CN104651509A - New drug target for osteosarcoma - Google Patents
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- CN104651509A CN104651509A CN201510075920.4A CN201510075920A CN104651509A CN 104651509 A CN104651509 A CN 104651509A CN 201510075920 A CN201510075920 A CN 201510075920A CN 104651509 A CN104651509 A CN 104651509A
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Abstract
The invention discloses application of C8orf59 gene in preparing drugs for diagnosing and treating osteosarcoma. The experiment proves that the expression level of the C8orf59 gene in osteosarcoma tissues is obviously higher than that in normal tissues, and therefore, the C8orf59 gene can be used as a specific marker for diagnosing osteosarcoma, so that the osteosarcoma diagnosis is more accurate and faster. The expression of the C8orf59 gene in the osteosarcoma cell line can be disturbed to obviously inhibit the cell proliferation, migration, invasion and tumor formation capacity and accelerate apoptosis. Therefore, the C8orf59 gene has potential application value for gene therapy of osteosarcoma, and is hopeful to be used for treating osteosarcoma and developing new anti-osteosarcoma drugs on such basis, thereby making contribution for preventing and treating osteosarcoma in clinic.
Description
Technical field
The invention belongs to biomedical sector, relate to a kind of osteosarcomatous medicine new target drone, be specifically related to the application of C8orf59 gene in preparation diagnosis osteosarcoma product and preparation treatment osteosarcoma medicine.
Background technology
Osteosarcoma is the malignant tumour deriving from mesenchymal tissue, and the tumour cell that its principal causative is characterized as proliferation in vivo directly forms prematurity bone or osteoid tissue.It is the modal primary malignant tumor of a kind of human skeletal system.Typical osteosarcoma is a kind of rare (accounting for 0.2% of whole malignant tumour) high carcinogenic malignant tumour, has three examples in its sickness rate each million people about annual.Osteosarcoma mainly appears at longer bone and the soft tissue of small part.Its principal pathogenetic crowd concentrates on 10 ~ 20 years old teenager, has sickness rate high, and the early stage rate of transform is high, cures the features such as survival rate is low.X-ray, tomography, nucleus magnetic resonance, Angiography and dynamic bone scintigraphy technology etc. are widely used in the diagnosis of this disease, tumorigenic degree and type of surgery and judge etc.But utilize above-mentioned clinical means to carry out osteosarcomatous diagnosis, usually cause the state of an illness of patient to be incured loss through delay, miss optimal treatment period.Therefore finding a kind of osteosarcoma early diagnosis marker is problem demanding prompt solution.
High-throughput transcript profile order-checking (RNA-sep) is a technology of carrying out degree of depth order-checking in transcript profile level, the application of sequencing technologies in transcriptome analysis mainly concentrates on the structure of gene expression profile, the discovery of the discovery of new gene, the structure of small molecules non-coding RNA express spectra and new micro RNA genes, protein coding gene annotation and study as transcript profile such as fusion transcript.Data are in initial process is similar substantially, next different bioinformatics softwares can be selected to calculate for different research purposes.
Large scale sequencing technology is utilized directly to check order to the cDNA sequence become by mRNA reverse transcription, produce number in reads quantity necessarily, thus the transcriptional level of one section of special genome area can directly be weighed by the reads number of comparison to this genome area.This technology is commonly used to the gene expression profile building a certain particular organization or cell, the analysis of gene expression profile is carried out by normal tissue and pathological tissues, obtain the gene of differential expression in two kinds of tissues, on the one hand for the generation of study of disease and the molecular mechanism of development provide new thinking, on the other hand for the Diagnosis and Treat of disease provides new diagnosis marker or drug target.
Utilize high-throughput transcript profile to check order to screen and the closely-related gene of osteosarcoma, be not only conducive to the pathomechanism studying osteosarcomatous generation and development, and new diagnosis marker or new drug target can be provided for Diagnosis and Treat osteosarcoma.
Summary of the invention
The present invention utilizes high throughput sequencing technologies to find, and the expression of C8orf59 gene in osteosarcoma tissue there are differences with the expression in normal bone tissues, compared with normal bone tissues, C8orf59 gene up-regulated in osteosarcoma tissue, by detecting the expression of C8orf59 gene, just can judge whether experimenter suffers from osteosarcoma.
An object of the present invention is to provide the application of C8orf59 gene in the osteosarcomatous product of preparation diagnosis.
Two of object of the present invention is to provide the application of C8orf59 gene in the osteosarcomatous medicine of preparation treatment.
To achieve these goals, present invention employs following technical scheme:
The invention provides the osteosarcomatous product of a kind of diagnosis, by the expression detecting C8orf59 gene in experimenter's scleroblast, the osteosarcomatous product of described diagnosis judges whether experimenter suffers from osteosarcoma.
Further, the osteosarcomatous product of described diagnosis comprises: with RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or the osteosarcomatous product of gene chip diagnosis.Described RT-PCR diagnoses osteosarcomatous product at least to comprise the primer of a pair specific amplified C8orf59 gene; Described real-time quantitative PCR diagnoses osteosarcomatous product at least to comprise the primer of a pair specific amplified C8orf59 gene; Described immunodetection diagnoses osteosarcomatous product to comprise: the antibody be combined with C8orf59 protein-specific; Described in situ hybridization diagnoses osteosarcomatous product to comprise: with the probe of the nucleic acid array hybridizing of C8orf59 gene; The osteosarcomatous product of described gene chip diagnosis comprises: with the probe of the nucleic acid array hybridizing of C8orf59 gene.
