CN106755372A

CN106755372A - A kind of application of molecular marker in OSCC is diagnosed and is treated

Info

Publication number: CN106755372A
Application number: CN201611136262.6A
Authority: CN
Inventors: 杨承刚; 任静; 程城
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2016-12-12
Filing date: 2016-12-12
Publication date: 2017-05-31
Anticipated expiration: 2036-12-12
Also published as: CN106755372B

Abstract

The invention discloses a kind of application of molecular marker in OSCC is diagnosed and is treated, the biomarker is GDPD2, tested by qPCR and found, GDPD2 genes express downward in oral squamous cell carcinoma and cell, and GDPD2 gene overexpressions can suppress the propagation of cell and reduce the invasion and attack rate of cancer cell.The invention provides the application of the accelerator in OSCC diagnosis and treatment of GDPD2, while the invention also discloses a kind of method for suppressing cell propagation.

Description

A kind of application of molecular marker in OSCC is diagnosed and is treated

Technical field

The invention belongs to biomedicine field, be related to a kind of molecular marker OSCC diagnose and treatment in Application, the specific molecular marker is GDPD2.

Background technology

Oral squamous cell carcinomas are the most commonly seen malignant tumours in oral cavity position and periphery, and it is total that its incidence of disease accounts for oral cavity region tumors More than the 80% of the incidence of disease, is to be worth causing one of malignant disease of people's highest attention and attention.Although in prosperities such as America and Europes The incidence of disease of national oral squamous cell carcinomas has declined, but the incidence of disease is still presented ascendant trend in worldwide.In recent years with The continuous improvement of medical level, the treatment aspect of many malignant tumours is made a lot of progress, and trouble is improve by treatment 5 years survival rates of person.But in terms of the treatment of oral squamous cell carcinomas, because the generation early stage of this disease is difficult to discover etc. special by patient Property, cause the treatment of the disease delayed, the treatment difficulty of cancer of late stage is increased.Thus it is necessary to increase oral cavity squamous cell carcinoma treatment Attention rate, searches out the more preferable treatment method of oral squamous cell carcinomas.

The treatment aspect of oral squamous cell carcinomas, conventional treatment method includes operative treatment, radiotherapy, chemotherapy, immune Treatment etc..Although these treatment methods can suppress tumour to a certain extent developing, but there is larger treatment disadvantage End.Such as radiotherapy, chemotherapy can inevitably cause serious side reaction and operative treatment is difficult the features such as carrying out in the presence of operation, single Pure operative treatment cannot effect a radical cure the defect of the aspects such as tumour.The curative effect of immunization therapy is not obvious.And gene therapy and molecular target It is oncotherapy means emerging in recent years to treatment, because the advantage of the aspects such as its side reaction is small, determined curative effect has obtained section Grind the favor of worker.

Molecular targeted therapy refers to the abnormal signal of modulate tumor related gene, intervention tumour on cellular and molecular level Path or blocking energy pathway, the targeted therapy of the molecular target oral cavity squamous cell carcinoma that searching can be intervened have great importance.

The content of the invention

In order to make up the deficiencies in the prior art, diagnosed for OSCC it is an object of the invention to provide one kind With the biomarker for the treatment of.

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

The invention provides GDPD2 genes or the purposes of its expression product, for preparing diagnosis OSCC Product.

Further, the product is included by by sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies or immune survey The change of fixed method detection GDPD2 genes and/or its expression product.

Further, the nucleic acid amplification technologies are selected from PCR, reverse transcriptase polymerase chain reaction, transcription Jie Amplification, ligase chain reaction, strand displacement amplification and the amplification based on nucleotide sequence led.

The invention provides a kind of product for diagnosing OSCC, during the product can be by detecting sample The change of GDPD2 genes and/or its expression product diagnoses OSCC.

Further, the product includes chip, kit or preparation.

Further, the product includes chip, kit or preparation.Wherein, the chip includes genetic chip, protein Chip；The genetic chip includes solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and the oligonucleotides is visited Pin includes the oligonucleotide probe for GDPD2 genes for detecting GDPD2 gene transcription levels；The protein-chip bag Include solid phase carrier and be fixed on the specific antibody or part of the GDPD2 albumen of solid phase carrier；The kit includes gene Detection kit and protein immunization detection kit；The gene detecting kit is included for detecting GDPD2 genetic transcription water Flat reagent；The protein immunization detection kit includes the specific antibody or part of GDPD2 albumen.

Genetic chip of the present invention or gene detecting kit can be used to detect including the multiple including GDPD2 genes The expression of gene (for example, multiple genes related to OSCC).The protein chip or protein immunization are examined Test agent box can be used to detect that (such as related to OSCC is more including the multiple protein including GDPD2 albumen Individual protein) expression.Multiple marks of OSCC are detected simultaneously, oral cavity squama is greatly improved The accuracy rate of shape cell cancer diagnosis.

