CN107988374A - A kind of and the relevant molecular marker of osteosarcoma and its application - Google Patents
A kind of and the relevant molecular marker of osteosarcoma and its application Download PDFInfo
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Abstract
The invention discloses a kind of and the relevant molecular marker of osteosarcoma and its application, molecular marker is AWAT2, the albumen of AWAT2 genes and its coding up-regulated expression in Patients with Osteosarcoma, the expression by detecting AWAT2 may determine that whether patient suffers from osteosarcoma；Disturb the expression of AWAT2 to suppress the propagation of cancer cell by being related to siRNA, prompt AWAT2 to be applied to as molecular target in the accurate medical treatment of Patients with Osteosarcoma.
Description
Technical field
The invention belongs to biomedicine field, is related to a kind of and the relevant gene of osteosarcoma and its application, specific described
Gene is AWAT2.
Background technology
Osteosarcoma is most common bone malignant tumour, originating from the interstitial cell that can secrete bone matrix or osteoid, good hair
In teenager, grade malignancy is high, often causes death due to the Bone tumour of early stage, and 5 annual death rates are often up to more than 80%.
With the raising of surgical technic, the development of chemotherapeutics and the application of new adjuvant chemotherapy, the combined therapy effect of osteosarcoma and pre-
After obtained obvious improvement, five year survival rate may be up to more than 60%, even can be with some advanced oncotherapy centers
Reach more than 80%, the life quality of patient is greatly improved also with the development of limb-sparing surgery.New adjuvant chemotherapy
The application of chemotherapeutics is highlighted, because staging tomography, which can not only reduce, both sends out tumor size, is created conditions for operation, and can disappear
Go out potential metastatic lesion, reduces patient's DISTANT METASTASES IN probability.However, the toxic reaction of high-dose chemotherapy medicine is brought to patient
Great pain, quality of life of patients be not good enough.Successive treatment of the drug resistance of tumour cell also to osteosarcoma is brought at the same time
Great unstability.The mechanism of molecular biology in terms of understanding tumour bone uptake and reconstruction, seeks preferably chemotherapeutics
Or biostatic agent is the key for further improving clinical treatment of osteosarcoma effect.
Novel gene targeted therapies are biotherapies relatively advanced at present, realize and are directed to for different tumours individual
The treatment of property, is one of important means of current oncotherapy.RNA interference (RNAi is a kind of inherent gene silent technology,
ShRNA (short hairpin RNA) also known as short hairpin RNA, are that a kind of can be cloned into expression vector and express short interference
The DNA molecular of RNA (siRNA), can induce the efficiently specific degraded of homologous mRNA, at present become gene studies, cancer,
The important means such as Familial Occurrence disease and infectious disease.As patent 201510075917.2,201510075920.4,
201510075918.7, disclose in 201510075919.1 with the relevant gene marker of osteosarcoma occurrence and development, and lead to
The expression of external interference gene is crossed to demonstrate the treatment that gene can be used for osteosarcoma.Although in the prior art it has been reported that
The relation of portion gene and disease, but far can not meet the needs of clinical osteosarcoma diagnose and treat, it is also necessary to excavate
It is new be applied to the relevant effective gene of osteosarcoma as marker it is clinical.
The content of the invention
In order to make up for the deficiencies of the prior art, it is an object of the invention to provide a kind of relevant with osteosarcoma occurrence and development
Gene, by the expression for detecting the gene in sample, it can be determined that whether patient suffers from osteosarcoma or suffer from osteosarcoma
The size of risk, by targeting the gene, changes the expression or activity of the gene, it is possible to achieve the essence of Patients with Osteosarcoma
Quasi- targeted therapy.
To achieve these goals, the present invention adopts the following technical scheme that：
The present invention provides detect application of the reagent of AWAT2 expressions in the product for preparing diagnosis osteosarcoma.
Further, the reagent includes：Pass through RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip skill
Art detects AWAT2 expression reagents.
