CN104611449B - Application of the WWP1 genes in osteosarcoma diagnostic products and medicine is prepared - Google Patents

Abstract

The invention discloses the application of WWP1 genes, WWP1 genes can be used for the product for preparing diagnosis osteosarcoma, and WWP1 genes make the diagnosis of osteosarcoma more accurate, quick as the specific marker gene of diagnosis osteosarcoma.The product of diagnosis osteosarcoma includes：With the product of RT PCR, real-time quantitative PCR, immune detection, in situ hybridization or gene chip diagnosis osteosarcoma.The WWP1 genes of the present invention can be additionally used in the medicine for preparing treatment osteosarcoma, and new therapy target and effective new drug is provided to prevent and treat osteosarcoma.

Description

Application of the WWP1 genes in osteosarcoma diagnostic products and medicine is prepared

Technical field

The invention belongs to biomedical sector, it is related to the new application of WWP1 genes, and in particular to WWP1 genes are examined in preparation Application in knochenbruch sarcoma product and preparation treatment bone and flesh tumor medicine.

Background technology

Osteosarcoma is derived from the malignant tumour of mesenchymal tissue, and its principal causative is characterized as that the tumour cell bred in vivo is straight Connect to form prematurity bone or osteoid tissue.It is a kind of most common primary malignant tumor of human skeletal system.Typical bone and flesh Knurl is a kind of rare (account for whole malignant tumours 0.2%) high carcinogenic malignant tumour, the about annual each million people of its incidence of disease In have three.Osteosarcoma mainly appears on longer bone and least a portion of soft tissue.Its principal pathogenetic crowd concentrates on 10 ~20 years old teenagers, high with the incidence of disease, the early stage rate of transform is high, cures the low feature of survival rate.X-ray, tomography skill Art, nuclear magnetic resonance, Angiography and dynamic scintigraphy technology etc., which are widely used in the sick diagnosis, tumour, to be occurred Degree and type of surgery judge etc..But the diagnosis of osteosarcoma is carried out using above-mentioned clinical means, frequently result in patient's The state of an illness is delayed, and misses optimal treatment period.Therefore it is urgent problem to be solved to find a kind of osteosarcoma early diagnosis marker.

High flux transcript profile sequencing (RNA-sep) is the technology that deep sequencing is carried out in transcript profile level, sequencing Application of the technology in transcriptome analysis is concentrated mainly on the structure of gene expression profile, the discovery of new gene, small molecule non-coding Discovery, protein coding gene annotation and such as fusion of the structure and new micro RNA genes of rna expression spectrum In the research of the transcript profiles such as transcript.It is substantially similar that data are in initial processing, is next ground for different Studying carefully purpose can be calculated from different bioinformatics softwares.

Using large scale sequencing technology directly to by mRNA reverse transcriptions into cDNA sequence be sequenced, produce number with necessarily The reads quantity of meter, so that the transcriptional level of one section of special genome area directly can arrive the gene by comparing The reads numbers in region are organized to weigh.The technology is commonly used to build the gene expression profile of a certain particular organization or cell, by aligning Often tissue and pathological tissues carry out the analysis of gene expression profile, obtain the gene of differential expression in being organized at two kinds, are on the one hand The molecular mechanism of the occurrence and development of study of disease provides new thinking, and the on the other hand diagnosis for disease and treatment is provided newly Diagnosis marker or drug target.

Using high flux transcript profile sequencing screening and the closely related gene of osteosarcoma, not only contribute to study osteosarcoma The pathomechanism of occurrence and development, and new diagnosis marker or new medicine target can be provided to diagnose and treating osteosarcoma Point.

The content of the invention

The present invention using high throughput sequencing technologies find expression of the WWP1 genes in osteosarcoma tissue with normal bone group Expression in knitting has differences, compared with normal bone tissues, WWP1 genes up-regulated expression in osteosarcoma tissue, passes through detection The expression of WWP1 genes, it is possible to judge whether subject suffers from osteosarcoma.

An object of the present invention is to provide application of the WWP1 genes in the product for preparing diagnosis osteosarcoma.

The second object of the present invention is to provide application of the WWP1 genes in the medicine for preparing treatment osteosarcoma.

To achieve these goals, present invention employs following technical scheme：

The invention provides a kind of product for diagnosing osteosarcoma, the product of the diagnosis osteosarcoma by detect subject into In osteocyte the expression of WWP1 genes come judge subject whether suffer from osteosarcoma.

Further, the product of the diagnosis osteosarcoma includes：It is miscellaneous with RT-PCR, real-time quantitative PCR, immune detection, original position Friendship or the product of gene chip diagnosis osteosarcoma.It is described at least to include a pair of specific amplifieds with the RT-PCR products for diagnosing osteosarcoma The primer of WWP1 genes；The product for diagnosing osteosarcoma with real-time quantitative PCR at least includes a pair of specific amplified WWP1 genes Primer；The product for diagnosing osteosarcoma with immune detection includes：The antibody combined with WWP1 protein-specifics；It is described to use former The product of position hybridization diagnosis osteosarcoma includes：With the probe of the nucleic acid array hybridizing of WWP1 genes；It is described to use gene chip diagnosis The product of osteosarcoma includes：With the probe of the nucleic acid array hybridizing of WWP1 genes.