The invention provides application of the GDPD2 genes in the pharmaceutical composition for preparing treatment OSCC.

Further, described pharmaceutical composition includes GDPD2 genes and/or the accelerator of its expression product.

Further, the accelerator include GDPD2 gene expression products, promoted type miRNA, promoted type transcriptional control because Son or promoted type targeting micromolecular compound

The invention provides a kind of method for suppressing cell propagation, methods described is to addition GDPD2 bases in cultivating system Cause, albumen or its accelerator.

The invention provides a kind of composition of medicine, the composition of medicine includes above-mentioned pharmaceutical composition and containing 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 NSC-241240, taxane or A kind of taxanes of classification, or both.

The advantages of the present invention：

Present invention firstly discovers that molecular marker GDPD2 and OSCC that development occurs is related, by detecting The change of subject GDPD2, can be used for the diagnosis of oral squamous cell carcinomas.

The invention provides a kind of Molecular tools for treating OSCC, by the molecular target for changing oral squamous cell carcinomas Target is expressed so as to intervene or suppress OSCC.

Brief description of the drawings

Expression of Fig. 1 displays using QPCR detection GDPD2 genes in oral squamous cell carcinoma；

Expression of Fig. 2 displays using QPCR detection GDPD2 genes in OSCC cell；

Transfected condition of Fig. 3 displays using QPCR detection GDPD2 genes in OSCC；

Influences of Fig. 4 display mtt assay detections GDPD2 to oral cavity epidermoid carcinoma cell proliferation activity；

Fig. 5 displays detect the influence of GDPD2 gene pairs OSCC cell invasions using transwell cells.

Specific embodiment

The present invention, by a large amount of screenings, is found that in OSCC first by in-depth study extensively GDPD2 is presented specific low expression.It is demonstrated experimentally that by the special activity for expressing flat or expression product for improving GDPD2, can Effectively suppress the growth and invasion and attack of OSCC, the effect of OSCC is suppressed so as to reach.In this base The present invention is completed on plinth.

GDPD2 albumen is a kind of phosphoglycerol diester phosphodiesterase, and 1- phosphoric acid is produced for hydrolyzing phosphoglycerol inositol Inositol and glycerine.The albumen plays an important role in Osteoblast Differentiation and growth.

Can be used for GDPD2 polynucleotides of the invention or albumen (polypeptide) sequence includes the GDPD2 of various sources and species, And the GDPD2 including wild type, saltant type, or its active fragment.In some embodiments of the present invention, the GDPD2 From the mankind, the coded sequence or amino acid sequence of a kind of representational people GDPD2 are respectively such as SEQ ID NO.1 and SEQ ID Shown in NO.2.

Polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.

Polypeptide of the invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide.It is of the invention many Peptide may also include or not include the methionine residues of starting.

The polynucleotides for encoding the mature polypeptide of GDPD2 include：The coded sequence of encoding mature polypeptide；Mature polypeptide Coded sequence and various additional coding sequences；The coded sequence (and optional additional coding sequence) and non-coding of mature polypeptide Sequence.Term " polynucleotides of coded polypeptide " can be included encoding the polynucleotides of this polypeptide, or also include attached Plus the polynucleotides of coding and/or non-coding sequence.

The invention further relates to the variant of above-mentioned polynucleotides, its coding has many of identical amino acid sequence with the present invention The fragment of peptide or polypeptide, analogs and derivatives.The variant of this polynucleotides can be the natural allelic variant for occurring or The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insert variation.Such as this Known to field, allelic variant is an alternative forms for polynucleotides, it be probably one or more nucleotides substitution, Missing is inserted, but will not be from the function of substantially changing its coded polypeptide.

The invention further relates to the nucleic acid fragment with above-mentioned sequence hybridization, including the just nucleic acid fragment with antisense.Such as this Literary used, the length of " nucleic acid fragment " at least contains 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 cores Thuja acid, more than preferably at least 100 nucleotides.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid determining and/or Separate the polynucleotides of coding GDPD2 albumen.

People GDPD2 nucleotides full length sequence of the invention or its fragment can generally use PCR TRAPs, recombination method or artificial The method of synthesis is obtained.For PCR TRAPs, can be according to published relevant nucleotide sequence, especially ORFs sequence Arrange to design primer, and made with commercially available cDNA storehouses or the cDNA storehouses as prepared by conventional method well known by persons skilled in the art It is template, expands and obtain relevant sequence.When sequence is more long, it is often necessary to carry out twice or multiple PCR is expanded, then again will be each The secondary fragment for amplifying is stitched together by proper order.Once obtain relevant sequence, it is possible to recombination method come large quantities of Amount ground obtains relevant sequence.This is typically to be cloned into carrier, then is transferred to cell, then by conventional method from after propagation Isolated relevant sequence in host cell.

