CN107893119A

CN107893119A - Applications of the ZCCHC12 in osteosarcoma

Info

Publication number: CN107893119A
Application number: CN201711445269.0A
Authority: CN
Inventors: 常鹏; 孙耀兰; 肖枫
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2017-12-27
Filing date: 2017-12-27
Publication date: 2018-04-10
Anticipated expiration: 2037-12-27
Also published as: CN107893119B

Abstract

The invention discloses applications of the ZCCHC12 in osteosarcoma.The invention discloses applications of the ZCCHC12 in osteosarcoma diagnoses and treats；The present invention discloses a kind of product for diagnosing osteosarcoma, the product includes the horizontal reagents of detection ZCCHC12；The invention also discloses a kind of pharmaceutical composition, described pharmaceutical composition includes ZCCHC12 inhibitor.

Description

Applications of the ZCCHC12 in osteosarcoma

Technical field

The invention belongs to biomedicine field, is related to applications of the ZCCHC12 in osteosarcoma.

Background technology

Osteosarcoma (Osteosarcoma, OS) belongs to one of common Primary Malignant Bone Tumor, is mainly in teenager (Durfee RA,Mohammed M,Luu HH et al.Rheumatol Ther.2016,3(2):221-243).Amputation The standard method that osteosarcoma was treated before the 1970s, but this kind operation the limb function of patient is influenceed it is big, to patient Bring larger menticide.With the fast development of medical technology, surgical operation, chemotherapeutics, chemicotherapy auxiliary treatment etc. Means achieve significant progress, and limb salvage treatment is increasingly becoming the primary treatments of osteosarcoma, especially new adjuvant chemotherapy Joint limb-sparing surgery achieves great success in clinical treatment osteosarcoma, eliminates the pain of patient's amputation, is survived in 5 years Rate brings up to more than 80% [5].Molecular targeted therapy, immunization therapy and gene therapy are increasingly becoming study hotspot after this, New chapter has been composed for clinical treatment of osteosarcoma.

Most osteosarcoma have become high-grade malignant tumour in diagnosis, and early stage transfer easily occurs. Most common DISTANT METASTASES IN position is lung, accounts for 90% (Osasan S, Zhang M, Shen F et al.Anticancer Res.2016 36(9):4391-4398).Therefore, how to suppress the transfer of osteosarcoma, study effective treatment method, improve and suffer from The survival rate of person is urgent problem.The osteosarcoma such as operative treatment, radiotherapy, chemotherapy, immunization therapy often with therapeutic modality it Outside, make great efforts to explore more effective osteosarcoma early diagnosis, early stage suppression bone and flesh tumor metastasis, reduce the new of the Patients with Osteosarcoma death rate Treatment method.

Cell factor and its acceptor play an important role in cell signalling, the propagation of cell and atomization, During the fast breeding of osteosarcoma cell, necessarily cause the change of cell factor and its expression of receptor and distribution (Saini Vl, Hose CD,Monks A,et al.PLoS One.2012；7(8):e41401).Therefore it is thin to be present in osteosarcoma for searching characteristic Born of the same parents, or by the extremely caused material of osteosarcoma cell, or host to the stimulate the reaction of osteosarcoma caused material, and accordingly Reflect tumorigenesis, monitoring tumour is current study hotspot, such as patent to the tumor markers of therapeutic response 201510075917.2,201510075920.4,201510075918.7,201510075919.1 just pair with osteosarcoma occur, The related gene of development is groped.

With going deep into osteosarcoma molecular biosciences research, the targeted therapy of osteosarcoma also more and more turns into research Emphasis, and represent the developing direction in future, finds new effective biomarker, early diagnosis for osteosarcoma and Targeted therapy has great importance.

The content of the invention

In order to make up the deficiencies in the prior art, an object of the present invention is, there is provided a kind of product for diagnosing osteosarcoma.

The second object of the present invention is, there is provided a kind of pharmaceutical composition for treating osteosarcoma.

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

A kind of product for diagnosing osteosarcoma, the product include the horizontal reagents of detection ZCCHC12.Wherein, ZCCHC12 exists Up-regulated expression in Patients with Osteosarcoma, the product include but is not limited to preparation, chip, kit.

Further, the reagent is selected from：

The probe of specific recognition ZCCHC12 genes；Or

The primer of specific amplification ZCCHC12 genes；Or

Specifically bind the antibody or part of the albumen of ZCCHC12 codings.

Further, the primer sequence of specific amplification ZCCHC12 genes is as shown in SEQ ID NO.1~2.

The invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes ZCCHC12 inhibitor.

Further, the inhibitor is selected from：Nucleic acid inhibitor, protein inhibitor, proteolytic enzyme, protein binding molecule.

Further, the inhibitor is nucleic acid inhibitor siRNA, it is preferred that siRNA sequence such as SEQ ID NO.9~ Shown in 10.

The invention provides the application described in ZCCHC12 following any one：

Applications of the a.ZCCHC12 in the product for preparing diagnosis osteosarcoma；

Applications of the b.ZCCHC12 in the pharmaceutical composition for preparing treatment osteosarcoma；

Applications of the c.ZCCHC12 in the pharmaceutical composition for preparing treatment bone and flesh tumor metastasis；

Applications of the d.ZCCHC12 in the drug candidate of screening treatment osteosarcoma.