RT-PCR is used to diagnose the primer of the specific amplified C8orf59 gene comprised in osteosarcoma product to be utilize Primer5.0 primer-design software or other conventional online software designs of primer to form.To those skilled in the art, the amplimer going out can be used in RT-PCR experiment according to known sequences Design does not need to pay creative work and attainable.Therefore the primer of the specific amplified C8orf59 gene of RT-PCR of the present invention refer to all can in RT-PCR experiment the primer sequence of Successful amplification C8orf59 gene.In specific embodiment of the invention scheme, the C8orf59 gene amplification primer sequence for RT-PCR experiment is as follows: forward primer: 5 '-TACGGCTCGTGAGCGGAAT-3 ' (SEQ ID NO:1); Reverse primer: 5 '-GGGATAAGTGATTTAATATCC-3 ' (SEQ ID NO:2).
The primer of the specific amplified C8orf59 gene that described real-time quantitative PCR diagnoses osteosarcomatous product to comprise forms according to the conventional online software design of QPCR primer.To those skilled in the art, the amplimer going out can be used in QPCR experiment according to known sequences Design does not need to pay creative work and attainable.Therefore the primer of the specific amplified C8orf59 gene of real-time quantitative PCR of the present invention refer to all can in QPCR experiment the primer sequence of Successful amplification C8orf59 gene.In specific embodiment of the invention scheme, the C8orf59 gene amplification primer sequence for QPCR experiment is as follows: forward primer: 5 '-ATTCCTCAGCAGCGTCAT-3 ' (SEQ ID NO:3); Reverse primer: 5 '-GCATCACCAGTATATTACAACAGA-3 ' (SEQ ID NO:4).
Can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative with the probe of the nucleic acid array hybridizing of C8orf59 gene.The length of described probe does not limit, if complete specific hybrid, with object nucleotide sequence specific binding, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe can grow to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different impacts to hybridization efficiency, signal specificity, the length of described probe is at least 14 base pairs usually, the longlyest generally be no more than 30 base pairs, best with 15-25 base pair with the length of object nucleotide sequence complementary.Described probe self-complementary sequences most preferably less than 4 base pairs, in order to avoid affect hybridization efficiency.
The antibody be combined with C8orf59 protein-specific can be polyclonal antibody, also can be monoclonal antibody.Above-mentioned antibody can obtain from commercial channels, also can use a series of methods known in the art to prepare.Such as, the people C8orf59 albumen of purifying or its antigen fragment are injected in animal body to produce polyclonal antibody.Equally, the cell of expressing people's C8orf59 albumen or its antigen fragment also can be used for causing immunity to animal and producing antibody.Prepared by monoclonal antibody using hybridoma techniques.The antibody of C8orf59 albumen comprises the antibody can preventing C8orf59 protein function, also can be the antibody not affecting people C8orf59 protein function.Each antibody-like can by causing immunity to the segment of people C8orf59 albumen or functional domain and produce, and people C8orf59 protein product and fragment thereof can produce with recombination method or synthesize with Peptide synthesizer.Antibody protein bound with the C8orf59 of non-modified form, can utilize the gene product produced in prokaryotic cell prokaryocyte such as E.coli carry out immune animal and obtain.Antibody with posttranslational modification form is combined as glycosylation or phosphorylation C8orf59 albumen or polypeptide, can utilize the gene product produced in eukaryotic cell is as yeast or insect cell carry out immune animal and obtain.
The invention provides the application of C8orf59 gene in the osteosarcomatous product of preparation diagnosis.
By the expression detecting C8orf59 gene in experimenter's scleroblast, the osteosarcomatous product of described diagnosis judges whether experimenter suffers from osteosarcoma.The osteosarcomatous product of described diagnosis comprises: with RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or the osteosarcomatous product of gene chip diagnosis.Described RT-PCR diagnoses osteosarcomatous product at least to comprise the primer of a pair specific amplified C8orf59 gene; Described real-time quantitative PCR diagnoses osteosarcomatous product at least to comprise the primer of a pair specific amplified C8orf59 gene; Described immunodetection diagnoses osteosarcomatous product to comprise: the antibody be combined with C8orf59 protein-specific; Described in situ hybridization diagnoses osteosarcomatous product to comprise: with the probe of the nucleic acid array hybridizing of C8orf59 gene; The osteosarcomatous product of described gene chip diagnosis comprises: with the probe of the nucleic acid array hybridizing of C8orf59 gene.
RT-PCR is used to diagnose the primer of the specific amplified C8orf59 gene comprised in osteosarcoma product to be utilize Primer5.0 primer-design software or other conventional online software designs of primer to form.To those skilled in the art, the amplimer going out can be used in RT-PCR experiment according to known sequences Design does not need to pay creative work and attainable.Therefore the primer of the specific amplified C8orf59 gene of RT-PCR of the present invention refer to all can in RT-PCR experiment the primer sequence of Successful amplification C8orf59 gene.In specific embodiment of the invention scheme, the C8orf59 gene amplification primer sequence for RT-PCR experiment is as follows: forward primer: 5 '-TACGGCTCGTGAGCGGAAT-3 ' (SEQ ID NO:1); Reverse primer: 5 '-GGGATAAGTGATTTAATATCC-3 ' (SEQ ID NO:2).
The primer of the specific amplified C8orf59 gene that described real-time quantitative PCR diagnoses osteosarcomatous product to comprise forms according to the conventional online software design of QPCR primer.To those skilled in the art, the amplimer going out can be used in QPCR experiment according to known sequences Design does not need to pay creative work and attainable.Therefore the primer of the specific amplified C8orf59 gene of real-time quantitative PCR of the present invention refer to all can in QPCR experiment the primer sequence of Successful amplification C8orf59 gene.In specific embodiment of the invention scheme, the C8orf59 gene amplification primer sequence for QPCR experiment is as follows: forward primer: 5 '-ATTCCTCAGCAGCGTCAT-3 ' (SEQ ID NO:3); Reverse primer: 5 '-GCATCACCAGTATATTACAACAGA-3 ' (SEQ ID NO:4).
