CN105671187A

CN105671187A - Set of genes for head and neck squamous cell carcinoma (HNSCC) molecular typing and application thereof

Info

Publication number: CN105671187A
Application number: CN201610212589.0A
Authority: CN
Inventors: 宁云山; 李妍
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2016-04-08
Filing date: 2016-04-08
Publication date: 2016-06-15
Anticipated expiration: 2036-04-08
Also published as: CN105671187B

Abstract

The invention provides a set of genes for head and neck squamous cell carcinoma (HNSCC) molecular typing. The set of genes comprises TP53 gene, CDKN2A gene, FAT1 gene, CASP8 gene, AJUBA gene, PIK3CA gene, NOTCH1 gene, KMT2D gene, NSD1 gene, HLA-A gene, HRAS gene, FBXW7 gene, RB1 gene, PIK3R1 gene, TRAF3 gene, NFE2L2 gene, CUL3 gene and PTEN gene. The invention also provides application of the genes in preparing a kit and gene chip for HNSCC molecular typing, and a kit and gene chip for HNSCC molecular typing prepared by using the genes. The genes can enhance the HNSCC typing rationality and accuracy, thereby providing key technical supports for implementing early diagnosis, effective interception and individualized treatment on HNSCC.

Description

One group of gene for squamous cell carcinoma of the head and neck molecule parting and application thereof

Technical field

The present invention relates to one group for the gene of squamous cell carcinoma of the head and neck molecule parting and preparing for the application in the test kit of squamous cell carcinoma of the head and neck molecule parting.

Background technology

Squamous cell carcinoma of the head and neck (headandnecksquamouscellcarcinoma, HNSCC) it is one of modal malignant tumor of incidence, mainly include lip cancer, gingival carcinoma, carcinoma of tongue, soft or hard carcinoma of palate, carcinoma of jaw, carcinoma of floor of mouth, oropharynx cancer, salivary gland carcinoma and carcinoma of maxillary sinus and betide the cancer etc. of facial area skin mucosa, the oral cancer of broad sense includes the cancer occurred in below eye socket, cervical region range above, the overwhelming majority belongs to squamous cell carcinoma, being referred to as squamous cell carcinoma of the head and neck, the developing country such as China is the hotspot of HNSCC. Whole world HNSCC fatality rate ranking the 6th in Cancer death rate, diagnose latter 5 years survival rates not as good as 50%, belong to prognosis disease (Petersen poor, disfiguring, P.E. (2009) .Oralcancerpreventionandcontrol--theapproachoftheWorldHe althOrganization.OralOncol45,454-460). Although the past three is during the decade, the oncotherapy means such as operation, radiotherapy and chemotherapy obtain significant progress, but the raising of HNSCC survival is extremely limited, this lasting high mortality rate is mainly attributed to transfer and recurrence (Leemans, C.R., the Braakhuis of HNSCC, B.J., andBrakenhoff, R.H. (2011) .Themolecularbiologyofheadandneckcancer.NatRevCancer11,9-22). HNSCC is the coefficient results of inside and outside many factors such as somatic cell gene sudden change, Nicotiana tabacum L., ethanol, HPV infection, and has height heterogeneity, causes clinical treatment effect and prognosis widely different.

At present, clinically the typing of HNSCC is still adopted the histological grade method being described by Broders and being adopted by WHO. The method is divided into 3 grades: 1 grade (differentiated), 2 grades (middle differentiation), 3 grades (low differentiation) according to the pleomorphism of tumor cell keratinization degree, cell and core, caryocinetic subjective assessment tumor histology, when Tumors display goes out different classification, highest ranked determines final classification (BarnsL, etal. (2005) .PathologyandgeneticsofheadandnecktumorsLyon:IARCPress, 168-175).But most authoritative institutions and scholar think at present, in individual patient, the dependency poor (WoolgarJA. (2006) .Histopathologicalprognosticatorsinoralandoropharyngeals quamouscellcarcinoma.OralOncol42:229-239) of Broders/WHO histological grade system and prognosis, therapeutic response, there is following open defect mainly due to this tissue typing method in this: the differentiation of tumor is careful not, has that histology is special-shaped and specimen of drawing materials comparatively is limited to; Classification depends on architectural feature rather than the function of tumor cell; Its interstitial supported with it and host tissue are not associated when evaluating tumor cell. Therefore, set up new, more accurately HNSCC typing be the urgent method set up in tumor of head and neck clinical treatment, be also basis and the premise of carrying out individualized treatment.