Further, the reagent includes the primer and/or probe for AWAT2 genes, or the antibody for AWAT2 albumen
And/or ligand.
The present invention provides a kind of product for diagnosing osteosarcoma, the product includes detecting AWAT2 expressions in sample
Chip, preparation or kit.
Further, the kit includes the primer of at least a pair of of specific amplification AWAT2 genes；Preferably, it is described to draw
Thing has the sequence shown in SEQ ID NO.1~2.
The present invention provides applications of the AWAT2 in the candidate compound of screening prevention or treatment osteosarcoma.
Further, the step of screening candidate compound is as follows：
The system for the albumen expressed or containing AWAT2 genes or its coding is handled with candidate substances；With
Detect the expression of the albumen of AWAT2 genes or its coding or activity in the system；
Wherein, if the candidate substances can reduce the expression or active preferably notable of AWAT2 genes or the albumen of its coding
Reduce, such as low more than 20%, preferably low more than 50%；More preferably low more than 80%), then show the candidate substances be prevention or
Treat the candidate compound of osteosarcoma.
In the present invention, the system includes but is not limited to：Cell system, subcellular fraction system, solution system, organizer
System, organ systems or animal system.The candidate compound includes but is not limited to：For AWAT2 genes or its upstream or under
Swim the disturbing molecule, nucleic acid inhibitor, micromolecular compound of gene design.
In the present invention, the step further includes：The candidate compound of acquisition is carried out further cell experiment and/
Or animal experiment, further to select and determine from candidate compound for preventing, alleviating or treating the useful thing of osteosarcoma
Matter.
Inhibitor the present invention provides AWAT2 functional expressions is preparing the pharmaceutical composition of prevention or treatment osteosarcoma
In application.The inhibitor of the AWAT2 functional expressions includes nucleic acid inhibitor, protein inhibitor, proteolytic enzyme, albumen
Binding molecule.Wherein nucleic acid inhibitor is selected from：AWAT2 gene expressions as target sequence and can be suppressed using AWAT2 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.Albumen
Binding molecule is selected from：The material combined with AWAT2 protein-specifics, can such as suppress the antibody or ligand of AWAT2 protein actives.
Further, the inhibitor is siRNA；Preferably, the siRNA sequence is as shown in SEQ ID NO.9~10.
When screening effective siRNA sequence, the present inventor is by largely comparing analysis, so as to find out optimal effective fragment.This hair
Person of good sense's design has synthesized a variety of siRNA sequences, and they are transfected osteosarcoma cell line by transfection reagent respectively and is verified,
Select the optimal siRNA of interference effect (in the present invention, optimal siRNA sequence is as shown in SEQ ID NO.9~10).
Nucleic acid inhibitors such as siRNA in the present invention can be with chemical synthesis, can also be by a recombinant nucleic acid structure
Expression cassette be transcribed into after single stranded RNA and prepared.The nucleic acid inhibitors such as siRNA, can be by using appropriate transfection reagent
It is transported into the cell, or can be also transported into the cell using multiple technologies known in the art.
The present invention provides a kind of pharmaceutical composition for treating osteosarcoma, described pharmaceutical composition includes AWAT2 features
The inhibitor of expression, and/or other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material with the inhibitor compatibility.
The inhibitor of the AWAT2 functional expressions refers to any activity for reducing AWAT2 albumen, reduces AWAT2 bases
Cause or the stability of albumen, lower the expression of AWAT2 albumen, reduce AWAT2 albumen effective acting times or suppress AWAT2 bases
The material of the transcription and translation of cause, these materials are used equally for the present invention, as the material useful for lowering AWAT2, so that
Available for preventing or treat osteosarcoma.Inhibitor includes nucleic acid inhibitor to example as mentioned, protein inhibitor, proteolytic enzyme,
Protein binding molecule.