Further, product described in a include with RT-PCR, real-time quantitative PCR, new-generation sequencing, in situ hybridization, chip or Immunoassay detects ZCCHC12 reagent.

Further, pharmaceutical composition described in b or c includes ZCCHC12 inhibitor, and/or with the inhibitor compatibility Other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material.

Pharmaceutically acceptable carrier and/or auxiliary material include but is not limited to diluent, excipient, adhesive, wetting agent, Sorbefacient, surfactant, Humectant, absorption carrier, lubricant, buffer, stabilizer, bacteriostatic agent, isotonic agent, chelating Agent, pH controlling agents.

The inhibitor is selected from：ZCCHC12 gene expressions as target sequence and can be suppressed using ZCCHC12 or its transcript Or the disturbing molecule of genetic transcription, including：It is shRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, anti- Phosphorothioate odn, or can express or be formed the construction of the shRNA, siRNA, dsRNA, Microrna, antisensenucleic acids；It is or special The opposite sex and the protein bound binding molecule (if suppressing the antibody or part of ZCCHC12 protein actives) of ZCCHC12 codings.

Further, include in d the step of the drug candidate of screening treatment osteosarcoma：

The system for the albumen expressed or containing ZCCHC12 genes or its coding is handled with candidate substances；With

Detect the expression of the albumen of ZCCHC12 genes or its coding or activity in the system.

The advantages of the present invention：

Present invention firstly discovers that ZCCHC12 expression is to the occurrence and development of osteosarcoma related, pass through detection ZCCHC12 level, it can be determined that whether patient suffers from osteosarcoma or suffer from the risk of osteosarcoma.

The invention provides a kind of product for diagnosing osteosarcoma, the product can fall ill in osteosarcoma to be diagnosed in early days, Instruct doctor to carry out early intervention, improve the survival rate and quality of life of patient.

The invention provides a kind of pharmaceutical composition and drug targets for treating osteosarcoma, can be realized by targeted therapy Precision is treated.

Brief description of the drawings

Fig. 1 is the expression figure in osteosarcoma tissue using QPCR detection ZCCHC12 genes；

Fig. 2 is the expression figure in osteosarcoma tissue using Western blot detection ZCCHC12 albumen；

Fig. 3 is transfected condition figures of the ZCCHC12 in osteosarcoma cell；Wherein, figure A is to bone using QPCR detection transfections The influence figure that ZCCHC12mRNA is expressed in sarcoma cell；Figure B is in osteosarcoma cell using Western blot detection transfections The influence figure of ZCCHC12 albumen；

Fig. 4 is the influence figure for detecting ZCCHC12 gene pairs human osteosarcoma cell proliferations；

Fig. 5 is to detect the influence figure that ZCCHC12 migrates to osteosarcoma cell；

Fig. 6 is to detect the influence figure that ZCCHC12 attacks to osteosarcoma cell.

Embodiment

The present invention, by high-flux sequence method, detects gene in osteosarcoma samples and existed by in-depth study extensively Tumor tissues and the expression of cancer beside organism, find differences expressing gene, inquires into its relation between the generation of osteosarcoma, so as to Early detection and targeted therapy for osteosarcoma find more preferable approaches and methods.By screening, present invention firstly discovers that bone ZCCHC12 conspicuousnesses raise in sarcoma.It is demonstrated experimentally that the expression by reducing ZCCHC12, can effectively suppress bone and flesh The growth and invasion and attack of oncocyte, prompt the expression of detection ZCCHC12 genes to turn into the auxiliary that osteosarcoma early diagnoses and examine One of severed finger mark, interference ZCCHC12 gene expressions, which can turn into, prevents or treats osteosarcoma or the new way of bone and flesh tumor metastasis.

(biology) mark

Mark (is used alone or such as bone and flesh tumor markers, osteosarcoma specificity marker is combined with other qualitative terms Thing, control mark, external source mark, endogenous mark) refer to it is measurable, can calculate or can otherwise obtain, with appointing What molecule or molecular combinations are related, can be used as the parameter of the indicant of biology and/or chemical state.In the present invention, " mark Thing " refers to the parameter related to one or more biomolecule (i.e. " biomarker "), such as natural or artificial synthesized caused Nucleic acid (i.e. genes of individuals, and coding and noncoding DNA and RNA) and albumen (such as peptide, polypeptide)." mark in the present invention Thing " also includes referring to what can be calculated or otherwise obtain by considering the expression data from two or more unlike signal things Single parameter.

Bone and flesh tumor markers, which refer to, may be used as and (combining individually or with other marks) osteosarcoma indicant in subject Certain types of mark, in specific embodiments of the present invention, bone and flesh tumor markers can be used for provide (individually or and other Mark combines) mark of osteosarcoma clinical assessment in subject.

ZCCHC12 genes

ZCCHC12 in the present invention includes wild type, saltant type or its fragment.A kind of representational ZCCHC12 genes sequence Row are as shown in ZCCHC12 genes (NC_000023.11) in current international public nucleic acid database GeneBank, people of the invention ZCCHC12 nucleotides full length sequence or its fragment can generally use PCR TRAPs, recombination method or artificial synthesized method to obtain.