In recent years, developing rapidly of high flux gene sequencing technology provides possibility for HNSCC molecule parting and accurate treatment. 2013, international cancer genome alliance (InternationalCancerGenomeConsortium) utilizes exon sequencing technologies that the gingiva in oral cancer-cheek squamous cell carcinoma is checked order, find that gingiva-cheek squamous cell carcinoma is except having head-neck malignant tumor common mutations gene TP53 and HRAS etc., also there is other special mutant gene, further demonstrate that the diversity of genovariation between different head-neck malignant tumor, carry out gene type for HNSCC and and guide its clinical treatment to provide bright prospects. Meanwhile, the targeted drug treatment based on molecule parting also provides possible for the individualized treatment of HNSCC.

Cetuximab (Cetuximab) is the first molecular target medicine for squamous cell carcinoma of the head and neck obtaining FDA approval, mainly acts on the extracellular space of EGFR. one multi-center clinical trial attracted people's attention (N=424) result shows, compared with radiation alone, Cetuximab combined radiotherapy can be effectively reduced H/N tumors local relapse, extend the survival of patients phase, significantly improve patient five year survival rate (Bonner, J.A., Harari, P.M., Giralt, J., Azarnia, N., Shin, D.M., Cohen, R.B., Jones, C.U., Sur, R., Raben, D., Jassem, J., etal. (2006) .Radiotherapypluscetuximabforsquamous-cellcarcinomaofthe headandneck.TheNewEnglandjournalofmedicine354, 567-578). in another multicenter III clinical trial phase (N=117), compared with cisplatin list medicine chemotherapy, the treatment of Cetuximab combination with cisplatin can significantly improve chemosensitivity (Burtness, B., Goldwasser, M.A., Flood, W., Mattar, B., Forastiere, A.A., andEasternCooperativeOncology, G (2005) .PhaseIIIrandomizedtrialofcisplatinplusplacebocomparedwi thcisplatinpluscetuximabinmetastatic/recurrentheadandnec kcancer:anEasternCooperativeOncologyGroupstudy.Journalof clinicaloncology:officialjournaloftheAmericanSocietyofCl inicalOncology23, 8646-8654). domestic scholars finds in the recent period: recombinant adenovirus p53 gene therapy is remarkably improved the survival rate of oral squamous cell carcinoma three phase patient in conjunction with conventional chemotherapy, but to oral cancer fourth phase survival without being obviously promoted effect, it is imperative for HNSCC molecule parting and individualized treatment thereof that prompting is set up.

Therefore, setting up the molecule parting based on molecular marker directiveness and effective personalized clinical treatment from gene aspect, individualized treatment and incidence thereof that HNSCC patient carries out clinic are precisely medical significant.

Summary of the invention

The present invention is directed to current HNSCC classifying method comparatively to limit to, research especially for the HNSCC molecule parting gene group of Chinese population is very limited, existing appraisal procedure accuracy is not high, the problem such as personalized treatment to HNSCC clinically cannot be instructed, it is provided that one group of gene for HNSCC molecule parting and detection kit thereof.

One aspect of the present invention provides one group of gene for squamous cell carcinoma of the head and neck molecule parting, specifically includes following 18 genes: TP53 gene, CDKN2A gene, FAT1 gene, CASP8 gene, AJUBA gene, PIK3CA gene, NOTCH1 gene, KMT2D gene, NSD1 gene, HLA-A gene, HRAS gene, FBXW7 gene, RB1 gene, PIK3R1 gene, TRAF3 gene, NFE2L2 gene, CUL3 gene and PTEN gene.