The primer that the specific amplified WWP1 genes included in osteosarcoma product are diagnosed using RT-PCR is to utilize Primer5.0 Primer-design software or other conventional primer online softwares are designed.To those skilled in the art, according to The amplimer that major gene sequences Design goes out can be used in RT-PCR experiments is that need not to pay creative work i.e. achievable. Therefore the present invention RT-PCR specific amplified WWP1 genes primer refer to it is all can RT-PCR experiment in successfully expand Increase the primer sequence of WWP1 genes.In specific embodiments of the present invention, the WWP1 gene amplification primers tested for RT-PCR Thing sequence is as follows：Forward primer：5’-CCAGAACAACAACGTGGCAG-3’(SEQ ID NO：1)；Reverse primer：5’- CTGCCGGGACACATTGATCT-3’(SEQ ID NO：2).

The primer for the specific amplified WWP1 genes that the product for diagnosing osteosarcoma with real-time quantitative PCR is included is according to often QPCR primer online softwares are designed.To those skilled in the art, energy is gone out according to known sequences Design The amplimer for being enough in QPCR experiments is that need not to pay creative work i.e. achievable.Therefore the real-time quantitative of the present invention The primer of PCR specific amplified WWP1 genes refer to it is all can QPCR experiment in Successful amplification WWP1 genes primer sequence Row.In specific embodiments of the present invention, the WWP1 gene magnification primer sequences tested for QPCR are as follows：Forward primer： 5’-AGAAGGACTTGATTATGGT-3’(SEQ ID NO：3)；Reverse primer：5’-TAATGGTTGATGCTGGAT-3’(SEQ ID NO：4).

Probe with the nucleic acid array hybridizing of WWP1 genes can be that DNA, RNA, DNA-RNA chimera, PNA or other spread out It is biological.The length of the probe is not limited, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence, is appointed What length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of the probe Degree can be grown to 60,80,100,150,300 base-pairs or longer, or even whole gene.Because different probe lengths is to hybridization Efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, most long to be usually no more than 30 base-pairs, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe self-complementary sequences Most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.

The antibody combined with WWP1 protein-specifics can be polyclonal antibody or monoclonal antibody.It is above-mentioned anti- Body can be obtained from commercial channels, and a series of methods known in the art can also be used to prepare.For example, by the people of purifying WWP1 albumen or its antigen fragment are injected into animal body to produce polyclonal antibody.Equally, expression people WWP1 albumen or it The cell of antigen fragment may also be used for causing animal immune and produce antibody.Monoclonal antibody can use hybridoma technology system It is standby.The antibody of WWP1 albumen includes that the antibody of WWP1 protein functions can be prevented or does not influence people's WWP1 protein functions Antibody.Each antibody-like can be caused immune and produced by the segment to people's WWP1 albumen or functional domain, and people's WWP1 eggs White product and its fragment can be produced with recombination method or synthesized with Peptide synthesizer.With the WWP1 albumen of non-modified form With reference to antibody, it is possible to use the gene outcome produced in prokaryotic such as E.coli obtains animal is immunized.With turning over Translate rear modified forms such as the antibody glycosylated or phosphorylation WWP1 albumen or polypeptide are combined, it is possible to use in eukaryotic such as yeast Or the gene outcome produced in insect cell obtains animal is immunized.

The invention provides application of the WWP1 genes in the product for preparing diagnosis osteosarcoma.

The product of the diagnosis osteosarcoma is by detecting that the expression of WWP1 genes in subject's Gegenbaur's cell is tested to judge Whether person suffers from osteosarcoma.The product of the diagnosis osteosarcoma includes：With RT-PCR, real-time quantitative PCR, immune detection, original position Hybridization or the product of gene chip diagnosis osteosarcoma.It is described at least to include a pair of special expansions with the RT-PCR products for diagnosing osteosarcoma Increase the primer of WWP1 genes；The product for diagnosing osteosarcoma with real-time quantitative PCR at least includes a pair of specific amplified WWP1 bases The primer of cause；The product for diagnosing osteosarcoma with immune detection includes：The antibody combined with WWP1 protein-specifics；It is described to use The product of in situ hybridization diagnosis osteosarcoma includes：With the probe of the nucleic acid array hybridizing of WWP1 genes；It is described to be examined with genetic chip The product of knochenbruch sarcoma includes：With the probe of the nucleic acid array hybridizing of WWP1 genes.

The primer that the specific amplified WWP1 genes included in osteosarcoma product are diagnosed using RT-PCR is to utilize Primer5.0 Primer-design software or other conventional primer online softwares are designed.To those skilled in the art, according to The amplimer that major gene sequences Design goes out can be used in RT-PCR experiments is that need not to pay creative work i.e. achievable. Therefore the present invention RT-PCR specific amplified WWP1 genes primer refer to it is all can RT-PCR experiment in successfully expand Increase the primer sequence of WWP1 genes.In specific embodiments of the present invention, the WWP1 gene amplification primers tested for RT-PCR Thing sequence is as follows：Forward primer：5’-CCAGAACAACAACGTGGCAG-3’(SEQ ID NO：1)；Reverse primer：5’- CTGCCGGGACACATTGATCT-3’(SEQ ID NO：2).

The primer for the specific amplified WWP1 genes that the product for diagnosing osteosarcoma with real-time quantitative PCR is included is according to often QPCR primer online softwares are designed.To those skilled in the art, energy is gone out according to known sequences Design The amplimer for being enough in QPCR experiments is that need not to pay creative work i.e. achievable.Therefore the real-time quantitative of the present invention The primer of PCR specific amplified WWP1 genes refer to it is all can QPCR experiment in Successful amplification WWP1 genes primer sequence Row.In specific embodiments of the present invention, the WWP1 gene magnification primer sequences tested for QPCR are as follows：Forward primer： 5’-AGAAGGACTTGATTATGGT-3’(SEQ ID NO：3)；Reverse primer：5’-TAATGGTTGATGCTGGAT-3’(SEQ ID NO：4).