Can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative with the probe of the nucleic acid array hybridizing of C8orf59 gene.The length of described probe does not limit, if complete specific hybrid, with object nucleotide sequence specific binding, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe can grow to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different impacts to hybridization efficiency, signal specificity, the length of described probe is at least 14 base pairs usually, the longlyest generally be no more than 30 base pairs, best with 15-25 base pair with the length of object nucleotide sequence complementary.Described probe self-complementary sequences most preferably less than 4 base pairs, in order to avoid affect hybridization efficiency.
The antibody be combined with C8orf59 protein-specific can be polyclonal antibody, also can be monoclonal antibody.Above-mentioned antibody can obtain from commercial channels, also can use a series of methods known in the art to prepare.Such as, the people C8orf59 albumen of purifying or its antigen fragment are injected in animal body to produce polyclonal antibody.Equally, the cell of expressing people's C8orf59 albumen or its antigen fragment also can be used for causing immunity to animal and producing antibody.Prepared by monoclonal antibody using hybridoma techniques.The antibody of C8orf59 albumen comprises the antibody can preventing C8orf59 protein function, also can be the antibody not affecting people C8orf59 protein function.Each antibody-like can by causing immunity to the segment of people C8orf59 albumen or functional domain and produce, and people C8orf59 protein product and fragment thereof can produce with recombination method or synthesize with Peptide synthesizer.Antibody protein bound with the C8orf59 of non-modified form, can utilize the gene product produced in prokaryotic cell prokaryocyte such as E.coli carry out immune animal and obtain.Antibody with posttranslational modification form is combined as glycosylation or phosphorylation C8orf59 albumen or polypeptide, can utilize the gene product produced in eukaryotic cell is as yeast or insect cell carry out immune animal and obtain.
The osteosarcomatous product of described diagnosis of the present invention is selected from following group: test kit, chip, filter paper.Mentioned reagent box can be the test kit detecting gene transcription level, as RT-PCR kit, QPCR test kit, also can be the test kit of gene expression detection level, as ELISA kit.Said chip can be gene chip or protein chip, and it can the expression level of testing goal gene.
Described RT-PCR kit, except comprising the primer of specific amplified C8orf59 gene, also can comprise PCR damping fluid, dNTPs, MgCl
2, Tap archaeal dna polymerase.Described RT-PCR kit also can comprise RNA and extract reagent, and RNA enzyme extraction pack is containing Trizol, chloroform, Virahol, 75% ethanol.
Described QPCR test kit, except comprising the primer of specific amplified C8orf59 gene, also can comprise SYBRGreen polymerase chain reaction system.SYBR Green polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBR Green fluorescence dye.
Preferably, PCR damping fluid comprises: 25mM KCl, 2.5mM MgCl2,200mM (NH
4)
2sO
4.
Described QPCR test kit also can comprise M-MLV reverse transcription system, and this reverse transcription system comprises: T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.
Preferably, reverse transcription reaction liquid comprises: the MgCl of the KCL of the Tris-HCL of 250mM pH8.3,375mM, 15mM
2, the DTT of 50mM.
The RNA enzyme inhibitors that RNA enzyme inhibitors can select this area conventional, is preferably the recombinant protein enzyme of the Noncompetition inhibition RNA enzyme of escherichia coli expression.
Described QPCR test kit also can comprise RNA and extract reagent, and RNA enzyme extraction pack is containing Trizol, chloroform, Virahol, 75% ethanol.
Described ELISA kit, except comprising the antibody that is combined with C8orf59 protein-specific, also comprises bag and is buffered liquid, washings, confining liquid, sample loading buffer, reaction terminating liquid, target protein standard substance.
Preferably, it is phosphate buffered saline buffer PBS that bag is buffered liquid, and its composition is 140mM NaCl, 2.7mMKCl, 10mM Na
2hPO
4, 1.8mM KH
2pO
4, pH value is 7.4.
Preferably, washings is the PBS containing 1%Tween-20, and be called 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, and 1%BSA, 0.05%Tween-20, pH value is 8.4.
Preferably, reaction terminating liquid composition is 2M H
2sO
4.
Present invention also offers the application of C8orf59 gene in the osteosarcomatous medicine of preparation treatment.
Further, the osteosarcomatous medicine of described treatment can be the medicine suppressing human osteosarcoma cell proliferation, it can be the medicine promoting apoptosis in osteosarcoma cells, can be the medicine suppressing osteosarcoma cell migration, can be the medicine of suppression osteosarcoma cell invasion and attack, can also be the medicine suppressing osteosarcoma cell to become knurl.
Further, the osteosarcomatous medicine for the treatment of of the present invention comprises: the double stranded RNA being suppressed C8orf59 genetic expression by RNA interfering, or based on the tumor vaccine of C8orf59 antigen protein or for suppressing the protein of C8orf59 protein-active.
In the present invention, described RNA disturbs (RNA interference, RNAi) refer to high conservative during evolution, brought out by double-stranded RNA (double-stranded RNA, dsRNA), the phenomenon of the efficient selective degradation of homologous mRNA.Use RNAi technology can specific depletion or close the expression of specific gene, this technology be widely used in the field of gene exploring gene function and communicable disease and malignant tumour.RNAi screening based on cell has many advantages in functional gene research, is mainly manifested in most cell types and can uses RNAi method, and the expression of relatively easy downward or reticent any goal gene.