Detection technique (method)

The expression of the gene of the present invention is examined using a variety of detection techniques known to persons of ordinary skill in the art Survey, these technologies include but is not limited to：Nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies, immunoassay technology.

The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but is not limited to chain terminator (Sanger) sequencing and dye Expect terminator sequencing.One of ordinary skill in the art it will be recognized that due to RNA in cell less stable and in an experiment Be more vulnerable to nuclease attack, thus before sequencing generally by RNA reverse transcriptions into DNA.

The another exemplary non-limiting examples of Nucleic acid sequencing techniques include sequencing of future generation, and (deep sequencing/high pass measures Sequence), high throughput sequencing technologies be it is a kind of based on unimolecule cluster in synthesis sequencing technologies, based on proprietary reversible termination chemistry Reaction principle.The DNA of genome random fragment is attached to optically transparent glass surface during sequencing, these DNA fragmentations warp After crossing extension and bridge amplification, hundreds of millions of clusters is formed in glass surface, each cluster is the list for having thousands of parts of same templates Molecular cluster, four kinds of special deoxyribonucleotides with fluorophor are then utilized, skill is sequenced in synthesis by reversible Template DNA to be measured is sequenced art.

The exemplary, non-limitative example of nucleic acid hybridization technique include but is not limited in situ hybridization (ISH), microarray and Southern or Northern traces.In situ hybridization (ISH) be it is a kind of use mark complementary DNA or RNA chains as probe with A position tissue part or section (original position) are the spy in whole tissue (full organization embedding ISH) if tissue is sufficiently small Different in nature DNA or RNA sequence hybridization.DNAISH can be used for the structure for determining chromosome.RNAISH is used to measure and position tissue MRNA and other transcripts (for example, ncRNA) in section or full organization embedding.Generally sample cell and tissue are handled With fixation in situ target transcript, and increase the entrance of probe.Probe hybridizes with target sequence at high temperature, then by unnecessary probe Wash off.Autoradiograph, fluorescence microscopy or immunohistochemistry are used respectively, to using radiation, fluorescence or antigen mark in tissue The probe of the kilobase marker of note is positioned and quantified.ISH can also be used two or more by radioactivity or other non-put Penetrating property marks the probe of substance markers, to detect two or more transcripts simultaneously.

The present invention can simultaneously expand before detection or with detection to nucleic acid (for example, ncRNA).Nucleic acid amplification technologies Exemplary, non-limitative example include but is not limited to：PCR (PCR), reverse transcriptase polymerase chain reaction (RT- PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleotide sequence Expand (NASBA).One of ordinary skill in the art will be it will be recognized that some amplification techniques (for example, PCR) needs will before amplification RNA reverse transcriptions are into DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).

Commonly referred to as PCR PCR uses annealing and the primer extend of denaturation, primer pair and opposite strand Multiple circulations, exponentially increase target nucleic acid sequence copy number；The amplification of TMA transcriptive intermediate is (in substantial constant Temperature, multiple copies of target nucleic acid sequence are autocatalytically synthesized under conditions of ionic strength and pH, wherein target sequence is more Individual RNA copies autocatalytically generate other copy；LCR ligase chain reaction uses miscellaneous with the adjacent area of target nucleic acid The two groups of complementary DNA oligonucleotides handed over；Other amplification methods are included for example：The commonly referred to as NASBA expansion based on nucleotide sequence Increase；Use rna replicon enzyme (the commonly referred to as Q β replicase) amplification of amplification probe molecule in itself；Amplification method based on transcription； And the sequence amplification of self―sustaining.

The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.

Chip, kit

In the present invention, " chip ", " microarray ", " array " can be included but is not limited to equivalent substitute：DNA microarray (for example, cDNA microarrays and oligonucleotide microarray), protein microarray, micro-array tissue, transfection or cell microarray, change Chemical combination thing microarray and Antibody microarray.The DNA microarray of commonly referred to as genetic chip, DNA chip or biochip is micro- The set of DNA points is seen, these points are connected on the surface of solids (for example, glass, plastics or silicon), are formed for thousands of kinds Gene carries out expression pattern analysis or the array of expression monitoring simultaneously.Fixed DNA fragmentation is referred to as probe, and its is thousands of available In single DNA microarray.Microarray can be used for identifying disease base by comparing the gene expression in disease and normal cell Cause or transcript (for example, ncRNA).Multiple technologies can be used to be manufactured for microarray, and include but is not limited to：Printed with apicule needle Photoetching is carried out on to slide, using prefabricated mask, carries out photoetching, ink jet printing or microelectrode battle array using dynamic micro mirror element Electrochemical method on row.

Kit in the present invention can be used for detection ZCCHC12 expression, it is preferred that described kit has including detection The reagent of the albumen of the detection ZCCHC12 genes of effect amount or its coding, the one or more materials being selected from the group：Container, use Specification, positive control, negative control thing, buffer, auxiliary agent or solvent.Such as being suspended or fixing the solution of cell, Detectable label or tag, nucleic acid is set to be easy to the solution of hybridization, for the solution of cell lysis, or for the molten of nucleic acid purification Liquid.

