CN107881240A - The diagnosis and treatment mark of osteosarcoma - Google Patents
The diagnosis and treatment mark of osteosarcoma Download PDFInfo
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Abstract
The invention discloses the diagnosis and treatment mark of osteosarcoma, the mark is FIBCD1, it is demonstrated experimentally that FIBCD1 mRNA and its albumen up-regulated expression in osteosarcoma tissue, prompt to be applied to FIBCD1 in the early diagnosis of osteosarcoma；Further, In vitro cell experiment confirms that FIBCD1 and osteosarcoma cell propagation, apoptosis, migration and invasion and attack are relevant, prompts FIBCD1 to be applied to the clinical treatment of osteosarcoma as drug targets.
Description
Technical field
The invention belongs to biomedicine field, the diagnosis and treatment mark of osteosarcoma, the specific mark is FIBCD1.
Background technology
Osteosarcoma (Osteosarcoma, OS) is also known as osteogenic sarcoma, to be mainly in 10-25 year teen-age most common original
Hair property malignant bone tumor, the rate of transform, recurrence rate and the death rate are higher, are often associated with the bad knot such as amputation, Lung metastases, death
Office.At present, it is still more based on operation to treat the method for osteosarcoma, appropriate combined radio chemotherapy (Kushnir I, KolanderY,
Bickels J,et al.[J].Med Oncol.,2014,31(5):936.).Though tumor resection tissue is in clinical treatment of osteosarcoma
Important step, but surgery alone cure rate is only 15%-20% (Archer NP, Napier TS, Villanacci JF.
[J].Cancer Causes Control,2016,27(7):863 1 868.).Continuous progressive, various guarantor's limb of iconography means
Technology step up and the fast development of associated bone substitute etc. so that the treatment method that limbs retain is increasingly in bone and flesh
Cuted a striking figure in the treatment of knurl (Wang TY, Dormans JP, Chang B. [J] .Ann Plast Surg., 2012,69 (5):
560-564.).On the other hand, it is to combine to consolidate after tumor resection tissue in good time to be in progress particularly critical for control osteosarcoma
The chemotherapy of solidity or radiotherapy.In recent years, though the basis such as gene therapy, immunization therapy, molecular targeted therapy and clinical research obtain
Certain progress, current curative effect are not affirmed still.
The occurrence and development of tumour are a complicated processes, are bodies under various factors effect, the cell of local organization
The normal regulation grown to it is lost on gene level, the neoformation for causing the paraplasm of cell and being formed.Tumour is
Genopathy, its Basic of Biology are the exceptions of gene.At present, on osteosarcoma occurrence and development molecular regulation mechanism so far still
Do not get across.Along with molecular genetics, cell biology, molecular biology, cytogenetics, proteomics and transcription
The further investigation that group is learned etc., the influencing each other of living environment and genetic mutation be increasingly becoming osteosarcoma field study hotspot it
One (Zhang G, Bai R, Zhang T, et al. [J] .Genet Mol Res., 2015,14 (3):8283-8289).
Recent studies have found that in osteosarcoma some genes present differential expression, as patent 201510075917.2,
201510075920.4th, the WWP1 disclosed in 201510075918.7,201510075919.1, C8orf59, CCT γ,
PLEKHA5 genes.With going deep into osteosarcoma molecular biosciences research, the targeted therapy of osteosarcoma, which also more and more turns into, to be ground
The emphasis studied carefully, and the developing direction in future is represent, find new effective biomarker, the 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 biomarker is in osteosarcoma
Application in diagnosis and treatment.
The second object of the present invention is, there is provided a kind of product for diagnosing osteosarcoma and the drug regimen for the treatment of osteosarcoma
Thing.
To achieve these goals, present invention employs following technical scheme：
The first aspect of the present invention provides a kind of reagent for detecting biomarker and prepared for diagnosing osteosarcoma
Application in product, including：
A) biomarker in the sample of subject is identified, wherein the biomarker is FIBCD1；And
B) by the biomarker compared with reference, wherein with the difference of the biomarker with reference to compared with
For detecting osteosarcoma.
Further, the sample is tissue.
Further, with reference to compared with, biomarker expression level raises.
The second aspect of the present invention provides a kind of product, and the product includes the horizontal reagents of detection FIBCD1.Wherein,
FIBCD1 up-regulated expressions 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 FIBCD1 genes；Or
The primer of specific amplification FIBCD1 genes；Or
Specifically bind the antibody or part of the albumen of FIBCD1 codings.
Further, the primer sequence of specific amplification FIBCD1 genes is as shown in SEQ ID NO.1~2.
The third aspect of the present invention provides a kind of pharmaceutical composition, and described pharmaceutical composition includes FIBCD1 suppression
Agent, and/or other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material with the inhibitor compatibility.
