rnas

Concrete steps are with embodiment 3.

3.2 reverse transcription step are with embodiment 2.

3.3 QPCR amplification step are with embodiment 2.

4th, statistical method

Experiment all to complete according to being repeated 3 times, and result data is all to be represented in the way of mean+SD, Statistical analysiss are carried out using SPSS18.0 statistical software, the difference between interference XIRP2 gene expression panel and matched group is adopted Checked it is believed that working as P with t<There is when 0.05 statistical significance.

5th, result

Result such as Fig. 3 shows, compares TCA8113, unloaded siRNA-NC, siRNA2-XIRP2 group of transfection, siRNA1- XIRP2 group can significantly reduce the expression of XIRP2 gene, and difference has statistical significance (P<0.05).

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

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

1st, configuration concentration is the standard substance of 70000pg/ml, after 10 times of dilutions, then carries out 2 times of doubling dilutions, have 7 dilute Degree of releasing.

2nd, it is loaded：Set blank well, gauge orifice, testing sample hole respectively.Blank well adds sample diluting liquid 50 μ l, and remaining hole is respectively Plus the standard substance of variable concentrations gradient and each 50 μ l of testing sample.Gently rock mixing, ELISA Plate adds lid, 37 DEG C of reaction 2h.

3rd, discard liquid, dry.Every hole adds 200 μ l VEGF-C conjugates.37 DEG C, after 120min, discard in the hole liquid, Dry, PBS washes plate 3 times.

4th, sequentially every hole adds substrate solution 200 μ l, 37 DEG C of lucifuges colour developing 30min.

5th, sequentially every hole adds stop bath 50 μ l, terminating reaction.

6th, join, with enzyme, the optical density (OD value) that instrument sequentially measures each hole in 450nm wavelength.All standard substance and testing sample OD value be both needed to deduct the OD value in zero hole to obtain corrected value.

7th, the actual concentrations of sample are calculated.

8th, result is as shown in table 3 below, the XIRP2 gene of siRNA silence Tca8113 cell, the protein content also phase of XIRP2 Should reduce, illustrate that silence XIRP2 gene can suppress the expression of XIRP2 gene.

The expression of the XIRP2 albumen in the different group cell of table 3 ELISA detection

Embodiment 6 mtt assay detects Tca8113 cell-proliferation activity

Application MTT (Methyl thiazolyl tetrazolium, methyl thiazolyl tetrazolium) method detection XIRP2 gene sinks Impact to Tca8113 cell-proliferation activity after silent.

1st, Tca8113 cell is pressed 1 × 10 by cell culture³/ hole is inoculated in 96 orifice plates, 1,37 DEG C of every hole 100 μ, 5%CO₂Incubate Incubation culture in case.

2nd, cell transfecting step is with embodiment 3.

3rd, MTT detection

1) when transfecting 1～7 day, discard each hole culture medium, add MTT (5mg/ml) 20 μ l.Continue cellar culture 4h.

2) suck mixed liquor, every hole adds DMSO 200 μ l, concussion 10min so that crystallization is fully dissolved.In enzyme linked immunological instrument Absorbance value at upper survey 490nm, records result.

4th, with the time as transverse axis, absorbance value (OD) draws cell growth curve for the longitudinal axis.

5th, result：

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

Embodiment 7 Transwell cells in vitro Matrigel

Collect the other Tca8113 cell of different group within the 6th day after RNA interference, be resuspended in culture fluid, make final concentration of cells For 2 × 10⁶/ ml, draws 100 μ l cell suspension and adds in Transwell cell.Application Transwell cell method is observed The impact to Tca8113 cellular invasiveness for the XIRP2 gene silencing.

1st, Matrigel (4 μ g/ μ l) is put into 4 DEG C of thawings, prepare ice chest (ice bath environment).Matrigel is diluted with DMEM Use after 8 times.Apply 8 μ g people's fibronectin in the outer surface of Transwell cell filter membrane (8 μm of apertures), put super-clean bench endogenous wind Dry.

2nd, the inner surface in 6 hole Transwell cell filter membranes spreads with the Matrigel glue in 100 μ 1/ hole, 37 DEG C, 5%CO₂ Incubation 1h in incubator, forms a substrate barrier layer standby.

3rd, add the DMEM culture fluid 2.5m1 containing 20%FBS in the every in the hole of 6 orifice plates.

4th, collect the cell of exponential phase, be resuspended in culture fluid, final concentration of 2 × 10⁶/ml.

5th, cell suspension is added in Transwell cell, every hole 100 μ 1, cell is dipped in the conditioned medium of 6 orifice plates In, 37 DEG C, 5%CO₂Incubation 24h in incubator.

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

7th, HE dyeing：Brazilwood extract dyeing 3min, washing；Eosin stains 10～30s, washing.And wiped with cotton swab and do not pass through The cell of film.