Can also have the operation instructions of kit in the kit of the present invention, how be entered wherein describing using kit Row detection, and how tumor development to be judged using testing result, therapeutic scheme is selected.

Screening prevention or the compound for the treatment of osteosarcoma

The present invention can utilize the compound of biomarker ZCCHC12 screenings prevention or treatment osteosarcoma, in the present invention A kind of embodiment in, including step：In test group, test compound is added in cultivating system, and observes the test ZCCHC12 expression quantity and/or activity in the cell of group；In control group, testization is not added in identical cultivating system Compound, and observe the expression quantity and/or activity of ZCCHC12 in the cell of control group；

Wherein, if the ZCCHC12 of cell expression quantity and/or activity are less than control group in test group, the survey is indicated that Examination compound is expression to ZCCHC12 and/or activity have inhibitory action treating cancer drug candidate.

As one embodiment of the present invention, described step also includes：Traveling one is entered to the candidate compound of acquisition The cell experiment and/or animal experiment of step, further to select and determine for preventing, alleviating or treat from candidate compound The useful material of osteosarcoma.

As embodiments of the present invention, the system of the candidate compound of screening prevention or treatment osteosarcoma is not limited to cell System, in addition to cell system, subcellular fraction system, solution system, organizational framework, organ systems or animal system etc., the body System is not limited to above-mentioned form, as long as the system can detect test compound and can reduce ASPRV1 expression and/activity .

Inhibitor and pharmaceutical composition

Discovery based on inventor, the invention provides a kind of purposes of ZCCHC12 inhibitor, suppresses bone for preparing The pharmaceutical composition of sarcoma.As used herein, described ZCCHC12 inhibitor includes but is not limited to inhibitor, antagonist, resistance Stagnant dose, blocking agent, nucleic acid inhibitor etc..

Described ZCCHC12 genes or the inhibitor of albumen refer to any activity for reducing ZCCHC12 albumen, reduced The stability of ZCCHC12 genes or albumen, lower ZCCHC12 albumen expression, reduce ZCCHC12 albumen effective acting times, Or suppressing the material of the transcription and translation of ZCCHC12 genes, these materials are used equally for the present invention, as lowering Material useful ZCCHC12, so as to for preventing or treating osteosarcoma.For example, described inhibitor is：Nucleic acid inhibitor, Protein inhibitor, antibody, part, proteolytic enzyme, protein binding molecule, as long as it can be lowered on albumen or gene level The expression of ZCCHC12 albumen or its encoding gene.

As a kind of selection mode of the present invention, described ZCCHC12 inhibitor is that a species specificity is tied with ZCCHC12 The antibody of conjunction.The specific antibody includes monoclonal antibody, polyclonal antibody；The present invention not only includes complete antibody point Son, also any fragment including antibody or modification, for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as described Section can retain the binding ability with ZCCHC12 albumen.Art technology during preparation for the antibody of protein level Known to personnel, and the present invention can prepare the antibody using any method.

As a kind of preferred embodiment of the present invention, the inhibitor of the ZCCHC12 is a kind of ZCCHC12 specific small dry Disturb RNA molecule.As used herein, described " siRNA " refers to a kind of short-movie section double stranded rna molecule, can be with homologous mutual The mRNA of complementary series is the target specific mRNA of degraded, and this process is exactly RNA interference (RNAinterference) processes. SiRNA can be prepared into the form of double-strandednucleic acid, and it contains a positive-sense strand and an antisense strand, and this two chains are only miscellaneous Double-strand is formed under conditions of friendship.One double-stranded RNA compound can be prepared by the positive-sense strand that is separated from each other and antisense strand.Cause This, for example, complementary positive-sense strand and antisense strand are chemical syntheses, can produce the double of synthesis by anneal thereafter Chain RNA compounds.

When screening effective siRNA sequence, the present inventor is optimal effective so as to find out by largely comparing analysis Fragment.The present inventor's design has synthesized a variety of siRNA sequences, and they are transfected into osteosarcoma cell line by transfection reagent respectively Verified, select the optimal siRNA of interference effect, they have the sequence shown in SEQ ID NO.9, SEQ ID NO.10 respectively Row, further tested in cellular level, as a result prove that suppression efficiency is very high for test cell line.

The nucleic acid inhibitor such as siRNA of the present invention can be with chemical synthesis, can also be by a recombinant nucleic acid structure Expression cassette is prepared after being transcribed into single stranded RNA.The nucleic acid inhibitors such as siRNA, can be by using appropriate transfection reagent quilt It is transported into the cell, or can be also transported into the cell using multiple technologies known in the art.