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 IDNO.9~10
It is shown.
The fourth aspect of the present invention provides the application described in following any one：
1) application of the product described in second aspect of the present invention in the instrument for preparing diagnosis osteosarcoma.
2) applications of the FIBCD1 in the medicine for preparing treatment tumour；
3) applications of the FIBCD1 in the pharmaceutical composition for preparing treatment bone and flesh tumor metastasis；
4) applications of the FIBCD1 in the drug candidate of screening treatment osteosarcoma.
Further, 1) product include with RT-PCR, real-time quantitative PCR, new-generation sequencing, in situ hybridization, chip or
Immunoassay detects FIBCD1 reagent；
Or 3) 2) pharmaceutical composition includes FIBCD1 inhibitor, and/or other medicines with the inhibitor compatibility described in
Class and pharmaceutically acceptable carrier and/or auxiliary material；
Wherein, the inhibitor is selected from：FIBCD1 gene tables as target sequence and can be suppressed using FIBCD1 or its transcript
Reach or the disturbing molecule of genetic transcription, including：ShRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna,
Antisensenucleic acids, or can express or be formed the construction of the shRNA, siRNA, dsRNA, Microrna, antisensenucleic acids；Or
Specificity and the protein bound binding molecule (if suppressing the antibody or part of FIBCD1 protein actives) of FIBCD1 codings.
4) include in the step of the drug candidate of screening treatment osteosarcoma：
The system for the albumen expressed or containing FIBCD1 genes or its coding is handled with candidate substances；With
Detect the expression of the albumen of FIBCD1 genes or its coding or activity in the system.
Brief description of the drawings
Fig. 1 is the expression figure in osteosarcoma tissue using QPCR detection FIBCD1 genes；
Fig. 2 is the expression figure in osteosarcoma tissue using Western blot detection FIBCD1 albumen；
Fig. 3 is transfected condition figures of the FIBCD1 in osteosarcoma cell；Wherein, figure A is to bone using QPCR detection transfections
The influence figure that FIBCD1mRNA is expressed in sarcoma cell；Figure B is in osteosarcoma cell using Western blot detection transfections
The influence figure of FIBCD1 albumen；
Fig. 4 is the influence figure for detecting FIBCD1 gene pairs human osteosarcoma cell proliferations；
Fig. 5 is to detect the influence figure that FIBCD1 migrates to osteosarcoma cell；
Fig. 6 is to detect the influence figure that FIBCD1 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, the gene for the expression that finds differences, its relation between the generation of osteosarcoma is inquired into, from
And it is that the early detection of osteosarcoma and targeted therapy find more preferable approaches and methods.By screening, present invention firstly discovers that
FIBCD1 conspicuousnesses raise in osteosarcoma.It is demonstrated experimentally that the expression by reducing FIBCD1, can effectively suppress bone and flesh
The growth and invasion and attack of oncocyte, the expression of detection FIBCD1 genes is prompted to turn into the auxiliary diagnosis of osteosarcoma early diagnosis
One of index, interference FIBCD1 gene expressions, which can turn into, prevents or treats osteosarcoma or the new way of bone and flesh tumor metastasis.
(biology) mark
" biomarker " refers to the molecule with specific biological characteristic, biochemical characteristics or aspect and indicated
Thing, it can be used for determining presence or absence of specified disease or situation and/or the order of severity of specified disease or situation.
In the present invention, " mark " refers to the parameter related to one or more biomolecule (i.e. " biomarker "),
Such as it is natural or artificial synthesized caused by 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 also includes finger can be by considering the expression number from two or more unlike signal things
According to the single parameter for calculating or otherwise obtaining.
" protein ", " peptide " and " polypeptide " is interchangeable and broadly refers to have two or more amino acid sub-
The compound of base, amino acid analogue or peptidomimetic.The subunit can be connected by peptide bond.Protein or peptide must include at least
Two amino acid and amino acid maximum number is unlimited, it can include the sequence of protein or peptide.Terms used herein " ammonia
Base acid " refers to natural and/or non-natural or the amino acid of synthesis, including both glycine and D and L optical isomers, amino
Acid-like substance and peptidomimetic.
FIBCD1 genes
FIBCD1 in the present invention includes wild type, saltant type or its fragment.A kind of representational FIBCD1 gene orders
As shown in FIBCD1 genes (NC_000009.12) in current international public nucleic acid database GeneBank, people of the invention
FIBCD1 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 the technology for detecting gene or protein expression level.
The expression of gene " detection " refer to determine biological sample marker gene mRNA exist and its expression with
Just predict the process of stomach cancer prognosis and the amount by measuring mRNA can be achieved.Analysis method is but not limited to for this purpose
RT-PCR, competitive RT-PCR (competitiveRT-PCR), real-time RT-PCR (Real-timeRTPCR), RNase protection are surveyed
Determine method (RPA；RNaseprotectionassay), northern blottings (northernblotting), DNA microarray chip
Deng.