Another aspect of the present invention additionally provides above-mentioned one group of gene for squamous cell carcinoma of the head and neck molecule parting in preparation for the application in the test kit of squamous cell carcinoma of the head and neck molecule parting. For this, the invention provides one group of PCR primer for squamous cell carcinoma of the head and neck molecule parting and one group of probe for squamous cell carcinoma of the head and neck molecule parting. The nucleotide sequence of described PCR primer is such as shown in SEQIDNo.1 to SEQIDNo.36, and the nucleotide sequence of described probe is such as shown in SEQIDNo.37 to SEQIDNo.54. Present invention also offers the application in preparing the test kit for squamous cell carcinoma of the head and neck molecule parting and gene chip of the above-mentioned PCR primer for squamous cell carcinoma of the head and neck molecule parting and probe.

Another aspect of the present invention additionally provides a kind of test kit for squamous cell carcinoma of the head and neck molecule parting, and it includes expanding one group of PCR primer for the gene of squamous cell carcinoma of the head and neck molecule parting described in claim 1 and/or can with a group described in claim 1 for the complementary probe of the gene order of squamous cell carcinoma of the head and neck molecule parting. The nucleotide sequence of above-mentioned PCR primer can as shown in SEQIDNo.1 to SEQIDNo.36. The nucleotide sequence of above-mentioned probe can as shown in SEQIDNo.37 to SEQIDNo.54.

Additionally, present invention also offers a kind of gene chip for squamous cell carcinoma of the head and neck molecule parting, it includes solid phase carrier and probe, and the nucleotide sequence of described probe can as shown in SEQIDNo.37 to SEQIDNo.54.

Molecule parting based on gene is introduced in the molecule parting of HNSCC by the present invention first, further enhances reasonability and the accuracy of HNSCC typing, promotes the ability of the clinical treatment instructing HNSCC. Simultaneously, China HNSCC crowd carries out checking and the optimization of method, found the molecule parting scheme being suitable for China HNSCC patients, thus for realizing HNSCC early diagnosis, effectively blocking-up, individualized treatment provides key technology and supports, in the precisely treatment of HNSCC, there is important clinical meaning.

Detailed description of the invention

Mode by the examples below further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments. The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.Unless otherwise defined, the same meaning that all specialties used in literary composition are familiar with one skilled in the art with scientific words. Additionally, any method similar or impartial to described content and material all can be applicable in the present invention. The use that preferably implementation described in literary composition and material only present a demonstration.

The present invention sets up gene molecule mark built-up pattern by the use in conjunction of gene test, molecular marker combination and data mining algorithm, utilizes polygenes assessment HNSCC molecule parting, mainly comprises the steps that

(1) collect clinical diagnosis data and the gene expression profile data of HNSCC, build the HNSCC gene expression profile data storehouse comprising known more than 20,000 genes of the mankind, 789 example samples;

(2) gene expression pattern is carried out statistical analysis, filter out 18 and the closely-related gene of HNSCC typing, be respectively as follows: TP53 gene, CDKN2A gene, FAT1 gene, CASP8 gene, AJUBA gene, PIK3CA gene, NOTCH1 gene, KMT2D gene, NSD1 gene, HLA-A gene, HRAS gene, FBXW7 gene, RB1 gene, PIK3R1 gene, TRAF3 gene, NFE2L2 gene, CUL3 gene and PTEN gene.

(3) by above-mentioned 18 gene expression patterns in the method detection HNSCC clinical samples such as PCR, gene expression chip and high-flux sequence, HNSCC clinical sample is carried out typing assessment.