As a kind of optional mode of the present invention, described ZCCHC12 inhibitor can also be a kind of " children purpura nephritis (Small hairpin RNA, shRNA) ", it is the non-coding small RNA molecular that can form hairpin structure, children purpura nephritis energy Enough by RNA interference channels come the expression of suppressor.As described above, shRNA can be expressed by double-stranded DNA template.Double-stranded DNA Template is inserted into a carrier, such as plasmid or viral vector, is then connected to a promoter carry out table in vitro or in vivo Reach.ShRN in the presence of DICER enzymes, can be cut into siRNA molecule in eukaryotic, hence into RNAi approach. " shRNA expression vectors " refers to plasmid of some this areas conventionally used for building shRNA structures, exist on the usual plasmid " Every sequence " and multiple cloning sites positioned at " intervening sequence " both sides or for replacing sequence, so as to people can by shRNA (or Analog) corresponding DNA sequence dna inserted by way of forward and reverse multiple cloning sites or replace thereon for replacing sequence, RNA after DNA sequence dna transcription can form shRNA (Short Hairpin) structure.Described " shRNA expression vectors " is current It can be bought and obtained by commercially available approach completely, such as some viral vectors.

Present invention also offers a kind of pharmaceutical composition, and it contains the described ZCCHC12 of effective dose inhibitor, and Pharmaceutically acceptable carrier.Described composition can be used for suppressing osteosarcoma.Any foregoing ZCCHC12 inhibitor Preparation for composition.The carrier includes but is not limited to diluent, excipient, adhesive, disintegrant, absorption enhancement Agent, surfactant, Humectant, absorption carrier, lubricant, buffer, stabilizer, bacteriostatic agent, isotonic agent, chelating agent, pH controls Preparation.

As used in the present invention, described " effective dose " refer to that people and/or animal can be produced function or activity and can be by people And/or the amount that animal is received.The effective dose of inhibitor can with the pattern of administration and the order of severity etc. of disease to be treated and Change.The selection of preferable effective dose can be determined (such as by facing by those of ordinary skill in the art according to various factors Bed experiment).Described factor includes but is not limited to：The pharmacokinetic parameter of the inhibitor of described ZCCHC12 genes is for example Bioavailability, metabolism, half-life period etc.；The order of severity of the disease to be treated of patient, the body weight of patient, the immune shape of patient Condition, approach of administration etc..

Pharmaceutical composition Orally-administrable of the present invention, parenteral administration, by suck spray delivery, it is local to Medicine, rectally, nasal administration, cheek administration, vagina administration are administered by the storage medicine device of implantation.It is preferred that it is administered orally or injects Administration.Pharmaceutical composition of the present invention contains any commonly employed nontoxic pharmaceutical acceptable carrier, auxiliary material or excipient.

The pharmaceutical composition of the present invention can also be with the drug combination of other treatment osteosarcoma, and other therapeutic compound can To be administered simultaneously with main active component, or even it is administered simultaneously in same composition.Can also with single composition or The dosage form different from main active component individually gives other therapeutic compounds.

Preferably, the means of gene therapy can be used to carry out.For example directly ZCCHC12 inhibitor can be passed through such as The methods of injection, delivers medicine to subject；Or the ceneme of ZCCHC12 inhibitor can will be carried by certain approach (such as expression vector or virus etc., or siRNA or shRNA) be delivered on target spot, and the ZCCHC12 for being allowed to expression activity suppresses Agent, concrete condition need to be depending on the types of described inhibitor, and these are well-known to those skilled in the art.

Experiment in the present invention is all at least completed according to being repeated 3 times, and result data is all with average value ± standard The mode of difference represented, statistical analysis is carried out using SPSS18.0 statistical softwares, and the paired comparisons of cancer and cancer beside organism are adopted Examined with t, it is believed that work as P<There is statistical significance when 0.05.

The present invention is further illustrated with reference to specific embodiment, embodiments of the invention are only used for explaining the present invention, It is not intended to limit protection scope of the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.

The screening of the gene marker related to osteosarcoma of embodiment 1

1st, sample collection

6 osteosarcoma tissues and cancer beside organism's sample are collected respectively, the acquirement of tissue samples obtains the informed consent of patient, And obtain and pass through the agreement of the committee of organizational ethics.

2nd, the preparation of RNA sample

RNA is extracted using the tissue RNA extracts kits of Invitrogen companies, concrete operations are with reference to specification.

3rd, the quality analysis of RNA sample

The RNA concentration and purity extracted are detected using Nanodrop2000, agarose gel electrophoresis detection RNA Integrality, Agilent2100 measure RIN values.Concentration >=200ng/ μ l, OD260/280 is between 1.8~2.2.

4th, rRNA is removed

The rRNA in total serum IgE is removed using Ribo-Zero kits.

5th, construction cDNA library

The structure of cDNA library, specific behaviour are carried out using the Truseq RNAsample Prep Kit using Illumina Make by specification progress.

6th, upper machine sequencing

CDNA library is sequenced using Hiseq4000 microarray datasets, concrete operations by specification is carried out.

7th, high flux transcript profile sequencing data is analyzed

Bioinformatic analysis and processing are carried out to sequencing result, the screening criteria of differential gene is fdr<0.05, two groups Fpkm average values difference be more than 5.

8th, result

RNA-seq results show that ZCCHC12 expressions significantly raise in Patients with Osteosarcoma, and difference has statistics Meaning (P<0.05).

The differential expression of embodiment 2QPCR sequence verification ZCCHC12 genes

1st, large sample QPCR checkings are carried out to ZCCHC12 gene differential expressions.According to the sample collection mode in embodiment 1 Select Patients with Osteosarcoma cancer beside organism and each 50 of osteosarcoma tissue.