It is of the present invention carry gene carrier be various carriers known in the art, such as commercially available carrier, including plasmid, Clay, bacteriophage, virus etc..Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast Cell；Or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, the bacterium of streptomyces is thin Born of the same parents；Fungal cell's such as yeast；Plant cell；The insect cell of fruit bat S2 or Sf9；The zooblast of CHO, COS or 293 cells Deng.

Converting host cell with recombinant DNA can be carried out with routine techniques well known to those skilled in the art.When host is original When core biology is such as Escherichia coli, the competent cell that can absorb DNA can be harvested after exponential phase of growth, use CaCl₂Method treatment, institute With the step of it is generally well-known in the art.Another method is to use MgCl₂.If desired, conversion can also use the side of electroporation Method is carried out.When host is eucaryote, following DNA transfection methods are can select：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in the cell or on cell membrane.Such as Fruit need, can utilize its physics, chemistry and other characteristics be separated by various separation methods and purification of Recombinant albumen.This A little methods are well-known to those skilled in the art.The example of these methods is included but is not limited to：Conventional renaturation process, use Protein precipitant processes (salting-out method), centrifugation, the broken bacterium of infiltration, super treatment, ultracentrifugation, sieve chromatography (gel filtration), suction Attached chromatography, ion-exchange chromatography, the combination of high performance liquid chroma- tography (HPLC) and other various LC technologies and these methods.

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

Herein comprising the methods availalbe any in the prior art for detecting biomarker expression.Biological marker of the present invention The expression of thing can be detected (e.g., RNA transcript) or protein level in nucleic acid level.It is intended to determine by " detection expression " The quantity of the expression product of RNA transcript or its biomarker genes or presence.Therefore, " detection is expressed " includes a biological mark Will thing is determined to be expressed, can not being detected expression, and expression is in low-level, expression in normal level or the reality of overexpression Example.In order to determine low expression, the examined body sample can compare with the corresponding body sample from Healthy People.That That is, " normal " level of the expression is the expression of biomarker, such as from human subject or not by mouth Biomarker expression level in the cervical tissue sample of the sufferer that chamber squamous cell carcinoma is tormented.This sample can be with standard Change form is presented.In certain embodiments, the determination of biomarker overexpression need to not compare body sample and corresponding from strong The body sample of health people.

Nucleic acid amplification technologies of the present invention are selected from PCR (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleic acid sequence The amplification (NASBA) of row.Wherein, PCR is needed RNA reverse transcriptions into DNA (RT-PCR) before amplification, and TMA and NASBA directly expand Increase RNA.

Generally, PCR is circulated using the annealing of denaturation, primer pair and opposite strand and the multiple of primer extend, with index side Formula increases the copy number of target nucleic acid sequence；Then be used for for reverse transcriptase (RT) to prepare complementary DNA (cDNA) from mRNA by RT-PCR, Then cDNA is expanded by PCR to produce multiple copies of DNA；TMA is in the temperature of substantial constant, ionic strength and pH Under the conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, multiple RNA copies of wherein target sequence are autocatalytically given birth to Into other copy, TMA optionally includes using blocking, part, dwell section and other modification parts, to improve TMA processes Sensitivity and the degree of accuracy；Two groups of complementary DNA oligonucleotides that LCR is hybridized using the adjacent area with target nucleic acid.DNA few nucleosides Acid is covalently attached in the multiple circulations of repetition of thermal denaturation, hybridization and connection by DNA ligase, to produce detectable double-strand Connection oligonucleotide product；SDA is circulated using the multiple of following steps：Primer sequence pair is moved back with the opposite strand of target sequence Fire, primer extend is carried out in the case where there is dNTP α S to produce (hemiphosphorothioated) of the thiophosphorylation of double-strand half Primer extension product, the nicking of endonuclease that semi-modified restriction enzyme enzyme recognition site is carried out mediation, and from cutting The polymerase-mediated thing extension drawn that mouth 3' ends are carried out is put with replacing existing chain and producing for next round primer annealing, nicking and chain The chain for changing, so as to the geometry for causing product is expanded.

Term " probe " refers to that what can be combined with the particular sequence of another molecule or subsequence or other parts divides in the present invention Son.Unless otherwise noted, " probe " is often referred to be matched and another polynucleotides (often referred to as " target multinuclear by complementary base Thuja acid ") combine polynucleotide probes.Lack sufficient sequence according to the preciseness of hybridization conditions, probe energy and with the probe mutual The target polynucleotide of benefit property is combined.Probe can make direct or indirect mark, and its scope includes primer.Crossing system, including, but It is not limited to：Solution, solid phase, mixed phase or in situ hybridization determination method.

As probe, it is possible to use fluorescence labeling, radio-labeled, biotin labeling etc. are carried out to cancer detection with polynucleotides The label probe of mark.The labeling method of polynucleotides is in itself known.Can check by the following method in sample whether There is subject nucleic acid：Fixed subject nucleic acid or its amplified matter, are hybridized with label probe, are washed, and then determine with The mark 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, many nucleosides are used in the cancer detection being incorporated into solid phase Acid is also referred to as probe.The method that subject nucleic acid is determined using polynucleotide probes is also known in this area.Can enter as follows Row the method：Polynucleotide probes are made to be connect (preferably within ± 4 DEG C) near Tm or its with subject nucleic acid in buffer solution Touch for hybridizing, wash, the template nucleic acid for then determining the label probe of hybridization or being combined with solid phase probe.