The pharmaceutically acceptable carrier includes but is not limited to diluent, excipient, adhesive, wetting agent, absorption
Accelerating agent, surfactant, Humectant, absorption carrier, lubricant, buffer, stabilizer, bacteriostatic agent, isotonic agent, chelating agent,
PH controlling agents.
Brief description of the drawings
Fig. 1 is the expression figure in osteosarcoma tissue using QPCR detection AWAT2 genes；
Fig. 2 is the expression figure in osteosarcoma tissue using Western blot detection AWAT2 albumen；
Fig. 3 is the influence figure transfected using QPCR detections siRNA to AWAT2 gene expressions in osteosarcoma cell；
Fig. 4 is the influence transfected using western blot detections siRNA to AWAT2 protein expressions in osteosarcoma cell
Figure；
Fig. 5 is the influence figure with mtt assay detection AWAT2 gene pairs human osteosarcoma cell proliferations.
Specific embodiment
The present invention, by high-flux sequence method, detects gene in osteosarcoma samples and exists by in-depth study extensively
Tumor tissues and the expression of cancer beside organism, find wherein there is the gene of obvious differential expression, inquire into its generation with osteosarcoma
Between relation, so as to find more preferable approaches and methods for the early detection and targeted therapy of osteosarcoma.Pass through screening, this hair
It is bright to be found that in osteosarcoma that AWAT2 conspicuousnesses raise first.It is demonstrated experimentally that the expression by reducing AWAT2, can be effective
Ground suppresses the propagation of osteosarcoma cell, prompts the expression of detection AWAT2 genes to become the auxiliary of osteosarcoma early diagnosis
One of diagnosis index, interference AWAT2 gene expressions, which can become, prevents or treats osteosarcoma or the new way of bone and flesh tumor metastasis.
AWAT2 genes
AWAT2 in the present invention includes wild type, saltant type or its fragment.A kind of representational AWAT2 gene orders are such as
At present in international public nucleic acid database GeneBank shown in AWAT2 genes (NC_000023.11)；People's AWAT2 cores of the present invention
Thuja acid full length sequence or its fragment can usually use PCR amplification method, recombination method or artificial synthesized method to obtain.
The gene of the present invention is detected using a variety of detection techniques known to persons of ordinary skill in the art, these technologies
Including but not limited to：Nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies, biochip technology, immunoassay technology.This area
It is to be understood by the skilled artisans that all detection techniques can all realize the technical side of the present invention as the supplementary means of the present invention
Case.
Chip, kit
In the present invention, " chip ", " microarray ", " array " can be included but 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 and are used for thousands of kinds
Gene is carried out at the same time expression pattern analysis or the array of expression monitoring.Fixed DNA fragmentation is known as probe, 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 not limited to：Printed with apicule needle
Photoetching is carried out on to glass 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 the expression of detection AWAT2, it is preferred that it is effective that the kit includes detection
The reagent of the detection AWAT2 genes of amount, one or more material selected from the group below：Container, operation instructions, positive control,
Negative control thing, buffer, auxiliary agent or solvent.Such as being suspended or the solution of fixed cell, detectable label or tag,
Nucleic acid is set to be easy to the solution of hybridization, for the solution of cell lysis, or the solution for nucleic acid purification.
The present invention kit in can also have kit operation instructions, wherein describe how using kit into
Row detection, and how tumor development to be judged using testing result, therapeutic scheme is made choice.
Kit using the present invention, can detect AWAT2 by various methods (including but not limited to) selected from the group below：It is real
When Quantitative Reverse Transcription PCR, biochip test method, southern blotting technique method or RNA blottings or hybridization in situ.The common skill in this area
Art personnel can be according to physical condition and needing to be adjusted detection mode and change.
Inhibitor and pharmaceutical composition
Discovery based on inventor, the present invention provides a kind of purposes of the inhibitor of AWAT2, is used to prepare suppression bone and flesh
The pharmaceutical composition of knurl.As used herein, the inhibitor of the AWAT2 includes but not limited to inhibitor, antagonist, retardance
Agent, blocking agent, nucleic acid inhibitor etc..