2nd, RNA extracts specific steps as described in Example 1.

3rd, reverse transcription

Using FastQuant cDNA the first chain synthetic agent box (article No.s：KR106 mRNA reverse transcriptions) are carried out.Specific steps It is as follows：

(1) the μ l of 5 × gDNABuffer 2.0 are added, the μ g of total serum IgE 1, add Rnase Free ddH₂O makes cumulative volume to 10 μ l, 42 DEG C of heating 3min in water-bath；

(2) 20 μ l reaction systems are built：10 × Fast RT Buffer, 2.0 μ L, RT Enzyme Mix 1.0 μ l, FQ- μ l, the RNase Free ddH of RT Primer Mix 2.0₂Add in the mixed liquor in (1) and mix after the μ l of O 5.0 mixing；

(3) 42 DEG C of heating 15min in water-bath, 95 DEG C of heating 3min, -20 DEG C store for future use.

4th, QPCR is expanded

(1) design of primers

QPCR amplifications are designed according to the coded sequence of ZCCHC12 genes in Genebank and house-keeping gene GAPDH genes to draw Thing, synthesized by Bo Maide companies.

The primer sequence of ZCCHC12 genes：

Forward primer sequence is 5 '-GTTAATCAGAATGGTAAG-3 ' (SEQ ID NO.1)；

Reverse primer sequences are 5 '-CATCTATCTCTATCACAT-3 ' (SEQ ID NO.2).

The primer sequence of GAPDH genes：

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

Reverse primer sequences are 5 '-GGCTGTTGTCATACTTCTCATGG-3 ' (SEQ ID NO.4).

(2) PCR reaction systems：Forward primer and each μ of 0.6 μ l, 2 × SuperReal PreMix Plus 10 of reverse primer L, DNA profiling 2 μ l, ddH₂μ l, the 50 × ROX Reference Dye of O 7.4^△2 μ l, the μ l of sterile purified water 4.8.

(3) PCR reaction conditions：95 DEG C of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 circulations, 95 DEG C 15s, 60 DEG C of 60s, 95 DEG C of 15s.In the enterprising performing PCR reaction of the type quantitative real time PCR Instruments of ABI 7300, analyzed by melt curve analysis Purpose band is determined with electrophoresis, Δ Δ CT methods carry out relative quantification.

5th, result

As a result as shown in figure 1, compared with cancer beside organism, ZCCHC12 up-regulated expressions in osteosarcoma tissue, difference has system Meter learns meaning (P<0.05) it is, consistent with high-flux sequence result.

The differential expression of the protein immunization imprinting of embodiment 3 experiment detection ZCCHC12 albumen

1st, the extraction of total protein is organized

Put it into and be placed in the glass homogenizer in ice after shredding tissue with scissors, RIPA lysates and PMSF are with 100: 1 ratio is mixed, and the RIPA lysates of the ratio addition respective amount of 100 μ l lysates, glass are added according to every 20mg tissue specimens Glass homogenizer pulverize tissue until its fully crack, the liquid after cracking is drawn in EP pipes, at 4 DEG C 14000rpm centrifuge 5min, collect supernatant.

2nd, total protein concentration determines

The measure of protein concentration is carried out according to the specification of BCA determination of protein concentration kits.

3rd, SDS-PAGE electrophoresis

According to the separation gel of the specification preparation 8% of PAGE gel reagent preparation box and 5% concentration glue and carry out Electrophoresis.

4th, western is detected

1) electrotransfer

Pvdf membrane is put into methanol solution and activates 5min, is put into transferring film buffer solution and balances 20min.PAGE glue is taken out to put Enter in transferring film buffer solution, cut corresponding PAGE glue, according to be followed successively by from down to up filter paper, pvdf membrane, PAGE glue, filter paper it is suitable Sequence is put into half-dried transferring film instrument, constant pressure 25V transferring films 1.5h；

2) immuning hybridization

Pvdf membrane is taken out, PBS is placed in 5%BSA solution after rinsing shakes closing 2h at room temperature, and pvdf membrane is put into hybridization In bag, add primary antibody and stay overnight, wash pvdf membrane with TBST buffer solutions, add corresponding secondary antibody, be incubated 2h at room temperature, TBST delays Fliud flushing is washed.

3) DAB develops the color

The slightly dry rear DAB nitrite ions that Fresh is added dropwise of pvdf membrane, record is scanned after pvdf membrane colour developing.Made with β-actin For internal reference, sxemiquantitative gray analysis is carried out to band using Quantity One Labworks image acquisition and analysis softwares, experiment repeats 3 It is secondary, as a result take average gray value.

5th, result

As a result as shown in Fig. 2 expression of the ZCCHC12 albumen in osteosarcoma tissue is significantly higher than cancer beside organism.

The silence of embodiment 4ZCCHC12 genes

1st, cell culture

Cell line of human osteosarcoma U-2OS, with the DMEM culture mediums containing 10% hyclone and 1%P/S 37 DEG C, 5% CO₂, relative humidity be 90% incubator in cultivate.Change within 2-3 days liquid 1 time, cell growth is good, is grown in monolayer adherence.Make Passed on the 0.25% trypsase conventional digestion containing EDTA.