The polynucleotides used as probe be preferably sized to 18 or more nucleotides, more preferably 20 or More nucleotides, and coding region total length or less.When being used as primer, the polynucleotides are preferably sized to 18 Or more nucleotides, 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 Comprising the various different nucleic acid or peptide probes that substrate surface is connected to according to different known locations.These arrays, also referred to as " microarray ", can generally produce these arrays using mechanical synthesis methods or light guiding synthetic method, and the light guiding is closed The combination of photolithography method and solid phase synthesis process is incorporated into method.Array can include flat surface, or can be pearl Nucleic acid or peptide in son, gel, polymer surfaces, the fiber of such as optical fiber, glass or any other suitable substrate.Can be with Certain mode carrys out array of packages, so as to allow to carry out the diagnosis of global function device or the manipulation of other manner.

In the present invention, term " antibody " refers to the natural or synthetic antibody of selective binding target antigen.The term bag Include polyclonal and monoclonal antibody.In addition to complete immunoglobulin molecules, the fragment of those immunoglobulin molecules or poly- It is " anti-that the mankind of the immunoglobulin molecules of compound and selective binding target antigen or humanization form are also included within term In the range of body ", as long as they show desired BA." monoclonal antibody " refers to from a group substantially homogeneity The antibody that obtains of antibody, that is, each antibody for constituting colony is identical and/or combine same epitope, except production monoclonal antibody During it is issuable may become external, such variant is general with indivisible presence.Such monoclonal antibody is typically wrapped The antibody comprising the polypeptide sequence for combining target is included, wherein target Binding peptide sequence is by including in many polypeptide sequences of comforming Select what single target Binding peptide sequence was obtained in interior process.

Monoclonal antibody also includes " chimeric " antibody, wherein a part for heavy chain and/or light chain and derived from particular species Or the corresponding sequence that belongs in the antibody of specific antibodies classification or subclass is identical or homologous, and the remainder of chain with derived from another One species or the corresponding sequence belonged in the antibody of another antibody isotype or subclass are identical or homologous, and this antibody-like piece Section, as long as they show desired BA.

Polyclonal antibody includes antibody obtained by the immune GDPD2 protein of animal (for example, mouse) to producing human antibody. After chimeric antibody or humanized antibody is prepared for, the amino acid in variable region (for example, FR) and/or constant region can be used Other amino acid substitutions etc..

GDPD2 accelerator in the present invention, when (administration) is administered in treatment, can promote GDPD2 genes and/or The expression of albumen or activity, so as to suppress OSCC.In specific embodiments of the present invention, the GDPD2 promotees Entering agent includes GDPD2 gene expression products, promoted type miRNA, promoted type transcription regulatory factor or promoted type targeting small molecule Compound.

These accelerator can be generally configured in nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, Wherein pH ordinarily be about 5-8, and preferably pH is about 6-8, although pH value can be with the property for being configured material and disease to be treated Disease and be varied from.The pharmaceutical composition for having configured can be administered by conventional route, including but be not limited to orally to Medicine, parenteral administration, it is administered or by implantation by sucking spray delivery, local administration, rectally, nasal administration, cheek Storage medicine device administration.In the present invention term parenteral route include subcutaneous, intracutaneous, intravenous, intramuscular, in joint, intra-arterial, synovial membrane In interior, breastbone, bring up in interior, damage location and intracranial injection or infusion techniques.As long as destination organization can be reached.

Pharmaceutical composition in the present invention, GDPD2 albumen of the present invention or its accelerator and medicine containing safe and effective amount Acceptable carrier or excipient on.This kind of carrier is included but is not limited to：Salt solution, buffer solution, glucose, water, glycerine, second Alcohol and combinations thereof.Pharmaceutical preparation should match with administering mode.Pharmaceutical composition of the invention can be made into injection form, example The aqueous solution such as with physiological saline or containing glucose and other assistant agents is prepared by conventional method.Such as tablet and capsule Etc pharmaceutical composition, may be carried out by conventional means preparation.Pharmaceutical composition such as injection, solution, tablet and capsule preferably exist Prepared under aseptic condition.The dosage of active component is the effective dose for the treatment of.

Pharmaceutical composition of the invention can be used for supplementing the missing or deficiency of endogenic GDPD2 albumen, by improving The expression of GDPD2 albumen or the function of enhancing GDPD2 albumen, so as to treat because oral cavity squamous thin caused by the reduction of GDPD2 albumen Born of the same parents' cancer.