Detection method for assessing HNSCC molecule parting provided by the invention mainly comprises the steps that

(1) RNA in clinical sample is extracted. Described clinical sample is taken from the tumor tissues of described object, it is possible to be fixing paraffin embedding (FFPE) sample of fresh sample or formalin, or the peripheral blood of described object or saliva;

(2) expression pattern and the expression of above-mentioned 18 genes in the clinical sample of described object is detected by gene expression detection method, and by the molecule parting of statistical method assessment HNSCC. Described gene expression detection method includes digital pcr, quantitative RT-PCR, gene chip and high throughput sequencing technologies etc., it is preferable that digital pcr and gene chip.

The test kit for HNSCC molecule parting of the present invention comprises primer and/or probe, described primer is preferably the synthesis oligonucleotide fragment that genespecific is high, as long as with the Gene Partial complementary in assessment HNSCC molecule parting gene group of the present invention, and the gene of assessment HNSCC molecule parting gene group of the present invention can be amplified. It is preferred that shown in the nucleotide sequence of described primer such as SEQ ID NO.1～SEQIDNO.36. Described probe is preferably the synthesis oligonucleotide fragment that genespecific is high, as long as can with the Gene Partial complementary in assessment HNSCC molecule parting gene group of the present invention. It is preferred that shown in the nucleotide sequence of described probe such as SEQ ID NO.37～SEQIDNO.54.

Embodiment 1The screening of assessment HNSCC molecule parting gene group

Delivering or be stored in the 789 example HNSCC oncogene full genome order-checkings of the data bases such as TCGA and COSMIC, exon order-checking, RNA order-checking and gene expression chip and clinical variable related data by the analysis of EPIG gene expression spectrum analysis program is disclosed, HNSCC molecule parting gene group is assessed in screening. Analysis includes non-supervisory cluster analysis recognition expression spectrum and related gene; The stability analysis of candidate gene. First pass through cluster analysis calculate each gene in 789 example samples be relative to each other coefficient and with this cluster formation gene specific express spectra (r > 0.7, P < 0.001), then the dependency of each gene and specifically expressing spectrum is calculated, candidate gene (r > 0.7, P < 0.001) is selected with this.Experimental result: screening obtains the gene of 18 the highest assessment HNSCC molecule partings of scoring, and list of genes is shown in table 1 below.

Table 1:18 can be used for assessing the gene of HNSCC molecule parting

Sequence number	Gene name	Gene identities number ID
			1	TP53	NM_001126118.1
2	CDKN2A	XM_011517679.1
			3	FAT1	NM_005245.3
4	CASP8	XM_006712793.2
			5	AJUBA	NM_198086.2
6	PIK3CA	NM_006218.2
			7	NOTCH1	NM_017617.4
8	KMT2D	NM_003482.3
			9	NSD1	NM_022455.4
10	HLA-A	NM_002116.7
			11	HRAS	NM_001130442.2
12	FBXW7	NM_033632.3
			13	RB1	NM_000321.2
14	PIK3R1	NM_001242466.1
			15	TRAF3	NM_145726.2
16	NFE2L2	NM_001145412.2
			17	CUL3	NM_001257198.1
18	PTEN	NM_000314.6

Embodiment 2The molecule parting of HNSCC is assessed by digital pcr method and gene expression chip

In the present embodiment, adopt digital pcr and gene expression chip method, detect embodiment 1 in the cancer beside organism of 100 groups of paraffin-embedded tumor tissues of China patient HNSCC and pairing thereof and screened the expression of 18 genes obtained. Primer and probe that the detection of digital pcr method is used are mankind's complete genome sequences disclosed in NCBI (US National Biotechnology Information center), use PrimerPremier5.0 design, by gene chemical synthesis company (the raw work in Shanghai) synthesis. Shown in the nucleotide sequence of primer such as table 2 below (SEQ ID No .1～SEQIDNo.36), the nucleotide sequence of probe such as table 3 below is (shown in SEQ ID No .37～SEQIDNo.54.