Can efficiently to be rejected in order to ensure C8orf59 gene or reticent, the siRNA specific fragment according to the mRNA sequences Design of C8orf59 gene.General design principle (the Elbashir et.al 2001 that the design consideration of siRNA has been delivered, 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, this online tool is: siRNASelectionProgram of Whitehead Institute (BingbingYuan et.al 2004, http://jura.wi.mit.edu/bioc/siRNAext/) and BLOCK-iTTM RNAi DesignerofINVITROGEN (winner of the 2004Frost & Sullivan Excellence in Research Award, https: //rnaidesigner.invitrogen.com/sirna/).In order to improve the validity of siRNA segment further, the advantage of comprehensive two Photographing On-line instruments is designed for the siRNA segment of screening.Finally, filter siRNA sequence by sequence analysis (NCBI BLAST), with improve siRNA segment specificity and reduce RNAi interference effect of missing the target.SiRNA oligonucleotide is by Shanghai JiMa pharmacy Technology Co., Ltd's chemosynthesis.In the specific embodiment of the present invention, specifying information for the siRNA sequence (siRNA-C8orf59) of C8orf59 is as follows: positive-sense strand 5 '-UGUUCUUGGCCAUUGUCAGAA-3 ', antisense strand 5 '-CUGACAAUGGCCAAGAACAAA-3 '.Design negative control siRNA (siRNA-NC): positive-sense strand: 5 '-CGUACGCGGAAUACUUCGA-3 ' (SEQ ID NO:5) simultaneously; Antisense strand: 5 '-UCGAAGUAUUCCGCGUACG-3 ' (SEQ ID NO:6).
Described tumor vaccine refers to the immunologic function excited with the particular matter in tumor tissues in patient body, enables the immunologic function killing tumor cell of patient.Tumor vaccine mainly refers to therapeutic vaccine, is to obtain patient to treat by the method for vaccine more after being ill, to control recurrence and the transfer of tumour.Tumor vaccine has individuality, because the tumor type of each patient's gained, by stages different, material expressed by tumour cell is also different, and tumor vaccine is made up of the tumour of patient itself, therefore the tumor vaccine prepared for the tumour that patient is concrete has individuation feature, its side effect is little, with strong points.
Time prepared by described tumor vaccine, with the tumor tissues of operation method excised tumor patient and aseptic freezen protective, to extract tumour antigen.Obtaining tumour antigen is the first step, then will obtain dendritic cell, namely obtain from the mononuclearcell of patient blood.Carry out again after extraction mononuclearcell screening and vitro culture, then dendritic cell is stimulated in vitro with tumour antigen, enable this dendritic cell premunition information, finally the dendritic cell trained is fed back in body, make the immunne response produced in body for tumour antigen.
Present invention also offers the above-mentioned application of siRNA in the osteosarcomatous medicine of preparation treatment for C8orf59.The osteosarcomatous medicine of described treatment can be the medicine suppressing human osteosarcoma cell proliferation, it can be the medicine promoting apoptosis in osteosarcoma cells, can be the medicine suppressing osteosarcoma cell migration, can be suppress the medicine of osteosarcoma cell invasion and attack, also suppression osteosarcoma cell to become the medicine of knurl.
Said medicine, except comprising siRNA, can also comprise pharmaceutically common pharmaceutical carrier.
The method of application of described medicine can be oral, systemic administration (such as, enter through skin, snuffing or with suppository) or parenteral administration (such as, intramuscular, intravenously or subcutaneous).
Described medicine can be made into multiple formulation, as tablet, injection, capsule etc.Medicine injection is prepared by ordinary method with physiological saline or the aqueous solution containing glucose and other assistant agents.The medicine of Tablet and Capsula and so on, also prepares by those skilled in the art's ordinary method.
Advantage of the present invention and beneficial effect:
(1) to make public for the first time C8orf59 gene relevant to osteosarcoma in the present invention, and C8orf59 gene is expected to become the osteosarcomatous molecular marker of diagnosis, and for studying the thinking that osteosarcomatous molecule mechanism provides new.
(2) utilize the mode of gene expression detection to diagnose osteosarcomatous existence whether method sensitiveer, be conducive to the diagnosis that disease is early stage.
Accompanying drawing explanation
Fig. 1 display utilizes RT-PCR to detect the differential expression of C8orf59 gene in osteosarcoma tissue and normal bone tissues;
Fig. 2 display utilizes real-time quantitative PCR to detect the differential expression of C8orf59 gene in osteosarcoma tissue and normal bone tissues;
Fig. 3 display utilizes real-time quantitative PCR on transcriptional level, to detect RNA interfering for C8orf59 to the impact of C8orf59 genetic expression;
The impact that Fig. 4 utilizes mtt assay detection C8orf59 genetic expression to breed osteosarcoma cell line U2OS.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring HarborLaboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1 screens the gene relevant to osteosarcoma
1.1 sample collection
The sample of the routine normal bone tissues of each collection 8, osteosarcoma tissue.
The preparation of 1.2 RNA sample and mass analysis
1.2.1 the preparation of RNA sample
Normal bone tissues and osteosarcoma tissue judge RNA sample quality through agarose gel electrophoresis after RNA extracting, and eligible may be used for further transcriptome analysis.
1.2.2 the mass analysis (NanoDrop1000 spectrophotometer) of RNA sample
NanoDrop1000 spectrophotometer detects RNA sample, the sample requirement of RNA-seq order-checking: OD260/OD280 is 1.8-2.2.