Medicine of the invention can also be with the drug combination of other treatment OSCC, and other therapeutic compound can It is administered simultaneously with main active component, or even is administered simultaneously in same composition.Can also with single composition or The dosage form different from main active component 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.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.

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 carried out to patient.The term includes active treatment, i.e., specially for the purpose of improving disease, pathological state or illness Treatment, and also including etiological treatment, the i.e. treatment for the purpose of removing relevant disease, pathological state or the cause of disease of illness. Additionally, the term also includes palliative treatment, that is, it is designed for controlling for relief of symptoms rather than cure diseases, pathological state or illness Treat；Prophylactic treatment, i.e., at utmost reducing or partially or completely suppress the development of relevant disease, pathological state or illness For the purpose for the treatment of；And supportive treatment, i.e., it is mesh to improve relevant disease, pathological state or illness for supplementing another kind Specific therapy treatment.

Term " sample " refers to the composition obtained from target patient in the present invention, and it includes cell and/or other molecules Body-will be characterized and/or be recognized, for example, according to physics, biochemistry, chemistry and/or physiological characteristic.For example, phrase is " clinical Sample " or " disease sample " and its variant, refer to any sample obtained from target patient, by the expected or known sample It is obtained in that cell and/or molecule body, the biomarker being for example characterized.

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 limitation 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) described in condition, or according to the condition proposed by manufacturer.

Embodiment 1 screens the gene marker related to OSCC

1st, sample collection

It is each to collect 6 surrounding normal mucosal tissues and oral squamous cell carcinoma, confirm own through pathological diagnosis Patient is preoperative not to receive any type for the treatment of.The sample that operation is cut freezes in liquid nitrogen, the equal informed consent of patient, above-mentioned institute There is the acquirement of sample by the agreement of the committee of organizational ethics.

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

Taking-up freezes the tissue samples in liquid nitrogen, tissue samples is put into the mortar of precooling and is ground, according to Specification in kit is extracted and separates RNA.It is specific as follows：

1) Trizol is added, room temperature places 5min；

2) chloroform 0.2ml is added, forced oscillation centrifuge tube is used, is fully mixed, 5-10min is placed at room temperature；

3) 12000rpm centrifugations 15min, (is careful not to be drawn onto two-layer water during upper strata aqueous phase is moved on into another new centrifuge tube Protein substance between phase), the isopropanol of -20 DEG C of isometric precoolings is added, it is fully reverse to mix, it is placed in 10min on ice；

4) 12000rpm carefully discards supernatant after 15min at a high speed, and 75% is added in the ratio of 1ml/ml Trizol DEPC ethanol washing precipitation (4 DEG C of preservations), washing precipitate, vibration is mixed, 4 DEG C, 12000rpm centrifugations 5min；

5) ethanol liquid is discarded, 5min is placed at room temperature, add DEPC water dissolves precipitation；

6) RNA purity and concentration are measured with Nanodrop2000 ultraviolet specrophotometers, is frozen in -70 DEG C of refrigerators.

3rd, reverse transcription and mark

With Low RNA Input Linear Amplification Kit by mRNA reverse transcriptions into cDNA, while using Cy3 Difference labelling experiment group and control group.

4th, hybridize

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

5th, data processing

Chip Agilent scanner scannings after hybridization, resolution ratio is 5 μm, and scanner is automatically with 100% and 10%PMT Each scanning 1 time, 2 times result Agilent softwares merge automatically.Scan image data using Feature Extraction at Reason analysis, the initial data application Bioconductor program bags for obtaining carry out follow-up data treatment.Last Ratio values are experiment Group and control 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 quantity of the GDPD2 genes in oral squamous cell carcinoma is significantly lowered.

The differential expression of the QPCR sequence verification GDPD2 genes of embodiment 2

1st, large sample QPCR checkings are carried out to GDPD2 gene differential expressions.Selected according to the sample collection mode in embodiment 1 Select normal mucosa tissue and each 80 of oral squamous cell carcinoma.

2nd, RNA extraction steps are as described in Example 1.

3rd, reverse transcription：

1) reaction system：

2) reverse transcription reaction condition

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

Coded sequence according to GDPD2 genes and GAPDH genes in Genebank designs QPCR amplimers, by Bo Maide Biotech firm synthesizes.Specific primer sequence is as follows：

GDPD2 genes：

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

Reverse primer is 5 '-CAAGTAGATGACCAGGACAA-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：

The PCR reaction systems of table 1

3) PCR reaction conditions：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, in the enterprising performing PCR reaction of Light Cycler quantitative real time PCR Instruments, is analyzed as fluorescent marker by melt curve analysis Determine purpose band with electrophoresis, Δ Δ CT methods carry out relative quantification, each sample carries out 3 repetitions and tests.

5th, statistical method

With GAPDH as internal reference, the reality of oral squamous cell carcinoma and normal mucosa histofluorescence quantitative RT-PCR is calculated Result is tested, statistical analysis is carried out using SPSS18.0 statistical softwares, difference between the two is checked using t, with P<0.05 tool There is significant difference.