Probe with the nucleic acid array hybridizing of WWP1 genes can be that DNA, RNA, DNA-RNA chimera, PNA or other spread out It is biological.The length of the probe is not limited, as long as completing specific hybrid, being specifically bound with purpose nucleotide sequence, is appointed What length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of the probe Degree can be grown to 60,80,100,150,300 base-pairs or longer, or even whole gene.Because different probe lengths is to hybridization Efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, most long to be usually no more than 30 base-pairs, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe self-complementary sequences Most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.

The antibody combined with WWP1 protein-specifics can be polyclonal antibody or monoclonal antibody.It is above-mentioned anti- Body can be obtained from commercial channels, and a series of methods known in the art can also be used to prepare.For example, by the people of purifying WWP1 albumen or its antigen fragment are injected into animal body to produce polyclonal antibody.Equally, expression people WWP1 albumen or it The cell of antigen fragment may also be used for causing animal immune and produce antibody.Monoclonal antibody can use hybridoma technology system It is standby.The antibody of WWP1 albumen includes that the antibody of WWP1 protein functions can be prevented or does not influence people's WWP1 protein functions Antibody.Each antibody-like can be caused immune and produced by the segment to people's WWP1 albumen or functional domain, and people's WWP1 eggs White product and its fragment can be produced with recombination method or synthesized with Peptide synthesizer.With the WWP1 albumen of non-modified form With reference to antibody, it is possible to use the gene outcome produced in prokaryotic such as E.coli obtains animal is immunized.With turning over Translate rear modified forms such as the antibody glycosylated or phosphorylation WWP1 albumen or polypeptide are combined, it is possible to use in eukaryotic such as yeast Or the gene outcome produced in insect cell obtains animal is immunized.

The product of the diagnosis osteosarcoma of the present invention is selected from the following group：Kit, chip, filter paper.Mentioned reagent box can Be detect gene transcription level kit, such as RT-PCR kit, QPCR kits or detection gene expression water Flat kit, such as ELISA kit.Said chip can be genetic chip or protein chip, and it being capable of testing goal base The expression of cause.

The RT-PCR kit in addition to the primer comprising specific amplified WWP1 genes, can also comprising PCR buffer solutions, dNTPs、MgCl₂, Tap archaeal dna polymerases.The RT-PCR kit can also include RNA extracts reagents, RNase extracts reagent bag Containing Trizol, chloroform, isopropanol, 75% ethanol.

The QPCR kits can also include SYBR Green polymerizations in addition to the primer comprising specific amplified WWP1 genes PCR system.SYBR Green PCRs system includes PCR buffer solutions, dNTPs, SYBR Green fluorescence Dyestuff.

Preferably, PCR buffer solutions are included：25mM KCI, 2.5mM MgCl₂, 200mM (NH₄)₂SO₄。

The QPCR kits can also include M-MLV reverse transcription systems, and the reverse transcription system is included：T repeats oligonucleotides Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptases, RNase inhibitor, dNTPs.

Preferably, reverse transcription reaction liquid is included：250mM pH8.3 Tris-HCL, 375mM KCL, 15mM MgCl₂, 50mM DTT.

RNase inhibitor can select RNase inhibitor commonly used in the art, and preferably Bacillus coli expression is noncompetitive Suppress the recombinant protease of RNase.

The QPCR kits can also include RNA extracts reagents, and RNase extracts reagent includes Trizol, chloroform, isopropyl Alcohol, 75% ethanol.

The ELISA kit except comprising in addition to the antibody that WWP1 protein-specifics are combined, also comprising coating buffer solution, Cleaning solution, confining liquid, sample loading buffer, reaction terminating liquid, destination protein standard items.

Preferably, coating buffer solution is phosphate buffer PBS, and its composition is 140mM NaCl, 2.7mM KCl, 10mM Na₂HPO₄, 1.8mM KH₂PO₄, pH value is 7.4.

Preferably, cleaning solution is the PBS containing 1%Tween-20, and referred to as PBST, its composition is PBS, 1%Tween-20.

Preferably, confining liquid is the PBS containing 1%BSA, and its composition is PBS, 1%BSA.

Preferably, the composition of sample loading buffer is 50mM Tris-HCl, 0.5M KCl, 1%BSA, 0.05%Tween- 20, pH value is 8.4.

Preferably, reaction terminating liquid composition is 2M H₂SO₄。

Present invention also offers application of the WWP1 genes in the medicine for preparing treatment osteosarcoma.

Further, the medicine of the treatment osteosarcoma can be the medicine for suppressing human osteosarcoma cell proliferation, can promote The medicine of apoptosis in osteosarcoma cells, can be the medicine for suppressing osteosarcoma cell migration, can suppress osteosarcoma cell invasion and attack Medicine, can be suppress osteosarcoma cell into knurl medicine.

Further, the medicine for the treatment of osteosarcoma of the present invention includes：WWP1 gene expressions are suppressed by RNA interfering Double stranded RNA, or the tumor vaccine based on WWP1 antigen proteins or the protein for suppressing WWP1 protein actives.

In the present invention, the RNA interference (RNA interference, RNAi) refers to be highly conserved during evolution , induced by double-stranded RNA (double-stranded RNA, dsRNA), the phenomenon of the efficient selective degradation of homologous mRNA.Make With RNAi technology can with specific depletion or close specific gene expression, the technology have been widely used for explore gene function and The field of gene of communicable disease and malignant tumour.RNAi based on cell is screened in terms of functional gene research With many advantages, RNAi methods can be used by being mainly manifested in most cell types, and is easier to lower or is sunk relatively The expression of silent any target gene.