Table 2:18 can be used for assessing the gene PCR primer sequence of HNSCC molecule parting

Table 3:18 can be used for assessing the gene probe sequence of HNSCC molecule parting

Digital P CR (digitalPCR, dPCR) it is the third generation round pcr that gets up of newly-developed, relative to Real-time quantitative PCR (quantitativereal-timePCR, qPCR), digital pcr technology can increase substantially the sensitivity of detection method, degree of accuracy, accuracy and repeatability, and achieves absolute quantitation truly. Current digital pcr technology mainly includes two classes, large-scale integrated micro-fluidic chip digital pcr (cdPCR) and microdroplet digital pcr (dropletdigitalPCR, ddPCR), both approaches is all by DNA sample random assortment to be measured to the microreactor or microdroplet of chip before pcr amplification, in microreactor or microdroplet or without nucleic acid target molecule to be measured, or at least contain a nucleic acid target molecule to be measured, and each microreactor or microdroplet are independent PCR reactors. After pcr amplification, each microreactor or microdroplet are detected by detector, have fluorescence signal to be then judged to " 1 ", and unstressed configuration signal is then judged to " 0 ", the final ratio according to Poisson distribution principle and positive microdroplet, calculates copy number or the concentration of target sequence in sample to be tested.

In the present embodiment, adopt TrizolRNA to extract test kit the paraffin-embedded sample confirming as HNSCC through pathological diagnosis and extract total serum IgE; Process with DNase to guarantee the pollution removing genomic DNA completely; CDNA is obtained after reverse transcription. Digital pcr is adopted to carry out the detection of 18 gene expressions. Digital pcr adopts and completes in the QX200 droplet type digital pcr system of Bio-Rad company, after PCR reaction terminates, according to the expression pattern of 18 genes in above-mentioned 100 groups of samples and expression, statistical analysis also carries out molecule parting in conjunction with corresponding clinical data, being divided into 4 types (I～IV), concrete outcome is shown in table 4 below.

Table 4: digital pcr method HNSCC molecule parting result

Embodiment 3The molecule parting of HNSCC is assessed by gene expression chip

In the present embodiment, adopt gene expression chip method, detect embodiment 1 in the cancer beside organism of 100 groups of paraffin-embedded tumor tissues of China patient HNSCC identical with embodiment 2 and pairing thereof and screened the expression of 18 genes obtained. Probe used by gene expression chip used is mankind's complete genome sequence disclosed in NCBI (US National Biotechnology Information center), uses PrimerPremier5.0 design, by gene chemical synthesis company (the raw work in Shanghai) synthesis.Shown in the nucleotide sequence of probe such as sequence table SEQ IDNo.37～SEQIDNo.54.

Gene expression chip is by microelectric technique and micro-processing technology, by the DNA fragmentation of a large amount of particular sequences or few core former times acid fragment, it is fixed on the carrier such as glass, silicon chip by matrix high density, testing sample is with after fluorescence molecule labelling, big DNA or oligonucleotide fragment hybridization with on chip, by obtaining a kind of detection method of gene information after fluorescent scanning and computer analysis. Its outstanding feature is in that the nucleic acid sequence information in micro-example can carry out detection quick, accurate, high-throughout and analysis, it is possible to the gene expression abundance simultaneously obtaining thousands of genes associates collection of illustrative plates with expression pattern.

In the present embodiment, adopt TrizolRNA to extract test kit the paraffin-embedded sample confirming as HNSCC through pathological diagnosis and extract total serum IgE; Process with DNase to guarantee the pollution removing genomic DNA completely; CDNA is obtained after reverse transcription. CDNA is mixed with gene chip, hybridization; By scanning the gene expression abundance of fluorescence signal detection gene. After gene chip reaction terminates, according to the expression pattern of 18 genes in above-mentioned 100 groups of samples and expression, statistical analysis also carries out molecule parting in conjunction with corresponding clinical data according to embodiment 2, and concrete outcome is in Table 5.