6th, result

As shown in figure 1, compared with control group, GDPD2 genes are expressed in oral squamous cell carcinoma and are remarkably decreased, poor It is different with statistical significance (P<0.05) it is, consistent with RNA-sep results.

Differential expression of the GDPD2 genes of embodiment 3 in OSCC cell line

1st, cell culture

OSCC cell line Tca8113, HN13, normal mucosa epithelial cell line HIOEC is handed over purchased from Shanghai 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, cultivate in the incubator that relative humidity is 90%.2-3 days Change liquid 1 time, passed on using the 0.25% trypsase conventional digestion containing EDTA.

2nd, the extraction of cell total rna

1) culture is terminated when cell is merged up to 80-90%, 0.25% Trypsin Induced is collected cell and managed in 1.5m1EP In, often add lm1Trizol slowly to shake smudge cells in pipe, 10min is placed on ice.

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

Remaining operation step is with RNA extraction process in tissue.

3rd, reverse transcription

Specific steps are with embodiment 2.

4th, statistical method

Experiment is all completed according to being repeated 3 times, and result data is represented in the way of mean+SD, Statistical analysis is carried out using SPSS18.0 statistical softwares, difference between the two is checked using t, it is believed that work as P<Have when 0.05 It is statistically significant.

5th, result

Result is as shown in Fig. 2 compared with normal mucosa epithelial cell, GDPD2 genes are in OSCC cell Expressed in Tca8113, HN13 and lowered, difference has statistical significance (P<0.05) it is, consistent with RNA-sep results.

The overexpression of the GDPD2 genes of embodiment 4

1st, cell culture

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

2nd, the overexpression of GDPD2 genes

The structure of GDPD2 gene overexpression carriers

Coded sequence (as shown in SEQ ID NO.1) design amplimer according to GDPD2 genes, primer sequence is as follows：

Forward primer：5’-CCGAAGCTTGCCACCATGGACTGGTCCCTGGCATT-3’(SEQ ID NO.7)

Reverse primer：5’-CGGCTCGAGCTCCATCATGAAATTGTTGATCC-3’(SEQ ID NO.8)

From cDNA library (clontech companies, article No. into Human fetal spleen：638831) the GDPD2 genes of amplification total length in Coded sequence, above-mentioned cDNA sequence is inserted into through restriction enzyme after restriction enzyme HindIII and XhoI double digestion In the eukaryotic expression vector pcDNA3.1 of HindIII and XhoI double digestions, the recombinant vector pcDNA3.1- for obtaining is connected GDPD2 is used for subsequent experimental.

3rd, transfect

OSCC cell is divided into three groups, respectively control group (Tca8113), blank control group (transfection ) and GDPD2 overexpression group (transfection pcDNA3.1-GDPD2) pcDNA3.1-NC.Turning for carrier is carried out using liposome 2000 Dye, specific transfection method instruction to specifications is carried out.The transfection concentrations of pcDNA3.1 empty carriers and pcDNA3.1-GDPD2 It is 0.5 μ g/ml.

4th, QPCR detects the transcriptional level of GDPD2 genes

The extraction of 4.1 cell total rnas

Specific steps are with embodiment 3.

4.2 reverse transcription steps are with embodiment 2.

4.3 QPCR amplification steps are with embodiment 2.

5th, statistical method

Experiment is all completed according to being repeated 3 times, and result data is represented in the way of mean+SD, Statistical analysis is carried out using SPSS18.0 statistical softwares, the difference between GDPD2 gene overexpressions group and control group uses t Inspection, it is believed that work as P<There is statistical significance when 0.05.

6th, result

Result such as Fig. 3 shows, with non-transfection group compared with empty plasmid group is transfected, overexpression in transfection GDPD2 groups GDPD2, difference has statistical significance (P<0.05).

The mtt assay of embodiment 5 detects Tca8113 cell-proliferation activities

Influence using mtt assay detection GDPD2 gene overexpressions to Tca8113 cell-proliferation activities.

Tca8113 cells are pressed 1 × 10 by the 1st, cell culture³/ hole is inoculated in 96 orifice plates, per 1,37 DEG C of 100 μ of hole, 5%CO₂ Culture is incubated in incubator.

2nd, cell transfecting step is with embodiment 3.

3rd, MTT detections

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

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

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

5th, result：

Result is as shown in figure 4, compared with the control, the cell propagation for transfecting pcDNA3.1-GDPD2 groups is significantly reduced.

The Transwell cells in vitro Matrigels of embodiment 6

Cell transfecting collects the Tca8113 cells of different groups on the 6th day, is resuspended in nutrient solution, makes final concentration of cells be 2 ×10⁶/ ml, 100 μ l cell suspensions of absorption are added in Transwell cells.GDPD2 is observed using Transwell cells method Influence of the gene overexpression to Tca8113 cellular invasiveness.