The AWAT2 genes or the inhibitor of albumen refer to any activity for reducing AWAT2 albumen, reduce AWAT2
The stability of gene or albumen, the expression for lowering AWAT2 albumen, reduce AWAT2 albumen effective acting times or suppress AWAT2
The material of the transcription and translation of gene, these materials are used equally for the present invention.
As a kind of selection mode of the present invention, the inhibitor of the AWAT2 is that a species specificity is combined with SPOCD
Antibody.The specific antibody includes monoclonal antibody, polyclonal antibody；The present invention not only includes complete antibody molecule,
Any fragment or modification including antibody, for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as the fragment can
Retain the binding ability with AWAT2 albumen.It is well known to those skilled in the art during preparation for the antibody of protein level
, 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 AWAT2 is a kind of specific small interference of AWAT2
RNA molecule.As used herein, " siRNA " refers to a kind of short-movie section double stranded rna molecule, can be with homologous complementary
The mRNA of sequence is the target specific mRNA of degraded, this process is exactly RNA interference (RNA interference) processes.It is small
RNA interfering can be prepared into the form of double-strandednucleic acid, it contains a positive-sense strand and an antisense strand, this two chains are only hybridizing
Under conditions of form double-strand.One double-stranded RNA compound can be prepared by the positive-sense strand that is separated from each other and antisense strand.Therefore,
For example, complementary positive-sense strand and antisense strand are chemical syntheses, by anneal, can produce the double-strand of synthesis thereafter
RNA compounds.
When screening effective siRNA sequence, the present inventor is by largely comparing analysis, so as to find out optimal effective
Fragment.The present inventor's design has synthesized a variety of siRNA sequences, and they are transfected osteosarcoma cell line by transfection reagent respectively
Verified, select the optimal siRNA of interference effect, they have the sequence shown in SEQID NO.9, SEQ ID NO.10 respectively
Row, are 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, the inhibitor of the AWAT2 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 above-mentioned, 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.ShRNA under the action 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 that people can by shRNA (or
Analog) corresponding DNA sequence dna be inserted into 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." the 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, it contains the inhibitor of a effective amount of AWAT2, and medicine
Acceptable carrier on.The composition can be used for suppressing osteosarcoma.The inhibitor of any foregoing AWAT2 is used equally for
The preparation of composition.The carrier includes but is not limited to diluent, excipient, adhesive, disintegrant, sorbefacient, table
Face activating agent, Humectant, absorption carrier, lubricant, buffer, stabilizer, bacteriostatic agent, isotonic agent, chelating agent, pH controlling agents.
As used herein, described " effective dose " refer to that people and/or animal can be produced function or activity and can by people and/
Or the amount that animal is received.The effective dose of inhibitor can become with the pattern of administration and the severity of disease to be treated etc.
Change.Preferable a effective amount of selection can be determined (such as to pass through clinic by those of ordinary skill in the art according to various factors
Experiment).The factor includes but not limited to：The pharmacokinetic parameter of the inhibitor of the AWAT2 genes is for example biological
Utilization rate, metabolism, half-life period etc.；The severity of disease that patient to be treated, the weight of patient, patient immune state,
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 ingredient, or even it is administered simultaneously in same composition.Can also with single composition or
The dosage form different from main active ingredient individually gives other therapeutic compounds.
Preferably, the means of gene therapy can be used to carry out.Such as can be directly by the inhibitor of AWAT2 by such as noting
The methods of penetrating delivers medicine to subject；Alternatively, can by certain approach will carry AWAT2 inhibitor ceneme (such as
Expression vector or virus etc., or siRNA or shRNA) it is delivered on target spot, and the AWAT2 inhibitor of expression activity is allowed to, specifically
Situation need to be depending on the type of the inhibitor, these are well-known to those skilled in the art.