Table 5: gene expression chip HNSCC molecule parting result

Comparing embodiment 2 and embodiment 3, adopt digital pcr and gene expression chip that same group of 100 groups of China HNSCC carry out 18 typing related gene expression detections, and two kinds of detection methods of result are basically identical to the result of HNSCC molecule parting. Show that the present invention passes through HNSCC genome sequencing, exon order-checking and RNA order-checking and may be used for assessment HNSCC molecule parting in conjunction with 18 HNSCC typing related genes that clinical data obtains.

Embodiment 4The molecule parting of HNSCC is assessed by high-flux sequence

In order to verify in embodiment 2 and embodiment 3 molecular typing methods to same group 100 groups Chinese's HNSCC samples further, the present invention adopt further high throughput sequencing technologies in embodiment 2 and embodiment 3 the sample of typing (I～IV) carry out stochastic sampling (each hypotype is 10 groups of samples, i.e. the cancer beside organism of 10 HNSCC cancerous tissues and 10 corresponding pairings) and carry out transcript profile sequence verification. Based on high throughput sequencing technologies transcript profile check order can analyze sample transcription this structure and expression, it is provided that the most comprehensive transcript profile information. Specifically comprise the following steps that

Step 1: extract total serum IgE in 100 groups of Chinese's HNSCC sample tissue (the RNA extraction agent box using Roche company the to produce RNA to extract in tissue).

Step 2: gained RNA is made the library being available for order-checking. The RNA of gained tissue makes the library being available for secondary order-checking, and the preparation method in library comprises the following steps:

The RNA extracting tissue is generated by reverse transcription under the guidance of single-minded primer the cDNA of the 18 kinds of molecule parting genes screened. End-filling also carries out 5 ' end phosphorylations, by 30 μ lDNA, 45 μ l pure water, 10 μ l have the T4DNA ligase buffer of 10mMATP, 4 μ l comprise 10mMdNTPMix, 5 μ lT4DNA polymerases, l μ lKlenow enzyme, 5 μ lT4 ligases mixing after, in 20 DEG C of temperature baths 30 minutes (reagent prepares test kit PE-102-1001 from Illumina sample), after temperature bath, adopt QIAGENQIAquickPCR Purification Kit DNA.End hangs A: be dissolved in by the product of upper step in 32 μ l buffer, add Klenow buffer 5 μ l, 1mMdATP10 μ l, KlenowE χ o-3 μ l, 30 minutes (reagent prepares test kit from Illumina sample) is kept at 37 DEG C, product is connected by QIAGENMinElutePCR purification kit: DNA is dissolved in 10 μ l buffer, add DNA ligase buffer 2 χ 25 μ l, PEAdapterOligoMix10 μ l, DNA ligase 5 μ l, 15 minutes (reagent is that Illumina sample prepares test kit) is kept at 20 DEG C, QIAGENQIAquickPCR Purification Kit DNA is adopted namely to obtain library after temperature bath.

Step 3: utilize the primer sequence shown in nucleotide sequence such as sequence table SEQ IDNo.1～SEQIDNo.36 of primer to carry out gained DNA library carrying out secondary order-checking with MiSeq. Both-end order-checking is carried out with IlluminaMiSeq sequenator. This process is automatically performed (Illumina company) by instrument itself.

Step 4: interpretation of result. Being analyzed by gained sequencing result, sequencing result shows: comparing with the cancer beside organism of pairing, the expression of each HNSCC hypotype Middle molecule parting gene exists significant difference.

Embodiment 5HNSCC molecule parting PCR kit

Round pcr utilizes specific primer and probe to realize the amplification in vitro of genes of interest and quantitative. Test kit provided by the invention can adopt Real-time quantitative PCR and numeral round pcr for assessing HNSCC molecule parting, it is preferable that digital pcr technology. The present invention provides HNSCC molecule parting PCR kit, including specific primer and/or probe, shown in the nucleotide sequence of primer such as SEQ ID No .1～SEQIDNo.36, shown in the nucleotide sequence of probe such as SEQ ID No .37～SEQIDNo.54.