In order to ensure WWP1 genes can be rejected efficiently or silence, devised according to the mRNA sequence of WWP1 genes SiRNA specific fragments.SiRNA design according to delivered general design principle (Elbashir et.al 2001, Schwarz et.al 2003, Khvorova et.al 2003, Reynolds et.al 2004, Hsieh et.al 2004, Ui-Tei et.al 2004), by online tool complete design, the online tool is：siRNASelectionProgram of Whitehead Institute (BingbingYuan et.al 2004, http：//jura.wi.mit.edu/bioc/ ) and BLOCK-iTTM RNAi Designer ofINVITROGEN (winner of the 2004Frost＆ siRNAext/ Sullivan Excellence in Research Award, https：//maidesigner.invitrogen.com/ sirna/).In order to further improve the validity of siRNA segments, integrate the advantage of two Photographing On-line instruments to be designed for The siRNA segments of screening.Finally, siRNA sequence is filtered by sequence analysis (NCBI BLAST), to improve siRNA pieces Disconnected effect of missing the target that is specific and reducing RNAi interference.SiRNA oligonucleotides is by Shanghai JiMa pharmacy Technology Co., Ltd Learn synthesis.The present invention embodiment in, for WWP1 siRNA sequence (siRNA-WWP1) specifying information such as Under：- the ACAAAGAUGAGUAAGACACGA-3 ' of positive-sense strand 5 ' ,-GUGUCUUACUCAUCUUUGUAU-3 ' of antisense strand 5 '.Set simultaneously Count negative control siRNA (siRNA-NC) positive-sense strand：5’-CGUACGCGGAAUACUUCGA-3’(SEQ ID NO：5)；Antisense Chain：5’-UCGAAGUAUUCCGCGUACG-3’(SEQ ID NO：6).

The tumor vaccine refers to excite the immunologic function in patient's body with the particular matter in tumor tissues, makes patient Immunologic function being capable of killing tumor cell.Tumor vaccine refers mainly to therapeutic vaccine, is to obtain to use vaccine again after being ill in patient Method is treated, to control the recurrence and transfer of tumour.Tumor vaccine has individuality, because swollen obtained by each patient Knurl type, by stages all different, the material expressed by tumour cell is also different, and tumor vaccine is the tumour by patient in itself It is made, therefore the tumor vaccine prepared for the specific tumour of patient has individuation feature, its Small side effects is with strong points.

When prepared by the tumor vaccine, with the tumor tissues and sterile freezen protective of operation method excised tumor patient, with Extract tumour antigen.It is the first step to obtain tumour antigen, then to obtain BMDC, the i.e. single core from patient blood thin Obtained in born of the same parents.Screened again and in vitro culture after extraction mononuclearcell, then stimulate dendron shape in vitro with tumour antigen Cell, enables the BMDC premunition information, finally feeds back to the BMDC trained in vivo, makes in vivo Produce the immune response for tumour antigen.

Present invention also offers applications of the above-mentioned siRNA for WWP1 in the medicine for preparing treatment osteosarcoma.It is described The medicine for the treatment of osteosarcoma can be the medicine for suppressing human osteosarcoma cell proliferation, can be the medicine for promoting apoptosis in osteosarcoma cells Thing, can be the medicine for suppressing osteosarcoma cell migration, can suppress the medicine of osteosarcoma cell invasion and attack, can also be suppression Medicine of the osteosarcoma cell into knurl.

Said medicine can also include pharmaceutically common pharmaceutical carrier in addition to comprising siRNA.

The method of application of the medicine can be oral, systemic administration (for example, through skin, nasal inhalation or use suppository) Or parenteral administration (for example, intramuscular, intravenously or subcutaneously).

The medicine can be made into a variety of formulations, such as tablet, injection, capsule.With physiological saline or containing glucose and The aqueous solution of other assistant agents prepares medicine injection by conventional method progress.The medicine of tablet and capsule etc, also can be by this It is prepared by art personnel conventional method.

The advantages of the present invention：

(1) present invention firstly discloses WWP1 genes are related to osteosarcoma, WWP1 genes are expected to turn into diagnosis osteosarcoma Molecular marker, and provide new thinking for the molecule mechanism for studying osteosarcoma.

(2) method for diagnosing the presence or absence of osteosarcoma using the mode for detecting gene expression is sensitiveer, is conducive to disease The diagnosis of sick early stage.

Brief description of the drawings

Fig. 1 displays detect differential expression of the WWP1 genes in osteosarcoma tissue and normal bone tissues using RT-PCR；

Fig. 2 displays detect that expression of the WWP1 genes in osteosarcoma tissue and normal bone tissues is poor using real-time quantitative PCR It is different；

Fig. 3 displays detect the RNA interfering for WWP1 to WWP1 gene expressions using real-time quantitative PCR on transcriptional level Influence；

Fig. 4 detects the influence that WWP1 gene expressions are bred to osteosarcoma cell line U2OS using mtt assay.

Embodiment

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 method 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 related to osteosarcoma

1.1 sample collection

It is each to collect 8 normal bone tissues, the samples of osteosarcoma tissue.

The preparation and quality analysis of 1.2 RNA samples

1.2.1 the preparation of RNA sample

Normal bone tissues and osteosarcoma tissue judge RNA sample quality after RNA is extracted through agarose gel electrophoresis, close Lattice person can be used for further transcriptome analysis.

1.2.2 the quality analysis (NanoDrop1000 spectrophotometers) of RNA sample

NanoDrop1000 spectrophotometers detect RNA sample, the sample requirement of RNA-seq sequencings：OD260/OD280 is 1.8-2.2。

1.2.3 the quality analysis (Agilent Technologies2100Bioanalyzer) of RNA sample

The Bioanalyzer of Agilent Technologies 2100 detect RNA sample quality, observation 28S rRNA and Substantially, without degraded, the RNA-seq that meets that RNA Perfection Index is qualified, concentration reaches requirement cDNA texts are sequenced in 18S rRNA master tapes The requirement that storehouse is built, can be used for library construction and sequencing.