In a specific embodiment of the present invention, experiment all completed according to being at least repeated 3 times, result data be all with
The mode of mean+SD represents, statistical analysis is carried out using SPSS18.0 statistical softwares, cancerous tissue with it is normal
The paired comparisons of mucosal tissue are examined using t by cancer, it is believed that work as P<There is statistical significance when 0.05.
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiments are merely to illustrate this
Invent rather than limit the scope of the invention.The experimental method of actual conditions is not specified in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
Embodiment 1 and the screening of the relevant gene marker of osteosarcoma
1st, sample collection
6 osteosarcoma tissues and cancer beside organism's sample are respectively collected, the acquirement of tissue samples obtains the informed consent of patient, and
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, high-flux sequence
The rRNA in total serum IgE is removed using Ribo-Zero kits, utilizes the Truseq using Illumina
RNA sample Prep Kit carry out the structure of cDNA library, and cDNA library is surveyed using Hiseq4000 microarray datasets
Sequence.
5th, high throughput transcript profile sequencing data is analyzed
Bioinformatic analysis and processing are carried out to sequencing result, using the expression quantity of cuffquant quantification of mrna,
Cuffdiff compares differential expression of the control group with tumor group, and the screening criteria of differential gene is with fdr<0.05, two groups
The difference of fpkm average values is more than 5.
6th, result
The results show that compared with cancer beside organism, the AWAT2 expressions in osteosarcoma tissue significantly raise RNA-seq.
The differential expression of 2 QPCR sequence verification AWAT2 genes of embodiment
1st, large sample QPCR verifications are carried out to AWAT2 gene differential expressions.Selected 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) 5 × gDNA Buffer, 2.0 μ l are added, 1 μ g of total serum IgE, 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-
2.0 μ l, RNase Free ddH of RT Primer Mix2Add in the mixed liquor in (1) and mix after 5.0 μ l of O 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 amplimers are designed according to the coded sequence of AWAT2 genes in Genebank and house-keeping gene GAPDH genes,
Synthesized by Bo Maide companies, the amplimer sequence of wherein AWAT2 as shown in SEQ ID NO.1~2, house-keeping gene GAPDH's
Amplimer sequence is as shown in SEQ ID NO3~4.
(2) PCR reaction systems：Forward primer and each 0.6 μ l, 2 × SuperReal PreMix Plus, 10 μ of reverse primer
L, DNA profiling 2 μ l, ddH27.4 μ l, 50 × ROX Reference Dye of O△2 μ l, 4.8 μ l of sterile purified water.
(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 of 15s,
60 DEG C of 60s, 95 DEG C of 15s.PCR reactions are carried out on 7300 type fluorescence quantitative PCR instruments of ABI, pass through melt curve analysis analysis and electrophoresis
Determine purpose band, Δ Δ CT methods carry out relative quantification.
5th, result
The results are shown in Figure 1, and compared with cancer beside organism, AWAT2 up-regulated expressions in osteosarcoma tissue, difference has statistics
Learn meaning (P<0.05) it is, consistent with high-flux sequence result.
The differential expression of 3 protein immunization imprinting of embodiment experiment detection AWAT2 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 mixes, 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, collects supernatant.
2nd, total protein concentration measures
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 blot are 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 rinses to be placed in 5%BSA solution 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 carries out 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
The results are shown in Figure 2, and AWAT2 protein expression levels are significantly higher than cancer beside organism in osteosarcoma tissue.
The silence of 4 AWAT2 genes of embodiment
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 plates5A 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) design of siRNA
RNA interfering, wherein control group are designed according to the gene order of AWAT2：The sequence of siRNA-NC such as SEQ ID NO.5
Shown in~6, experimental group：The sequence of siRNA1 is as shown in SEQ ID NO.7~8, sequence such as SEQ ID NO.9~10 of siRNA2
It is shown.
Experiment is divided into three groups：Control group (U-2OS), negative control group (siRNA-NC) and experimental group (siRNA1,
SiRNA2), the sequence of wherein negative control group siRNA and AWAT2 genes is without homology.