2nd, transfect

1) precellular processing is transfected

The day before transfection, 3~5 × 10 are planted on 6 well culture plates⁵Individual cells/well, one is cultivated in antibiotic-free culture medium My god, cell density is 30~50% during transfection, changes serum free medium into before transfection.

2) siRNA design

Negative control siRNA sequence (siRNA-NC)：

Positive-sense strand is 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.5)

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

siRNA1：

Positive-sense strand is 5 '-ACAACUUCAUGUUUCUGUCUG-3 ' (SEQ ID NO.7)

Antisense strand is 5 '-GACAGAAACAUGAAGUUGUUC-3 ' (SEQ ID NO.8)

siRNA2：

Positive-sense strand is 5 '-UCCGUUUAAUAAAAGCAUCAU-3 ' (SEQ ID NO.9)

Antisense strand is 5 '-GAUGCUUUUAUUAAACGGAAG-3 ' (SEQ ID NO.10)

siRNA3：

Positive-sense strand is 5 '-AUCUAUCUCUAUCACAUUGCA-3 ' (SEQ ID NO.11)

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

Experiment is divided into three groups：Control group (U-2OS), negative control group (siRNA-NC) and experimental group (siRNA1, SiRNA2, siRNA3), the sequence of wherein negative control group siRNA and ZCCHC12 genes is without homology.

3) transfect

Transfected using the liposome Lipofectamine 3000 of Invitrogen companies, by specification is operated

5th, QPCR detects the transcriptional level of ZCCHC12 genes

The extraction of 5.1 cell total rnas

The RNA in cell is extracted using Qiagen cell RNA extracts kit, experimental implementation is to specifications Carry out.

5.2 reverse transcription steps are the same as embodiment 2.

5.3QPCR amplification steps are the same as embodiment 2.

6th, Western detects the expression of ZCCHC12 albumen

The cell of the different disposal group in logarithmic phase is collected, cell is washed with the PBS of precooling, adds RIPA lysates, 30min is placed on ice, is scraped off the cell of cracking using cell scraper, and the liquid after cracking is drawn to EP pipes using pipettor In, 14000rpm centrifuges 5min at 4 DEG C, collects the supernatant after centrifugation, remaining step is the same as embodiment 3.

7th, result

As a result such as Fig. 3 is shown, with non-transfection group compared with transfecting siRNA-NC groups, experimental group siRNA2 expressions reduce Significantly, difference has statistical significance (P<0.05), siRNA2 is selected to carry out subsequent experimental.

The influence of embodiment 5ZCCHC12 gene pairs human osteosarcoma cell proliferations

ZCCHC12 gene pairs human osteosarcoma cell proliferation capacities are detected using MTT experiment

1st, the good cell of upgrowth situation is taken, conventional digestion counts cell, cell is diluted into conjunction into after single cell suspension The cell suspension of suitable concentration.

2nd, in 96 well culture plates, the different disposal group cell per well after dilution is inoculated with 2000 cells, 3 is at least set and puts down Row hole and cell-free medium control, 37 DEG C, 5%CO₂Cultivate 24h.

3rd, 1 after inoculation, 2,3,4,5 days daily OD values taken out 3 hole cells and its 490nm is detected with mtt assay, counted Number, calculate average value.

4th, abandoning supernatant before detecting, nutrient solution are washed 3 times, and MTT free serum cultures based sols (5mg/ml) 10 μ is added per hole L, continue in 37 DEG C of incubators to cultivate 4h, terminate culture.

5th, 100 μ l Formanzan lysates are added per hole, shaking table shakes 1min slowly.With wavelength it is 490nm on ELIASA Measure measure optical density (OD) value, using the time as transverse axis, OD value is that the longitudinal axis draws cell growth curve.

6th, result

As a result as shown in figure 4, compared with the control, for experimental group after siRNA2 is transfected, the propagation of cell substantially receives suppression System, difference have statistical significance (P<0.05) illustrate that the propagation of ZCCHC12 and osteosarcoma cell is closely related.

The influence of embodiment 6ZCCHC12 gene pairs apoptosis in osteosarcoma cells

Use the influence of flow cytomery ZCCHC12 gene pairs Apoptosis.

1st, cell culture and transfection procedure are the same as embodiment 3.

2nd, flow cytomery

1) cell of the different disposal group in exponential phase is broken into cell suspension through pancreatin digestion after-blow and counted. Take 10⁶The cell suspension of amount, 1000rpm centrifugations 5min；

2) supernatant is abandoned, 195 μ l Annexin V-FITC combination liquid is added and cell is gently resuspended；

3) 5 μ l Annexin V-FITC are added, soft to mix, lucifuge is incubated 10min at room temperature；

4) 1000rpm centrifuges 5min, abandons supernatant, and cell is gently resuspended in the Annexin V-FITC combination liquid for adding 190 μ l；

5) 10 μ l propidium iodides (PI) dyeing liquors are added, it is soft to mix, ice bath avoid light place, carry out flow cytomery Apoptosis situation, all experiments are repeated 3 times, results averaged.