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

2nd, the inner surface in 6 hole Transwell cells filter membranes is spread with the Matrigel glue in the holes of 100 μ 1/, 37 DEG C, 5%CO₂ 1h is incubated in incubator, a matrix barrier layer is formed standby.

3rd, the DMEM nutrient solutions 2.5m1 containing 20%FBS is added in 6 orifice plates are per hole.

4th, the cell of exponential phase is collected, is resuspended in nutrient solution, final concentration of 2 × 10⁶/ml。

5th, by cell suspension addition Transwell cells, per the μ 1 of hole 100, cell is dipped in the conditioned medium of 6 orifice plates In, 37 DEG C, 5%CO₂24h is incubated in incubator.

6th, Transwell cells are taken out, filter membrane methyl alcohol fixes 1 minute.

7th, HE dyeing：Brazilwood extract dyeing 3min, washing；10~30s of eosin stains, 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, 5 different visuals field is saturating in being counted up and down per film Cell number is crossed, average value is calculated.Every group parallel to set 3 filter membranes.

9th, data processing

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

10th, result

Result is as shown in figure 5, Tca8113, pcDNA3.1-GDPD2, pcDNA3.1-GDPD2 group cell are in transwell After cultivating 24h in cell, the cell number in room face is substantially reduced under pcDNA3.1-GDPD2 group polycarbonate membranes.

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 are improved and modification will be also fallen into the protection domain of the claims in the present invention.

SEQUENCE LISTING

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

<120>A kind of application of molecular marker in OSCC is diagnosed and is treated

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 1383

<212> DNA

<213>People source

<400> 1

atggactggt ccctggcatt cctgctggtc atctctctac tggtcacata tgcatccttg 60

ctattggtcc tggccctgct cctgcggctt tgtagacagc ccctgcatct gcacagcctc 120

cacaaggtgc tgctgctcct cattatgctg cttgtggcgg ctggccttgt gggactggac 180

atccaatggc agcaggagtg gcatagcttg cgtgtgtcac tgcaggccac agccccattc 240

cttcatattg gagcagccgc tggaattgcc ctcctggcct ggcctgtggc tgataccttc 300

taccgtatcc accgaagagg tcccaagatt ctgctactgc tcctattttt tggagttgtc 360

ctggtcatct acttggcccc cctatgcatc tcctcaccct gcatcatgga acccagagac 420

ttaccaccca agcctgggct ggtgggacac cgaggggccc ccatgctggc tcccgagaac 480

accctgatgt ccttgcggaa gacagctgaa tgcggagcta ctgtgtttga gactgatgtg 540

atggtcagct ccgatggggt ccccttcctc atgcatgatg agcacctcag caggaccacg 600

aatgtagcct ctgtattccc aacccgaatc acagcccaca gcagtgactt ctcctggact 660

gaactgaaga gactcaatgc tggatcctgg ttcctagaga ggcgaccctt ctggggggcc 720

aaaccgctgg caggccctga tcagaaagag gctgagagtc agacggtacc agcattagaa 780

gagctattgg aggaagctgc agccctcaac ctttccatca tgttcgactt gcgccgaccc 840

ccacagaacc acacatacta tgacactttt gtgatccaga cattggagac tgtgctgaat 900

gcaagggtgc cccaagccat ggtcttttgg ctaccagatg aagatcgggc taatgtccaa 960

cgacgggcac ctggaatgcg ccagatatat ggacgtcagg gaggcaacag aacggagagg 1020

ccccagtttc ttaacctccc ctatcaagat ctgccactat tggatatcaa ggcattgcat 1080

aaggataatg tctcggtgaa cctatttgta gtgaacaagc cctggctctt ctctctgctt 1140

tggtgtgcag gggtggattc ggtcaccacc aacgactgcc agctgctgca gcagatgcgt 1200

taccctatct ggcttattac ccctcaaacc tacctaatca tatgggtcat taccaattgt 1260

gtttccacca tgctgctttt gtggaccttc ctcctccaaa gaagatttgt taagaagaga 1320

gggaaaactg gcttagaaac agcagtgctg ctgacaagga tcaacaattt catgatggag 1380

tga 1383

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Met Asp Trp Ser Leu Ala Phe Leu Leu Val Ile Ser Leu Leu Val Thr