8th, basis of microscopic observation, take a picture and count invasion cell number, every film count up and down in 5 different visuals field saturating Cross cell number, calculate meansigma methodss.Every group parallel to set 3 filter membranes.

9th, data processing

With SPSS18.0 software, statistical analysis are carried out to data.Measurement data mean ± standard deviation represents.Multiple samples This mean compares and adopts one factor analysis of variance, P<0.05 is that difference is statistically significant.

10th, result

Result is as shown in figure 5, Tca8113, siRNA1-XIRP2, siRNA-NC group cell is trained in transwell cell After foster 24h, under siRNA1-XIRP2 group polycarbonate membrane, the cell number in room face substantially reduces.

The explanation of above-described embodiment is only intended to understand the method for the present invention and its core concept.It should be pointed out that for this For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention And modification, these improve and modify also by the protection domain falling into the claims in the present invention.

daSEQUENCE LISTING

<110>Beijing Yang Shen biology information technology company limited

<120>Oral squamous cell carcinoma biomarker and its application

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 2817

<212> DNA

<213>People source

<400> 1

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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Met Phe Pro Met Gln Lys Gly Ser Leu Asn Leu Leu Arg Gln Lys Trp

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Glu Ser Cys Asp Tyr Gln Arg Ser Glu Cys His Pro Arg Asp Ser His

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Cys Thr Ile Phe Gln Pro Gln Glu Ser Lys Leu Leu Ala Pro Glu Gly

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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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Lys Glu Asp Ser Leu Ser Ser Arg Arg Arg Ile Glu Arg Phe Ser Ile

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

195 200 205

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

225 230 235 240

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

260 265 270

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

275 280 285

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

290 295 300

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

420 425 430

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

465 470 475 480

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

Lys Arg Asn Arg Cys Tyr Ser Asp Thr Glu

930 935

<210> 3

<211> 18

<212> DNA

<213>Artificial sequence

<400> 3

gcagccttat ctacagtc 18

<210> 4

<211> 18

<212> DNA

<213>Artificial sequence

<400> 4

tcttccttcc ttccttct 18

<210> 5

<211> 21

<212> DNA

<213>Artificial sequence

<400> 5

ctctggtaaa gtggatattg t 21

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence

<400> 6

ggtggaatca tattggaaca 20

<210> 7

<211> 19

<212> RNA

<213>Artificial sequence

<400> 7

uucuccgaac gugucacgu 19

<210> 8

<211> 19

<212> RNA

<213>Artificial sequence

<400> 8

acgugacacg uucggagaa 19

<210> 9

<211> 21

<212> RNA

<213>Artificial sequence

<400> 9

uguugauauu gguauugagc a 21

<210> 10

<211> 21

<212> RNA

<213>Artificial sequence

<400> 10

cucaauacca auaucaacau c 21

<210> 11

<211> 21

<212> RNA

<213>Artificial sequence

<400> 11

auaacauccu cuuucugaca a 21

<210> 12

<211> 21

<212> RNA

<213>Artificial sequence

<400> 12

gucagaaaga ggauguuaua g 21

Claims

Application in the product of preparation diagnosis oral squamous cell carcinoma for the 1.XIRP2 gene.
2. according to claim 1 application it is characterised in that described product include by chip, blotting, RT-PCR, Real-time quantitative PCR, FISH method, CGH method or array CGH method, bisulfite sequencing, the change of COBRA method detection XIRP2 gene Change to diagnose the product of oral squamous cell carcinoma.
3. application according to claim 2 is it is characterised in that described real-time quantitative PCR diagnoses oral squamous cell carcinoma Product at least include the primer of a pair of specific amplification XIRP2 gene, described primer such as SEQ ID NO.3 and SEQ ID Shown in NO.4.
4. a kind of product of diagnosis oral squamous cell carcinoma is it is characterised in that described product can be by detecting XIRP2 in sample The change of gene is diagnosing oral squamous cell carcinoma.
5. product according to claim 4 is it is characterised in that described product includes chip, test kit or preparation.
Application in the pharmaceutical composition of preparation treatment oral squamous cell carcinoma for the 6.XIRP2 gene.
7. application according to claim 6 is it is characterised in that described pharmaceutical composition includes XIRP2 gene and/or its table Reach the inhibitor of product.
8. a kind of pharmaceutical composition treating oral squamous cell carcinoma it is characterised in that described pharmaceutical composition include right will Seek the inhibitor described in 7.
9. a kind of method of suppression cell proliferation is it is characterised in that by the siRNA of XIRP2 gene, shRNA, antisense oligonucleotide Or Loss-of-function gene imports in tumor cell in vitro.
10. a kind of composition of medicine is it is characterised in that include the pharmaceutical composition described in claim 8 and the medicine containing antitumor agent Compositions.