Utilizing the probe of known nucleotide sequence and primer to prepare the technology of detection kit well known for the person skilled in the art, therefore for the concrete steps of the test kit for HNSCC molecule parting provided by the invention, therefore not to repeat here.

Embodiment 6HNSCC molecule parting gene chip

Gene chip is the gene expression analysis instrument utilizing a kind of higher flux, and HNSCC molecule parting gene chip provided by the invention is a kind of chip gene expression profile, mainly adopts oligonucleotide fragment to make probe, is solidificated on chip. Testing sample (process group) is carried out labelling from the mRNA of control sample with fluorescence molecule two kinds different, then hybridize with chip simultaneously, by analyzing the ratio of two kinds of samples and the fluorescence intensity of probe hybridization, detect the differential expression of HNSCC molecule parting related gene, thus assessing the molecule parting of HNSCC. The present invention provides HNSCC molecule parting gene chip, including specific probe, shown in the nucleotide sequence of probe such as SEQ ID No .37～SEQIDNo.54.

The technology utilizing the probe preparation gene chip of known nucleotide sequence is well known for the person skilled in the art, and therefore for the concrete steps of the gene chip for HNSCC molecule parting provided by the invention, therefore not to repeat here.

The above, be only the exemplary embodiments of the present invention. Protection scope of the present invention is not limited thereto, and any those of ordinary skill in the art, in the technical scope that disclosed herein, can expect change without creative work or replace, should be encompassed within protection scope of the present invention.

Claims

1. one group of gene for squamous cell carcinoma of the head and neck molecule parting, it is characterized in that, including following 18 genes: TP53 gene, CDKN2A gene, FAT1 gene, CASP8 gene, AJUBA gene, PIK3CA gene, NOTCH1 gene, KMT2D gene, NSD1 gene, HLA-A gene, HRAS gene, FBXW7 gene, RB1 gene, PIK3R1 gene, TRAF3 gene, NFE2L2 gene, CUL3 gene and PTEN gene.

2. one group of gene for squamous cell carcinoma of the head and neck molecule parting as claimed in claim 1 is used for the application in the test kit of squamous cell carcinoma of the head and neck molecule parting in preparation.

3. one group of PCR primer for squamous cell carcinoma of the head and neck molecule parting, it is characterised in that the nucleotide sequence of described PCR primer is such as shown in SEQIDNo.1 to SEQIDNo.36.

4. one group of PCR primer for squamous cell carcinoma of the head and neck molecule parting as claimed in claim 3 is used for the application in the test kit of squamous cell carcinoma of the head and neck molecule parting in preparation.

5. one group of probe for squamous cell carcinoma of the head and neck molecule parting, it is characterised in that the nucleotide sequence of described probe is such as shown in SEQIDNo.37 to SEQIDNo.54.

6. the one group of probe for squamous cell carcinoma of the head and neck molecule parting as claimed in claim 5 application in preparing the test kit for squamous cell carcinoma of the head and neck molecule parting and gene chip.

7. the test kit for squamous cell carcinoma of the head and neck molecule parting, it is characterized in that, including the gene for squamous cell carcinoma of the head and neck molecule parting that can expand described in claim 1 PCR primer and/or can be complementary with the gene order for squamous cell carcinoma of the head and neck molecule parting described in claim 1 probe.

8. the test kit for squamous cell carcinoma of the head and neck molecule parting as claimed in claim 7, it is characterised in that described PCR primer is the PCR primer for squamous cell carcinoma of the head and neck molecule parting described in claim 3.

9. the test kit for squamous cell carcinoma of the head and neck molecule parting as claimed in claim 7, it is characterised in that described probe is the probe for squamous cell carcinoma of the head and neck molecule parting described in claim 5.

10. the gene chip for squamous cell carcinoma of the head and neck molecule parting, it is characterised in that including solid phase carrier and probe, described probe is the probe for squamous cell carcinoma of the head and neck molecule parting described in claim 5.