1.3 high flux transcript profiles are sequenced

1.3.1 RNA-seq reads are positioned

First by low-quality read remove obtain clean read, then using TopHat v1.3.1 will clean fragment and UCSC H.sapiens reference genes groups (hg19) are matched, the index of H.sapiens UCSC hg19 editions advance structure Downloaded from TopHat homepages, and as reference gene group, when being matched using TopHat with genome, it is allowed to each read (acquiescence To 20) having multiple matching sites, most 2 mispairing.TopHat sets up possible according to exon region and GT-AG shear signals Shearing site storehouse, navigates to the read for not navigating to genome on genome according to these shearing site storehouses.We use The system default parameter of TopHat methods.

1.3.2 transcript abundance is assessed

The read file matched is by Cuffiinks v1.0.3 processing, and Cuffiinks v1.0.3 are by RNA-seq pieces Hop count mesh is standardized the relative abundance for calculating transcript.FPKM values refer to it is every 1,000,000 sequencing fragment in match it is specific The segment number of the exon region of gene 1kb length.The confidential interval of FPKM estimates is calculated by Bayesian inference method. The GTF comment files for the reference that Cuffiinks is used download (Homo_ from Ensembl databases sapiens.GRCh37.63.gtf)。

1.3.3 the detection of difference expression gene

The original document matched by the Ensembl GTF files of download and by TopHat is transferred to Cuffdiff, Cuffdiff re-evaluates the gene expression abundance for the transcript listed in GTF files using original matching files, detects difference table Reach.The only q values ＜ 0.01 in Cuffidff outputs, test display is considered as successfully more just differential expression.

1.4 result

The RNA-seq sequencing results of early stage filter out the gene of 979 difference expression genes, wherein expression up-regulation altogether 472, the gene 507 that expression is lowered.

The large sample PCR of embodiment 2 verifies the relation of candidate gene and osteosarcoma

Consideration yet there are no the gene studied on the gene with osteosarcoma correlation as candidate's base in the prior art Cause, while considering the result of early stage high flux transcript profile deep sequencing, WWP1 gene (its table is selected according to P value size Raised up in osteosarcoma tissue) verified.30 osteosarcoma tissues are collected, while 20 normal bone tissues samples are collected, Classical molecular biology experiment checking is carried out using PCR, concrete operation step is as follows：

1st, RNA is extracted

Collect to freeze after sample and be ground in tissue is put into the mortar of precooling after liquid nitrogen, taking-up, treat tissue sample This is into after powdered：

1. Trizol, room temperature preservation 5min are added；

2. chlorination imitates 0.2ml, uses forced oscillation centrifuge tube, fully mixes, 5min-10min is placed at room temperature；

3. upper strata aqueous phase (inhaling 70%) is drawn after 12000rpm high speed centrifugations 15min into another new centrifuge tube, is noted not It is drawn onto the protein substance between two layers of aqueous phase.New pipe is moved into, -20 DEG C of isometric pre- cold isopropanols are added, it is fully reverse mixed It is even, it is placed in 10min on ice；

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

5. ethanol liquid is discarded, 5min is placed at room temperature fully to dry precipitation, the treated water dissolvings of DEPC are added heavy Form sediment；

6. with Nanodrop2000 ultraviolet specrophotometers measurement RNA purity and concentration, freeze in -70 DEG C.

2nd, reverse transcription

Reverse transcription synthesis cDNA is carried out to 1 μ g total serum IgEs with RT Buffer.Using 25 μ L reaction systems, each sample Take 1 μ g total serum IgEs as template ribonucleic acid, following components is separately added into PCR pipe：DEPC water, 5 × RT Buffer, 10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations 1h, 72 DEG C of 10min, of short duration centrifugation.

3rd, RT-PCR is expanded

WWP1：Forward primer is 5 '-CCAGAACAACAACGTGGCAG-3 ' (SEQ ID NO：1)；

WWP1：Reverse primer is 5 '-CTGCCGGGACACATTGATCT-3 ' (SEQ ID NO：2).

β-actin：Forward primer is 5 '-CATCCTGCGTCTGGACCT-3 ' (SEQ ID NO：7)；

β-actin：Reverse primer is 5 '-GTACTTGCGCTCAGGAGGAG-3 ' (SEQ ID NO：8).

Each composition in reactant mixture：Forward primer, reverse primer, 10 × PCR buffer solutions, MgCl₂、dNTP、Taq DNA Polymerase, the amount of cDNA templates are respectively 0.2,0.2,0.4,0.4,1.0,1.0,0.2,0.1 and 5 μ L, finally supplement double steam Water makes reaction system be 10 μ L.PCR reaction condition is as follows：94 DEG C, 5min pre-degenerations；94 DEG C, 30s denaturation；55 DEG C, 30s is moved back Fire；72 DEG C, 30s extensions；Totally 35 circulations, electrophoresis detection pcr amplification product.Amount of DNA, bar are weighed with the brightness of DNA bands Band is brighter, and representation DNA amount is more.Internal reference control is made with β-actin, the brightness of the band of WWP1 gene amplification products carried out equal One change is handled, and compares the ratio of the brightness of WWP1 gene amplification products in normal bone tissues and osteosarcoma tissue.As a result such as Fig. 1 institutes Show, compared with normal bone tissues, WWP1 genes osteosarcoma tissue up-regulated expression, it is consistent with RNA-sep results.