3) transfect
Transfected using the liposome Lipofectamine 2000 of Invitrogen companies, by specification is grasped
Make, the silence effect of RNA interfering is observed after transfection.
5th, QPCR detects the transcriptional level of AWAT2 genes
The extraction of 5.1 cell total rnas
The RNA in cell is extracted using the cell RNA extracts kit of Qiagen, 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, result
As a result such as Fig. 3 is shown, with non-transfection group compared with transfecting siRNA-NC groups, experimental group siRNA1's and siRNA2
MRNA level in-site has all declined, and the wherein effect of siRNA2 is the most notable, therefore selects siRNA2 to carry out follow-up experiment.
Influences of 5 Western blot of the embodiment detection transfections siRNA to AWAT2 protein expressions
1st, cell culture and transfection procedure are the same as embodiment 4
2nd, the extraction of total protein of cell
The cell of the different disposal group in logarithmic phase is collected, cell is washed with the PBS of precooling.By RIPA cell pyrolysis liquids
With PMSF with 100:1 ratio mixes, and above-mentioned 150 μ l of lysate are added into cell, 30min is placed on ice, uses cell scraper
Knife scrapes off the cell of cracking, and the liquid after cracking is drawn in EP pipes using pipettor, and 14000rpm is centrifuged at 4 DEG C
5min.Supernatant after careful collection centrifugation.
3rd, total protein concentration measures
The measure of protein concentration is carried out according to the specification of BCA determination of protein concentration kits.
4th, 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.
5th, western detecting steps detailed in Example 3.
6th, result
The results are shown in Figure 4, compared with control group, transfects the AWAT2 albumen tables in the cell of siRNA1 and siRNA2 groups
All lowered up to amount, wherein siRNA2 groups are the most notable, therefore selection siRNA2 does the research of subsequent experimental.
The influence of 6 AWAT2 gene pairs human osteosarcoma cell proliferations of embodiment
1st, the good cell of upgrowth situation is taken, conventional digestion counts cell, cell is diluted to 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, calculates 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, continues in 37 DEG C of incubators to cultivate 4h, terminates culture.
5th, 100 μ l Formanzan lysates are added per hole, shaking table shakes 1min slowly.With wavelength it is 490nm in microplate reader
Measure measure optical density (OD) value, using the time as transverse axis, OD value draws cell growth curve for the longitudinal axis.
6th, result
The results are shown in Figure 5, and 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 during the occurrence and development of osteosarcoma, AWAT2 can promote cell
Propagation.
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, without departing from the principle of the present 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 (10)
1. detect application of the reagent of AWAT2 expressions in the product for preparing diagnosis osteosarcoma.
2. application according to claim 1, it is characterised in that the reagent includes：By RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization or chip technology detection AWAT2 expression reagents.
3. application according to claim 2, it is characterised in that the reagent include for AWAT2 genes primer and/or
Probe, or antibody and/or ligand for AWAT2 albumen.
4. a kind of product for diagnosing osteosarcoma, it is characterised in that the product includes the core that AWAT2 expressions are detected in sample
Piece, preparation or kit.
5. product according to claim 4, it is characterised in that the kit includes at least a pair of of specific amplification
The primer of AWAT2 genes；Preferably, the primer has the sequence shown in SEQ ID NO.1~2.
Applications of the 6.AWAT2 in the candidate compound of screening prevention or treatment osteosarcoma.
Application of the inhibitor of 7.AWAT2 functional expressions in the pharmaceutical composition for preparing prevention or treatment osteosarcoma.
8. application according to claim 7, it is characterised in that the inhibitor is siRNA.
9. application according to claim 8, it is characterised in that the siRNA sequence is as shown in SEQ ID NO.9~10.
10. a kind of pharmaceutical composition for treating osteosarcoma, it is characterised in that described pharmaceutical composition includes AWAT2 feature tables
The inhibitor reached, and/or other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material with the inhibitor compatibility.
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