1.2.3 the mass analysis of RNA sample (Agilent Technologies 2100 Bioanalyzer)
Agilent Technologies 2100 Bioanalyzer detects RNA sample quality, observation 28S rRNA and 18S rRNA master tape are obviously, nothing is degraded, RNA Perfection Index is qualified, concentration meets the requirements of the requirement meeting RNA-seq order-checking cDNA library structure, may be used for library construction and order-checking.
1.3 high-throughput transcript profile order-checkings
1.3.1 the RNA-seq section of reading location
First the low-quality section of reading is removed and obtain the clean section of reading, then utilize TopHat v1.3.1 will clean fragment to mate with reference to genome (hg19) with UCSC H.sapiens, the index built in advance of H.sapiens UCSC hg19 version is downloaded from TopHat homepage, and as reference genome, when utilizing TopHat to mate with genome, each section of reading (defaulting to 20) is allowed to have multiple coupling site, maximum 2 mispairing.TopHat sets up possible shearing site storehouse according to exon region and GT-AG shear signal, will not navigate to the genomic section of reading navigate on genome according to these shearing site storehouses.We use the system default parameter of TopHat method.
1.3.2 transcript abundance assessment
What match reads segment file by Cufflinks v1.0.3 process, and RNA-seq segment number is carried out the relative abundance of standardized calculation transcript by Cufflinks v1.0.3.FPKM value refers in each 1,000,000 sequenced fragments the segment number matching the long exon region of specific gene 1kb.The fiducial interval of FPKM estimated value is calculated by Bayesian inference method.The GTF comment file of the reference that Cufflinks uses downloads (Homo_sapiens.GRCh37.63.gtf) from Ensembl database.
1.3.3 the detection of difference expression gene
By the Ensembl GTF file of download be transferred to Cuffdiff by the source document that TopHat mates, Cuffdiff uses original matching files again to estimate the gene expression abundance of the transcript listed in GTF file, and checkout discrepancy is expressed.In Cuffidff exports, only have q value < 0.01, test display is successfully more just considered to differential expression.
1.4 result
The RNA-seq sequencing result in early stage filters out 979 difference expression genes altogether, the gene 472 of wherein expression level rise, the gene 507 that expression level is lowered.
Embodiment 2 large sample PCR verifies candidate gene and osteosarcomatous relation
Consider to yet there are no in prior art and carry out about this gene and osteosarcoma dependency the gene alternatively gene studied, consider the result of high-throughput transcript profile degree of depth order-checking in early stage simultaneously, select C8orf59 gene (its expression is raised in osteosarcoma tissue) to verify according to the size of P value.Collect 30 osteosarcoma tissue, collect 20 normal bone tissues samples simultaneously, adopt PCR to carry out classical molecular biology experiment checking, concrete operation step is as follows:
1, RNA extracts
Frozen in liquid nitrogen after collecting sample, organizing the mortar putting into precooling to grind, after tissue samples is powdered after taking-up:
1. Trizol is added, room temperature preservation 5min;
2. add chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5min-10min;
3. draw upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15min in another new centrifuge tube, note the proteic substance be not drawn onto between two-layer aqueous phase.Move into new pipe, add isopyknic-20 DEG C of pre-cold isopropanols, fully put upside down mixing, be placed in 10min on ice;
4. 12000rpm carefully discards supernatant liquor at a high speed after 15min, adds 75%DEPC washing with alcohol precipitation (4 DEG C of preservations), washing precipitate in the ratio of 1ml/ml Trizol, vibration mixing, 12000rpm high speed centrifugation 5min at 4 DEG C;
5. discard ethanol liquid, ambient temperatare puts 5min fully to dry precipitation, adds DEPC treated water dissolution precipitation;
6. RNA purity and concentration is measured with Nanodrop2000 ultraviolet spectrophotometer, frozen in-70 DEG C.
2, reverse transcription
With RT Buffer, reverse transcription synthesis cDNA is carried out to 1 μ g total serum IgE.Adopt 25 μ l reaction systems, each sample gets 1 μ g total serum IgE as template ribonucleic acid, adds following component respectively: DEPC water in PCR pipe, 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.Hatch 1h for 42 DEG C, 72 DEG C of 10min, of short duration centrifugal.
3, RT-PCR amplification
C8orf59: forward primer is 5 '-TACGGCTCGTGAGCGGAAT-3 ' (SEQ ID NO:1);
C8orf59: reverse primer is 5 '-GGGATAAGTGATTTAATATCC-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 reaction mixture: forward primer, reverse primer, 10 × PCR damping fluid, MgCl
2, dNTP, Taq archaeal dna polymerase, cDNA template amount for being respectively 0.2,0.2,0.4,0.4,1.0,1.0,0.2,0.1 and 5 μ L, finally supplement distilled water make reaction system be 10 μ L.The reaction conditions of PCR is as follows: 94 DEG C, 5min denaturation; 94 DEG C, 30s sex change; 55 DEG C, 30s anneals; 72 DEG C, 30s extends; Totally 35 circulations, electrophoresis detection pcr amplification product.Weigh DNA amount with the brightness of DNA band, band is brighter, and representation DNA amount is more.Do internal reference contrast with β-actin, homogenization process is carried out in the brightness of the band of C8orf59 gene amplification product, compares the ratio of the brightness of C8orf59 gene amplification product in normal bone tissues and osteosarcoma tissue.As shown in Figure 1, compared with normal bone tissues, C8orf59 gene is at the up-regulated of osteosarcoma tissue, consistent with RNA-sep result for result.