3rd, result：

As a result show, compared with control group, the apoptosis rate of the cell of experimental group illustrates ZCCHC12 to bone without significant changes The apoptosis of sarcoma has no significant effect.

The influence of embodiment 7ZCCHC12 cell migrations

1st, the μ g/ml of 1ml 50 fibronectin is added per hole into 6 orifice plates, is placed in 4 DEG C of refrigerator overnights.

2nd, remaining Fibronectin solution is discarded, is cleaned with serum free medium, by not existing together in exponential phase Reason group cell is inoculated in 6 orifice plates for being covered with fibronectin after pancreatin digestion is resuspended, and every group of cell sets 2 multiple holes, per hole 5 ×10⁵Individual cell, 37 DEG C, 5%CO₂Overnight incubation in incubator.

3rd, when cell length to about 90% fusion, an acellular cut is marked with 10 μ l Tip heads, PBS solution is washed The cell to come off is removed, serum free medium is added and continues to cultivate.

4th, 0h, 48h observe the healing state at cell cut and taken pictures after cut.Experiment is repeated 3 times, and is as a result taken Average value.

5th, result

As a result as shown in figure 5, substantially being reduced compared to the cell migration distance after siRNA2 for control group, is transfected, and it is right It can promote the migration of osteosarcoma cell according to without significant difference, illustrating that ZCCHC12 is overexpressed between group.

Influences of the embodiment 8ZCCHC12 to cell invasion

1st, prepared by Transwell cells

1) by 50mg/L Matrigel glue with the serum free medium of 4 DEG C of precoolings with 1:8 dilution proportion, mix；

2) the Matrigel glue (3.9 μ g/ μ l) of the μ l of 60 μ l~80 dilutions is taken to be placed in the Transwell upper chambers that aperture is 8 μm Polycarbonate membrane on, all micropores on film is covered by Matrigel, being placed in 37 DEG C of 30min polymerize Matrigel Into gel.

2nd, cell suspension is configured

The cell of different disposal group in exponential phase is digested through pancreatin, after serum free medium is resuspended, adjustment Cell concentration is 5 × 10⁴Individual/ml.

3rd, cell is inoculated with

2ml cell suspension is added in Transwell upper chambers, lower room adds the 1ml complete training containing 10% hyclone Base is supported, is positioned on 6 supporting orifice plates, 37 DEG C, 5%CO₂Under the conditions of cultivate 20-24h；Transwell cells are taken out, cotton swab is wiped The Matrigel glue in most upper chamber face and the cell for not penetrating film.

4th, dye

After cell culture terminates, Transwell cells are taken out, cotton swab wipes the Matrigel glue in upper chamber face to the greatest extent and do not penetrate film Cell, lower room face is with after 95% alcohol fixation 15min, haematoxylin dyeing 2min, and 5 high power lenses are taken at random under inverted microscope Visual field observation, count and take pictures.The cell number for counting cell Xia Shi faces is to penetrate the cell number of Matrigel glue, is taken the mean As experimental result, and the invasiveness of tumour cell is represented with the cell number, experiment is repeated 3 times, and every group of cell sets 3 multiple holes.

5th, result

As a result as shown in fig. 6, compared with control group, the cell for transfecting siRNA2 experimental group passes through Transwell cells The cell number of polycarbonate membrane significantly reduces, and no significant difference between control group, and as a result explanation interference ZCCHC12 expression can To reduce the invasion and attack of osteosarcoma cell.

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 improvement can also be carried out to the present invention And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.

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Claims

1. a kind of product for diagnosing osteosarcoma, it is characterised in that the product includes the horizontal reagents of detection ZCCHC12.

2. product according to claim 1, it is characterised in that the reagent is selected from：

The probe of specific recognition ZCCHC12 genes；Or

The primer of specific amplification ZCCHC12 genes；Or

Specifically bind the antibody or part of the albumen of ZCCHC12 codings.

3. product according to claim 2, it is characterised in that the primer sequence such as SEQ of specific amplification ZCCHC12 genes Shown in ID NO.1~2.

4. a kind of pharmaceutical composition, it is characterised in that described pharmaceutical composition includes ZCCHC12 inhibitor.

5. pharmaceutical composition according to claim 4, it is characterised in that the inhibitor is selected from：Nucleic acid inhibitor, albumen Inhibitor, proteolytic enzyme, protein binding molecule.

6. pharmaceutical composition according to claim 5, it is characterised in that the inhibitor is nucleic acid inhibitor siRNA, excellent Choosing, siRNA sequence is as shown in SEQ ID NO.9~10.

7.ZCCHC12 following any one described in application：

8. application according to claim 7, it is characterised in that product described in a include with RT-PCR, real-time quantitative PCR, New-generation sequencing, in situ hybridization, chip or immunoassay detect ZCCHC12 reagent.

9. application according to claim 7, it is characterised in that pharmaceutical composition described in b or c suppresses including ZCCHC12 Agent, and/or other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material with the inhibitor compatibility.

10. application according to claim 7, it is characterised in that wrapped in d the step of the drug candidate of screening treatment osteosarcoma Include：