1 5 10 15

Tyr Ala Ser Leu Leu Leu Val Leu Ala Leu Leu Leu Arg Leu Cys Arg

20 25 30

Gln Pro Leu His Leu His Ser Leu His Lys Val Leu Leu Leu Leu Ile

35 40 45

Met Leu Leu Val Ala Ala Gly Leu Val Gly Leu Asp Ile Gln Trp Gln

50 55 60

Gln Glu Trp His Ser Leu Arg Val Ser Leu Gln Ala Thr Ala Pro Phe

65 70 75 80

Leu His Ile Gly Ala Ala Ala Gly Ile Ala Leu Leu Ala Trp Pro Val

85 90 95

Ala Asp Thr Phe Tyr Arg Ile His Arg Arg Gly Pro Lys Ile Leu Leu

100 105 110

Leu Leu Leu Phe Phe Gly Val Val Leu Val Ile Tyr Leu Ala Pro Leu

115 120 125

Cys Ile Ser Ser Pro Cys Ile Met Glu Pro Arg Asp Leu Pro Pro Lys

130 135 140

Pro Gly Leu Val Gly His Arg Gly Ala Pro Met Leu Ala Pro Glu Asn

145 150 155 160

Thr Leu Met Ser Leu Arg Lys Thr Ala Glu Cys Gly Ala Thr Val Phe

165 170 175

Glu Thr Asp Val Met Val Ser Ser Asp Gly Val Pro Phe Leu Met His

180 185 190

Asp Glu His Leu Ser Arg Thr Thr Asn Val Ala Ser Val Phe Pro Thr

195 200 205

Arg Ile Thr Ala His Ser Ser Asp Phe Ser Trp Thr Glu Leu Lys Arg

210 215 220

Leu Asn Ala Gly Ser Trp Phe Leu Glu Arg Arg Pro Phe Trp Gly Ala

225 230 235

Lys Pro Leu Ala Gly Pro Asp Gln Lys Glu Ala Glu Ser Gln Thr Val

245 250 255

Pro Ala Leu Glu Glu Leu Leu Glu Glu Ala Ala Ala Leu Asn Leu Ser

260 265 270

Ile Met Phe Asp Leu Arg Arg Pro Pro Gln Asn His Thr Tyr Tyr Asp

275 280 285

Thr Phe Val Ile Gln Thr Leu Glu Thr Val Leu Asn Ala Arg Val Pro

290 295 300

Gln Ala Met Val Phe Trp Leu Pro Asp Glu Asp Arg Ala Asn Val Gln

305 310 315 320

Arg Arg Ala Pro Gly Met Arg Gln Ile Tyr Gly Arg Gln Gly Gly Asn

325 330 335

Arg Thr Glu Arg Pro Gln Phe Leu Asn Leu Pro Tyr Gln Asp Leu Pro

340 345 350

Leu Leu Asp Ile Lys Ala Leu His Lys Asp Asn Val Ser Val Asn Leu

355 360 365

Phe Val Val Asn Lys Pro Trp Leu Phe Ser Leu Leu Trp Cys Ala Gly

370 375 380

Val Asp Ser Val Thr Thr Asn Asp Cys Gln Leu Leu Gln Gln Met Arg

385 390 395 400

Tyr Pro Ile Trp Leu Ile Thr Pro Gln Thr Tyr Leu Ile Ile Trp Val

405 410 415

Ile Thr Asn Cys Val Ser Thr Met Leu Leu Leu Trp Thr Phe Leu Leu

420 425 430

Gln Arg Arg Phe Val Lys Lys Arg Gly Lys Thr Gly Leu Glu Thr Ala

435 440 445

Val Leu Leu Thr Arg Ile Asn Asn Phe Met Met Glu

450 455 460

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taccgtatcc accgaaga 18

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caagtagatg accaggacaa 20

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ctctggtaaa gtggatattg t 21

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ggtggaatca tattggaaca 20

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ccgaagcttg ccaccatgga ctggtccctg gcatt 35

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cggctcgagc tccatcatga aattgttgat cc 32

Claims

The purposes of 1.GDPD2 genes or its expression product, it is characterised in that the product for preparing diagnosis OSCC Product.
2. application according to claim 1, it is characterised in that the product is included by miscellaneous by sequencing technologies, nucleic acid The method detection GDPD2 genes of friendship technology, nucleic acid amplification technologies or immunoassays and/or the change of its expression product.
3. application according to claim 2, it is characterised in that the nucleic acid amplification technologies be selected from PCR, Reverse transcriptase polymerase chain reaction, the amplification of transcriptive intermediate, ligase chain reaction, strand displacement amplification and based on nucleotide sequence Amplification.
4. a kind of product for diagnosing OSCC, it is characterised in that the product can be by detecting GDPD2 in sample The change of gene and/or its expression product diagnoses OSCC.
5. product according to claim 4, it is characterised in that the product includes chip, kit or preparation.
6. product according to claim 5, it is characterised in that the chip, kit or preparation include being directed to GDPD2 bases The antibody or part of the specific primer, probe or the albumen for GDPD2 codings of cause.
Application of the 7.GDPD2 genes in the pharmaceutical composition for preparing treatment OSCC.
8. application according to claim 6, it is characterised in that described pharmaceutical composition includes GDPD2 genes and/or its table Up to the accelerator of product.
9. a kind of pharmaceutical composition for treating OSCC, it is characterised in that described pharmaceutical composition GDPD2 genes And/or the accelerator of its expression product.
10. a kind of method for suppressing cancer cell multiplication, it is characterised in that to added in cultivating system GDPD2 genes, albumen or its Accelerator.