4, QPCR amplification
Adopt 25 μ L reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are above to ensure the reliability of result all in triplicate.Prepare following reaction system: SYBR Green polymerase chain reaction system 12.5 μ L, forward primer (5 μm of ol/ μ l) 1 μ L, reverse primer (5 μm of ol/ μ l) 1 μ L, template cDNA2.0 μ L, distilled water 8.5 μ L; The forward primer sequence of amplification C8orf59 gene is 5 '-ATTCCTCAGCAGCGTCAT-3 ' (SEQ ID NO:3), and reverse primer sequences is 5 '-GCATCACCAGTATATTACAACAGA-3 ' (SEQ ID NO:4); The preferred GAPDH of house-keeping gene, the forward primer sequence of amplification house-keeping gene is 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQ IDNO:9), and reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO:10).Operations is all in carrying out on ice.Amplification program is: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) * 45 circulation.Using SYBR Green as fluorescent marker, in the enterprising performing PCR reaction of Light Cycler fluorescence real-time quantitative PCR instrument, by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification.Result as shown in Figure 2, compared with normal bone tissues, the up-regulated of C8orf59 gene in osteosarcoma tissue, consistent with RNA-sep result.
The expression change of C8orf59 albumen in embodiment 3 immunodetection sample
1. antigen protein obtains
Utilize gene engineering expression: the cDNA sequence that can obtain people C8orf59 gene from Genbank database, encoder block is obtained by pcr amplification, insert in prokaryotic organism or eukaryotic expression vector, express C8orf59 albumen, and press the purification system protein purification of gene engineering expression product.
2. antibody preparation
Following several method Dispersal risk can be adopted:
(1) cytogamy method: with the C8orf59 protein immune animal (comprising rabbit, goat etc.) of above-mentioned preparation, obtains spleen cell, then with myeloma cell fusion, and monoclonal antibody preparation technology prepares monoclonal antibody routinely.
(2) utilize phage display storehouse, clone the spleen IgG variable region of immune animal and be expressed as gene engineering monoclonal antibody.
(3) utilize the Western Immuno animal of purifying, prepare polyvalent antibody.
3. detect
(1) with the antibody (many anti-or monoclonal antibodies) of preparation, carry out osteosarcomatous pathology detection with histochemical method, positive signal is osteosarcoma.
(2) get patients serum, detect by ELISA method, positive reaction is the suspicious patient of osteosarcoma.
(3) using the probe one of of C8orf59 antibody as protein chip, diagnose for osteosarcoma.
Embodiment 4 C8orf59 genetic expression is on the impact of osteosarcoma cell physiological activity
The detection of 1.siRNA jamming effectiveness
1.1 cell cultures
Be research object with human osteosarcoma U2OS cell, human osteosarcoma U2OS clone is purchased from Shanghai cell bank, and the IMDM substratum of cell cultures containing 10% foetal calf serum, under the environment not containing penicillin, Streptomycin sulphate, is the CO of 5% in volume fraction
2, cellar culture under 37 DEG C of conditions.U2OS cell puts basis of microscopic observation to cell attachment growth, and volume is medium, and size is even, cell ovalize or irregular shape, and kytoplasm is less, and nucleus is comparatively large, and kernel is clear.
1.2 siRNA disturb C8orf59 genetic expression
SiRNA sequence (siRNA-C8orf59) for C8orf59:
Positive-sense strand 5 '-UGUUCUUGGCCAUUGUCAGAA-3 ' (SEQ ID NO:5);
Antisense strand is 5 '-CUGACAAUGGCCAAGAACAAA-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).
By cell by 1 × 10
4/ hole is inoculated in 24 porocyte culture plates, at 37 DEG C, 5%CO
2cell cultures 24h in incubator, without dual anti-, containing in the IMEM substratum of 10%FBS, transfection is according to the specification sheets transfection of lipofectamine 2000 (purchased from Invitrogen company), experiment is divided into negative control group and experimental group (20nM), wherein the sequence of negative control group siRNA and C8orf59 gene is without homology, and concentration is 20nM/ hole.Transfection respectively simultaneously.
1.3 QPCR detect the transcriptional level of C8orf59 gene
1.3.1 the extraction of cell total rna
Adopt TRIzol Reagent (Invitrogen Cat.No.15596-018) total RNA extraction reagent, by specification supplying method extracts the total serum IgE of U2OS cell.Concrete grammar is: get cell, rinses 3 times with the PBS that concentration is 0.01M, adds appropriate TRIzol reagent, and room temperature places 5min lysing cell, is filled in 1.5mL Eppendorf pipe after piping and druming evenly with 1mL/ pipe point.Often pipe adds 0.2mL chloroform, concuss 15s, and room temperature places 2-3min, 4 DEG C, the centrifugal 15min of 12000r/min, move to upper water mutually in clean Eppendorf pipe, add 0.5mL Virahol, mix gently, room temperature places 10min, 4 DEG C, the centrifugal 10min of 7500r/min.Abandon supernatant, 75% washing with alcohol RNA precipitation, the centrifugal 5min of 7500r/min, drying at room temperature RNA precipitate, and are dissolved in appropriate DEPC water after 5-10min.Massfraction is the integrity of the agarose gel electrophoresis detection RNA sample of 1.0%, and application Bio-Photometer carries out quantitative assay to the RNA extracted.
1.3.2 reverse transcription step is with embodiment 2.
1.3.3 QPCR amplification step is with embodiment 2.
1.4 result
Result such as Fig. 3 shows, and siRNA-C8orf59 effectively can suppress the expression of C8orf59 gene.
The impact of 2.C8orf59 gene pairs human osteosarcoma cell proliferation
Cell proliferation in vitro uses mtt assay to detect.