4th, QPCR is expanded

Using 25 μ L reaction systems, each sample sets 3 parallel pipes, all amplified reactions in triplicate more than to protect Demonstrate,prove the reliability of result.Prepare following reaction system：The μ L of SYBR Green PCRs system 12.5, forward primer (5 μm ol/ μ l) 1 μ L, reverse primer (5 μm of ol/ μ l) 1 μ L, template cDNA2.0 μ L, the μ L of distilled water 8.5；Expand WWP1 genes just It is 5 '-AGAAGGACTTGATTATGGT-3 ' (SEQ ID NO to primer sequence：3), reverse primer sequences be 5 '- TAATGGTTGATGCTGGAT-3’(SEQ ID NO：4)；The preferred GAPDH of house-keeping gene, expands the forward primer sequence of house-keeping gene It is classified as 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQ ID NO：9), reverse primer sequences be 5 '- GGTGGAATCATATTGGAACA-3’(SEQ ID NO：10).Operations are carried out on ice.Amplification program is：95℃ 10min, (95 DEG C of 15s, 60 DEG C of 60s) * 45 circulations.It is glimmering in Light Cycler using SYBR Green as fluorescent marker The enterprising performing PCR reaction of light real-time PCR, determines purpose band, Δ Δ CT methods are carried out by melt curve analysis analysis and electrophoresis Relative quantification.As a result as shown in Fig. 2 compared with normal bone tissues, up-regulated expression of the WWP1 genes in osteosarcoma tissue, together RNA-sep results are consistent.

The expression change of WWP1 albumen in the immune detection sample of embodiment 3

1. antigen protein is obtained

Utilize gene engineering expression：The cDNA sequence of people's WWP1 genes can be obtained from Genbank databases, passes through PCR Amplification is obtained in encoder block, insertion prokaryotes or eukaryotic expression vector, expresses WWP1 albumen, and by gene engineering expression The purification system protein purification of product.

2. Antibody preparation

Antibody can be prepared using following several method：

(1) cell fusion method：With the WWP1 protein immune animals of above-mentioned preparation (including rabbit, goat etc.), spleen is obtained Cell, then merged with myeloma cell, and routinely monoclonal antibody technology of preparing prepares monoclonal antibody.

(2) using phage display storehouse, the spleen IgG variable regions of the immune animal of clone are simultaneously expressed as genetic engineering list Clonal antibody.

(3) using the Western Immuno animal of purifying, polyvalent antibody is prepared.

3. detection

(1) with the antibody (many anti-or monoclonal antibodies) prepared, the pathological examination of osteosarcoma, positive letter are carried out with histochemical method Number be osteosarcoma.

(2) patients serum is taken, is detected with ELISA method, positive reaction is the suspicious patient of osteosarcoma.

(3) using WWP1 antibody as one of probe of protein-chip, for osteosarcoma diagnosis.

Influence of the WWP1 gene expressions of embodiment 4 to osteosarcoma cell physiological activity

The detection of 1.siRNA jamming effectiveness

1.1 cell culture

Using human osteosarcoma U2OS cell lines as research object, human osteosarcoma U2OS cell lines are purchased from Shanghai cell bank, cell Culture with containing 10% hyclone IMDM culture mediums, in the environment of without penicillin, streptomysin, in volume fraction be 5% CO₂, cellar culture under the conditions of 37 DEG C.U2OS cells, which are put, observes cell attachment growth under microscope, volume is medium, and size is equal Even, cell is oval or irregular shape, and kytoplasm is less, and nucleus is larger, and kernel is clear.

1.2 siRNA disturb WWP1 gene expressions

For WWP1 siRNA sequence (siRNA-WWP1)：

Positive-sense strand is 5 '-ACAAAGAUGAGUAAGACACGA-3 ' (SEQ ID NO：5)：

Antisense strand is 5 '-GUGUCUUACUCAUCUUUGUAU-3 ' (SEQ ID NO：6).

Negative control siRNA sequence (siRNA-NC)：

Positive-sense strand is 5 '-CGUACGCGGAAUACUUCGA-3 ' (SEQ ID NO：11)；

Antisense strand is 5 '-UCGAAGUAUUCCGCGUACG-3 ' (SEQ ID NO：12).

Cell is pressed 1 × 10⁴/ hole is inoculated into 24 porocyte culture plates, in 37 DEG C, 5%CO₂Cell culture in incubator 24h, in without IMEM culture mediums dual anti-, containing 10%FBS, transfection (is purchased from according to lipofectamine 2000 Invitrogen companies) specification transfection, experiment be divided into negative control group and experimental group (20nM), wherein negative control group The sequence of siRNA and WWP1 genes is without homology, and concentration is 20nM/ holes.Transfect respectively simultaneously.

1.3 QPCR detect the transcriptional level of WWP1 genes

1.3.1 the extraction of cell total rna

Using TRIzol Reagent (Invitrogen Cat.No.15596-018) total RNA extraction reagent, by specification Offer method extracts the total serum IgE of U2OS cells.Specific method is：Cell is taken, the PBS for being 0.01M with concentration is rinsed 3 times, added Appropriate TRIzol reagents, room temperature places 5min cell lysis, is dispensed to 1.5mL Eppendorf and managed with 1mL/ pipes after piping and druming is uniform In.Often pipe adds 0.2mL chloroforms, acutely shakes 15s, and room temperature places 2-3min, 4 DEG C, 12000r/min centrifugation 15min, will be upper Layer aqueous phase is moved in clean Eppendorf pipes, is added 0.5mL isopropanols, is gently mixed, and room temperature places 10min, 4 DEG C, 7500r/ Min centrifuges 10min.Abandon supernatant, 75% ethanol washing RNA precipitate, 7500r/min centrifugation 5min, drying at room temperature RNA precipitate, 5- Appropriate DEPC water is dissolved in after 10min.Mass fraction is the integrality of 1.0% agarose gel electrophoresis detection RNA samples, application Bio-Photometer is quantitative determined to the RNA of extraction.