2.1 steps:
By 5 × 10
3individual cell spreads into 96 orifice plates and overnight incubation, transfection next day negative control RNA or siRNA-C8orf59, and 0h, 24h, 48h, 72h, 96h, 120h, 144h carry out MTT detection after transfection.First, discard culture supernatant, change to the fresh culture of 100 μ L containing MTT 0.5mg/ml (Sigma company); Then 4h is hatched for 37 DEG C; Finally change to 100 μ L DMSO (Sigma company) and the 10min that vibrates.Final absorbancy uses 490nm wavelength detecting, draws cell growth curve.
2.2 result
As shown in Figure 4, after suppressing C8orf59 to express, the growth of osteosarcoma U 2OS is obviously suppressed result, shows that C8orf59 gene take part in the breeding of osteosarcoma cell.
The apoptotic impact of 3.C8orf59 gene pairs
Use the apoptotic impact of flow cytomery C8orf59 gene pairs.
3.1 step
Carry out cell transfecting according to previously described method, after transfection 72h, use precooling PBS washed cell, then use 0.25% trypsin digestion cell, stop digestion, using PBS resuspended in the cell of collected by centrifugation, is 1 × 10 by cell quantification
6individual/ml, gets the above-mentioned cell suspension of 200 μ L and is placed in Appendorf pipe, adds 10 μ L Annexin-V-FITC and mixes, and dyeing 15min is hatched in room temperature dark place, and before upper machine, 5min adds 10mg/L iodate third ingot (PI) and to dye 5 μ L.The cell of untransfected siRNA is used for standard quantitative with Annexin-V-FITC and PI dyeing respectively.Two Colour Fluorescence cell cytometry is carried out, observing apoptosis cell percentages with FACS flow cytometer.
3.2 statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, the t inspection that difference between the two adopts, think to there is statistical significance as P < 0.05.
3.3 result
The apoptosis rate of C8orf59 Gene interfere group is (45.23+2.53) %, 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 shows to suppress the expression of C8orf59 gene obviously can promote the apoptosis of osteosarcoma U 2OS.
The impact of 4.C8orf59 gene pairs osteosarcoma cell transfer ability
Cut Healing Experiments is adopted to detect the impact of C8orf59 gene pairs osteosarcoma cell transfer ability.
4.1 concrete steps
(1) select and spend the night containing the coat culture dish of 5cm size of the fibrinogenic PBS of 15 μ g/ml.
Within (2) second days, add the IMDM containing 10%FBS, use after sterilizing a few hours under ultraviolet light.
(3) U2OS cell is carried out cell transfecting according to previously described method, after transfection 72h, according to 1 × 10
6the standard of individual cell carries out inoculation culture to cell, and after 24h, whether observation of cell grows evenly.
(4) scrape at culture dish cell with cell in the middle of scratch a vestige, place it in incubator and cultivate the transfer ability of observation of cell in cut after 24h, select 3 samples when observing at every turn, observe 3 times.
4.2 statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, the t inspection that difference between the two adopts, think to there is statistical significance as P < 0.05.
4.3 result
After Matrigel starts 24h, the mean value of the cut healing rate of C8orf59 Gene interfere group and negative control group is respectively 28%, 75%, above-mentioned difference has statistical significance (P < 0.05), show the expression suppressing C8orf59 gene, the transfer ability of osteosarcoma U 2OS declines.
The impact of 5.C8orf59 gene pairs osteosarcoma cell invasive ability
Transwell is used to attack the impact of cell assay method detection C8orf59 gene pairs osteosarcoma cell invasive ability.
5.1 concrete operation steps are:
(1) in advance Matrigel (Beeton-Diekinson, FranklnLakes, NJ) is diluted to 200 μ g/ml with the IMDM of 4 DEG C, getting IMDM that 200 μ L dilute, to be placed on aperture be on the Transwell of 8 μm.
(2) make micropores whole on polycarbon resin film film all will be covered by Matrigel, spend the night under then it being placed on the condition of 37 DEG C.
(3) drying treatment is carried out to it.
(4) irradiate 2h with ultraviolet to the Transwell got ready and carry out sterilizing, the IMDM that pre-irradiation adds the serum-free that a small amount of sterilising treatment is crossed carries out aquation to it.
(5) carry out cell transfecting according to previously described method, after transfection 72h, get U2OS cell then by cell count 3 × 10
4join in each cell, substratum selects IMDM.
(6) be placed in 46 orifice plates and cultivated.
(7) 5%CO
2, hatch 24h in 37 DEG C of incubators.
(8) will cell take out after to wipe clean Matrigel and the cell in that face, upper room with paper handkerchief or cotton swab, after that face methyl alcohol of lower room fixes 10min, carry out conventional H E dyeing, use microscopic examination.
(9) observe according to Stochastic choice 3 high power lens visuals field, and under carefully counting cell, the number of cells in face, room is as the cell number penetrating Matrigel, this process carries out 3 times, calculates the reference frame that its mean number is used as calculating.
5.2 statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, the t inspection that difference between the two adopts, think to there is statistical significance as P < 0.05.
5.3 result
After 24h, C8orf59 Gene interfere group, it is 47 by the mean value of the cell count of Transwell film each visual field cell count under the microscope, and negative control group is 324 by the mean value of the cell count of Transwell film each visual field cell count under the microscope, above-mentioned difference has statistical significance (P < 0.05), show the expression suppressing C8orf59 gene, the invasive ability of osteosarcoma U 2OS declines.
6.C8orf59 gene pairs osteosarcoma cell becomes the impact of knurl ability
6.1 steps:
(1) carry out cell transfecting according to previously described method, by after the enzymic digestion of U2OS cell tryptase after transfection 72h, be prepared into cell suspension;
(2) be inoculated in after cell counting (200 cells/well) in 6 orifice plates, being continued by the cell inoculated to cultivate in incubator is greater than 50 to cell count in 14 days or most single clones, and midway carried out changing liquid and observation of cell state every 3 days; Under fluorescent microscope, cell clone is taken pictures before experiment stops;
(3) use paraformaldehyde fixed cell when experiment stops, after PBS washed cell, Giemsa dyes, and takes pictures.