1.3.2 reverse transcription step be the same as Example 2.

1.3.3 QPCR amplification steps be the same as Example 2.

1.4 result

As a result as Fig. 3 shows that siRNA-WWP1 can effectively suppress the expression of WWP1 genes.

2.WWP1 the influence of gene pairs human osteosarcoma cell proliferation

Cell proliferation in vitro is detected using mtt assay.

2.1 step：

By 5 × 10³Individual cell is spread into 96 orifice plates and overnight incubation, next day transfection negative control RNA or siRNA-WWP1, and MTT detections are carried out in 0h, 24h, 48h, 72h, 96h, 120h, 144h after transfection.First, culture supernatant is discarded, 100 μ L of change contain MTT0.5mg/ml (Sigma companies) fresh culture；Then 37 DEG C are incubated 4h；Finally changing to 100 μ L DMSO, (Sigma is public Department) and vibrate 10min.Final absorbance uses 490nm wavelength detectings, draws cell growth curve.

2.2 result

As a result as shown in figure 4, suppressing after WWP1 expression, the growth of osteosarcoma U 2OS is substantially suppressed, and is shown WWP1 genes take part in the breeding of osteosarcoma cell.

3.WWP1 the influence of gene pairs Apoptosis

Use the influence of flow cytomery WWP1 gene pairs Apoptosis.

3.1 step

Method as described above is carried out after cell transfecting, transfection 72h, and cell, Ran Houyong are washed using precooling PBS 0.25% trypsin digestion cell, stops digestion, the cell being collected by centrifugation is resuspended using PBS, is 1 × 10 by cell quantification⁶Individual/ Ml, takes the 200 above-mentioned cell suspensions of μ L to be placed into Appendorf pipes, adds 10 μ L Annexin-V-FITC and mixes, room temperature is dark Place is incubated 5min before dyeing 15min, upper machine and adds 5 μ L of 10mg/L propidium iodides (PI) dyeing.Untransfected siRNA cell difference Dyed with Annexin-V-FITC and PI for standard quantitative.Two Colour Fluorescence cell streaming meter is carried out with FACS flow cytometers Number, observing apoptosis cell percentages.

3.2 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 SPSS13.0 statistical softwares, the t of difference use between the two is examined, it is believed that as P ＜ 0.05 When there is statistical significance.

3.3 result

The apoptosis rate of WWP1 gene interference groups is (41.23+2.12) %, and the apoptosis rate of negative control group is (7.33+0.26) %, above-mentioned difference has statistical significance (P ＜ 0.05), and the above results show to suppress the expression of WWP1 genes The apoptosis of osteosarcoma U 2OS can be obviously promoted.

The influence of 4.WWP1 gene pairs osteosarcoma cell transfer abilities

The influence of WWP1 gene pairs osteosarcoma cell transfer abilities is detected using cut Healing Experiments.

4.1 specific steps

(1) stayed overnight from the coat culture dishes of 5cm sizes of the PBS containing 15 μ g/ml fibrinogens.

Add the IMDM containing 10%FBS within (2) second days, used after a few hours that sterilize under ultraviolet light.

(3) method of U2OS cells as described above is carried out after cell transfecting, transfection 72h, according to 1 × 10⁶It is individual thin The standard of born of the same parents is carried out after inoculated and cultured, 24h to cell, and whether observation cell grows uniformly.

(4) scraped with cell and a vestige is scratched in the middle of the cell of culture dish, placed it in incubator and cultivate after 24h Transfer ability of the cell in cut is observed, 3 samples are selected when observation every time, observed 3 times.

4.2 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 SPSS13.0 statistical softwares, the t of difference use between the two is examined, it is believed that as P ＜ 0.05 When there is statistical significance.

4.3 result

Matrigel starts after 24h, the average value difference of the cut healing rate of WWP1 gene interference groups and negative control group For 30%, 75%, above-mentioned difference has statistical significance (P ＜ 0.05), shows to suppress the expression of WWP1 genes, osteosarcoma cell U2OS transfer ability declines.

The influence of 5.WWP1 gene pairs osteosarcoma cell invasive abilities

The influence that cell determination method detects WWP1 gene pairs osteosarcoma cell invasive abilities is attacked using Transwell.

5.1 concrete operation steps are：

(1) Matrigel (Beeton-Diekinson, FranklnLakes, NJ) is diluted to 4 DEG C of IMDM in advance 200 μ g/ml, the IMDM for taking 200 μ L to dilute are placed on the Transwell that aperture is 8 μm.

(2) micropores whole on polycarbon resin film film is covered by Matrigel, then it is placed on 37 DEG C of condition It is lower to stay overnight.

(3) processing it is dried.

(4) irradiate 2h to the Transwell got ready to sterilize with ultraviolet, pre-irradiation adds what a small amount of sterilization treatment was crossed The IMDM of serum-free carries out aquation to it.

(5) method as described above is carried out after cell transfecting, transfection 72h, U2OS cells is taken, then by cell number 3 ×10⁴It is added in each cell, culture medium selects IMDM.

(6) it is placed into 46 orifice plates and cultivates.

(7) 5%CO₂, be incubated 24h in 37 DEG C of incubators.

(8) Matrigel and cell in that face of upper chamber, that face of lower room are wiped clean after cell is taken out with paper handkerchief or cotton swab Fixed with methanol after 10min, carry out conventional H E dyeing, use micro- sem observation.

(9) observed according to 3 high power field of view of random selection, and carefully count the number of cells in cell Xia Shi faces As the cell number for penetrating Matrigel, this process is carried out 3 times, calculates the reference frame that its average is used as calculating.

5.2 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 SPSS13.0 statistical softwares, the t of difference use between the two is examined, it is believed that as P ＜ 0.05 When there is statistical significance.

5.3 result

After 24h, WWP1 gene interference groups pass through the cell numbers of Transwell films each visual field cell number under the microscope Average value be 52, and negative control group passes through the cell numbers of Transwell films each visual field cell number under the microscope Average value is 324, and above-mentioned difference has statistical significance (P ＜ 0.05), shows to suppress the expression of WWP1 genes, osteosarcoma is thin Born of the same parents U2OS invasive ability declines.

The influence of 6.WWP1 gene pairs osteosarcoma cell one-tenth knurl abilities

6.1 step：

(1) method as described above is carried out after U2OS cell tryptase enzymic digestions after cell transfecting, transfection 72h, is prepared Into cell suspension；

(2) (200 cells/wells) is inoculated in 6 orifice plates after cell count, the cell being inoculated with is continued in incubator Cultivate by 14 days or untill cell number is more than 50 in most single clones, carried out changing liquid and observing cellular every 3 days halfway State；Cell clone is taken pictures under fluorescence microscope before experiment is terminated；

(3) fixed when experiment is terminated with paraformaldehyde after cell, PBS washing cells, Giemsa dyeing is taken pictures.

6.2 result

Plate clone experimental result is shown, compared with negative control group, the osteosarcoma cell of WWP1 gene expression interference groups The volume that clone's spot number of U2OS formation substantially reduced, cloned spot is obviously reduced, and the above results show that WWP1 silences cause bone The ability of sarcoma cell formation clone declines.

The WWP1 antigen proteins of embodiment 5 as osteosarcoma vaccine application

1. antigen protein is obtained

Utilize gene engineering expression：The cDNA sequence of people's WWP1 genes can be obtained from Genbank databases, passes through PCR Amplification obtains encoder block, and WWP1 antigen polypeptides are synthesized using Peptide synthesizer according to above-mentioned sequence.

2.DC (BMDC) in-vitro separation and culture

Detection in peripheral blood of patients underwent separating interface mononuclearcell after lymphocytes separating solution Ficoll gradient centrifugations.Cell After scrubbed 2 times, suspended with serum free medium AIM-V nutrient solutions (Gibco BRL companies), adjustment cell concentration to 3 × 10⁶ Individual/ml, is inoculated with into 6 well culture plates.In 37 DEG C, 5%CO₂In incubator after overnight incubation, gently blow and beat suspension cell and reclaim jelly Deposit, to prepare effector cell.Added in attached cell nutrient solution and contain 1000U/ml single colony stimulating factors and 500U/ml The AIM-V of interleukins.When culture was to the 7th day, the DC suspended is harvested.

The people B of 3.T2 cells or HLA-A2 EBV (Epstein Barr Virus, african lymphoma virus) transfections The WWP1 antigen polypeptides of lymphocyte and final concentration of 20 μ g/ml are incubated 4h altogether, and target cell is used as after washing.

4. identifying the WWP1 antigen vaccines of present invention treatment osteosarcoma in the following manner, pass through DC submission WWP1 Antigenic Peptides Activating effect T cell, produces the immune response that specificity is directed to WWP1 epitopes：

(1) processing of DC cells：DC is resuspended in after centrifugation in 2ml AIM-V, and it is more to this to add WWP1 Antigenic Peptides The final concentration of 40ug/ml of peptide.37 DEG C, 5%CO₂18h is cultivated, after being irradiated through 3000rad radioactive ray, is washed stand-by.

(2) preparation of effector cell：Target cell prepared by step 3 mixes incubation altogether with the DC cells after irradiation with 20: 1 6-7 days, DC is added according still further to same ratio, after 6-7 days, repetitive stimulation once, is used as effector T cell again.In 0,7,10, 14th, 19 days, culture supernatant is left and taken respectively, for cytokines measurement.

(3) cytokines measurement：Using in cytokine detection kits (being purchased from Endogen companies of the U.S.) detection culture The cell factor of secretion in clear.

5. test result indicates that, the effector T cell that the DC stimulated through WWP1 Antigenic Peptides of the present invention is activated can be produced special Property be directed to WWP1 epitopes immune response so that specific killing is combined with the target cell of the identical WWP1 Antigenic Peptides, say Bright WWP1 antigen proteins of the present invention can be used as potential clinical treatment of osteosarcoma vaccine.

The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.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 improve 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.

Claims (4)

1. Application of the 1.WWP1 genes in the medicine for preparing treatment osteosarcoma.
2. 2. application according to claim 1, it is characterised in that the medicine is selected from the following group：Suppress osteosarcoma cell to increase The medicine grown, the medicine for promoting apoptosis in osteosarcoma cells, the medicine of suppression osteosarcoma cell migration, suppression osteosarcoma cell invasion and attack Medicine, suppress osteosarcoma cell into knurl medicine.
3. 3. application according to claim 2, it is characterised in that the medicine includes suppressing WWP1 genes by RNA interfering The double stranded RNA of expression, or the tumor vaccine based on WWP1 antigen proteins or the albumen for suppressing WWP1 protein actives Matter.
4. 4. a kind of applications of siRNA of suppression WWP1 gene expressions in the medicine for preparing treatment osteosarcoma, it is characterised in that institute Stating siRNA sequence is：Positive-sense strand such as SEQ ID NO：Shown in 5, antisense strand such as SEQ ID NO：Shown in 6.