6.2 result
Plate clone experimental result shows, compared with negative control group, the volume that clone's spot number that the osteosarcoma U 2OS of C8orf59 genetic expression interference group is formed significantly reduces, clone spot obviously reduces, and the above results shows that C8orf59 silence causes osteosarcoma cell to form the ability decline of clone.
Embodiment 5 C8orf59 antigen protein is as the application of osteosarcoma vaccine
1. antigen protein obtains
Utilize gene engineering expression: the cDNA sequence that can obtain people C8orf59 gene from Genbank database, obtains encoder block by pcr amplification, use Peptide synthesizer synthesis C8orf59 antigenic peptide according to above-mentioned sequence.
The in-vitro separation of 2.DC (dendritic cell) and cultivation
Detection in peripheral blood of patients underwent is separating interface mononuclearcell after lymphocytes separating solution Ficoll gradient centrifugation.Cell, after washing 2 times, suspends with serum free medium AIM-V nutrient solution (Gibco BRL company), and adjustment cell concn is to 3 × 10
6individual/ml, inoculates into 6 well culture plates.At 37 DEG C, 5%CO
2in incubator after overnight incubation, blow and beat suspension cell gently and reclaim frozen, in order to prepare effector cell.The AIM-V containing 1000U/ml grain single colony stimulating factor and 500U/ml interleukin-is added in attached cell nutrient solution.When being cultured to the 7th day, the DC that results suspend.
The human B lymphocyte of EBV (Epstein Barr Virus, the epstein-barr virus (EB)) transfection of 3.T2 cell or HLA-A2 and final concentration are that the C8orf59 antigenic peptide of 20 μ g/ml hatches 4h, altogether as target cell after washing.
4. identify that the present invention treats osteosarcomatous C8orf59 antigen vaccine in the following manner, by DC submission C8orf59 antigen peptide activating effect T cell, produce the immunne response of specificity for C8orf59 epitope:
(1) process of DC cell: DC is resuspended in 2ml AIM-V after centrifugal, and to add C8orf59 antigen peptide to this polypeptide final concentration be 40ug/ml.37 DEG C, 5%CO
2cultivate 18h, after 3000rad radiation exposure, wash stand-by.
(2) preparation of effector cell: the target cell of step 3 being prepared and postradiation DC cell mix with 20: 1 hatches 6-7 days altogether, then adds DC according to same ratio, and after 6-7 days, repetitive stimulation once again, action effect T cell.In 0,7,10,14,19 day, leave and take culture supernatant respectively, for cytokines measurement.
(3) cytokines measurement: adopt cytokine detection kits (purchased from American Endogen company) to detect the cytokine of secreting in culture supernatant.
5. experimental result shows, through the effector T cell that the DC of C8orf59 antigen peptide stimulation of the present invention activates, the immunne response of specificity for C8orf59 epitope can be produced, thus specific killing is combined with the target cell of this identical C8orf59 antigen peptide, illustrate that C8orf59 antigen protein of the present invention can as potential clinical treatment of osteosarcoma vaccine.
The explanation of above-described embodiment is just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.
Claims (10)
1. diagnose an osteosarcomatous product, it is characterized in that, by the expression detecting C8orf59 gene in experimenter's scleroblast, described product judges whether experimenter suffers from osteosarcoma.
2. product according to claim 1, is characterized in that, described product comprises: with RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or the osteosarcomatous product of gene chip diagnosis.
3. product according to claim 2, is characterized in that, described RT-PCR diagnoses osteosarcomatous product at least to comprise the primer of a pair specific amplified C8orf59 gene; Described real-time quantitative PCR diagnoses osteosarcomatous product at least to comprise the primer of a pair specific amplified C8orf59 gene; Described immunodetection diagnoses osteosarcomatous product to comprise: the antibody be combined with C8orf59 protein-specific; Described in situ hybridization diagnoses osteosarcomatous product to comprise: with the probe of the nucleic acid array hybridizing of C8orf59 gene; The osteosarcomatous product of described gene chip diagnosis comprises: with the probe of the nucleic acid array hybridizing of C8orf59 gene.
4. the product according to any one of claim 1-3, is characterized in that, described product is selected from following group: test kit, chip, filter paper.
The application of 5.C8orf59 gene in the osteosarcomatous product of diagnosis according to any one of preparation claim 1-3.
The application of 6.C8orf59 gene in the osteosarcomatous medicine of preparation treatment.
7. application according to claim 6, it is characterized in that, described medicine is selected from following group: suppress the medicine of human osteosarcoma cell proliferation, suppress the medicine of apoptosis in osteosarcoma cells, suppress the medicine of osteosarcoma cell migration, suppress the medicine of osteosarcoma invasion and attack, suppress osteosarcoma cell to become the medicine of knurl.
8. application according to claim 7, it is characterized in that, described medicine comprises the double stranded RNA being suppressed C8orf59 genetic expression by RNA interfering, or based on the tumor vaccine of C8orf59 antigen protein or for suppressing the protein of C8orf59 protein-active.
9. suppress a siRNA for C8orf59 genetic expression, it is characterized in that, described siRNA sequence for: positive-sense strand is as shown in SEQ ID NO:5, and antisense strand is as shown in SEQ ID NO:6.
10. the application of siRNA according to claim 9 in the osteosarcomatous medicine of preparation treatment.
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