" expression of detection albumen " refers to the protein presence for determining expression in biological sample marker gene and its table
Up to level to predict the process of osteosarcoma, and can be combined by using the protein specific with being expressed in said gene
Antibody determine the amount of protein.Analysis method is but not limited to western blottings for this purpose
(westernblotting), ELISA (enzyme linked immunosorbent assay (ELISA)), radioimmunoassay (Radioimmunoassay), put
Penetrating property SRID (Radioimmunodiffusion), Auchterlonie (Ouchterlony) SRID, rocket
(Rocket) electrophoresis, histogenic immunity decoration method, immunoprecipitation assay (immunoprecipitationassay), complement knot
Close determination method (completefixationassay), FACS, protein-chip (proteinchip) etc..
The nucleic acid or albumen 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 FIBCD1 expression, it is preferred that described kit has including detection
The reagent of the albumen of the detection FIBCD1 genes of effect amount or its coding, the one or more materials being selected from the group：Container, using saying
Bright book, positive control, negative control thing, buffer, auxiliary agent or solvent.Such as being suspended or fixing the solution of cell, can
The label or tag of detection, 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 FIBCD1 screenings prevention or treatment osteosarcoma, the present invention's
In a kind of embodiment, including step：In test group, test compound is added in cultivating system, and observes the test group
Cell in FIBCD1 expression quantity and/or activity；In control group, test chemical combination is not added in identical cultivating system
Thing, and observe the expression quantity and/or activity of FIBCD1 in the cell of control group；
Wherein, if the FIBCD1 of cell expression quantity and/or activity are less than control group in test group, the test is indicated that
Compound is expression to FIBCD1 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 FIBCD1 inhibitor, suppresses bone for preparing
The pharmaceutical composition of sarcoma.As used herein, described FIBCD1 inhibitor includes but is not limited to inhibitor, antagonist, resistance
Stagnant dose, blocking agent, nucleic acid inhibitor etc..
Described FIBCD1 genes or the inhibitor of albumen refer to any activity for reducing FIBCD1 albumen, reduced
The stability of FIBCD1 genes or albumen, the expression for lowering FIBCD1 albumen, reduce FIBCD1 albumen effective acting times or suppression
The material of the transcription and translation of FIBCD1 genes processed, these materials are used equally for the present invention, as useful for lowering FIBCD1
Material, 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 lower FIBCD1 albumen on albumen or gene level
Or the expression of its encoding gene.
As a kind of selection mode of the present invention, described FIBCD1 inhibitor is that a species specificity is combined with FIBCD1
Antibody.The specific antibody includes monoclonal antibody, polyclonal antibody；The present invention not only includes complete antibody molecule,
Also any fragment including antibody or modification, for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as the fragment energy
Enough binding abilities retained with FIBCD1 albumen.Those skilled in the art are public during preparation for the antibody of protein level
Know, 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 FIBCD1 is a kind of specific small interference of FIBCD1
RNA molecule.As used herein, described " 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, and this process is exactly RNA interference (RNA interference) processes.It is small
RNA interfering 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 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 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 FIBCD1 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 FIBCD1 of effective dose inhibitor, and
Pharmaceutically acceptable carrier.Described composition can be used for suppressing osteosarcoma.Any foregoing FIBCD1 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.
Pharmaceutical composition can be various oral or parenteral dosage forms.Using including filler, filler, adhesive,
Wetting agent, conventional thinner or excipient pharmaceutical composition including disintegrant, and surfactant.Solid orally ingestible
Including tablet, pill, pulvis, granule, capsule etc..These solid pharmaceutical preparations can by by least one compound with it is a kind of or more
Kind of excipient, for example, prepared by the mixing such as starch, calcium carbonate, sucrose, lactose, gelatin.Except simple excipient, can also make
With lubricator such as magnesium stearate or talcum.In addition, liquid oral medicine includes suspension, solution, emulsion and syrup etc..Except logical
It is commonly used for outside the water and atoleine of simple diluent, may also include various excipient, for example, wetting agent, sweetener, spices,
Preservative etc..The preparation of parenteral includes sterile water solution, and nonaqueous solvents, suspending agent, emulsion is freeze-dried, suppository etc..Third
Glycol, polyethylene glycol, vegetable oil such as olive oil, injectable esters such as ethyl oleate etc. may be used as nonaqueous solvents and suspending agent.Bolt
Agent main component can include witepsol, polyethylene glycol, Tween61, cocoa butter, laurel tallow, glycerin gelatine etc..
Pharmaceutical composition can have any one preparation being selected from the group：Tablet, pill, powder, granule, capsule, suspend
Liquid, solution, emulsion, syrup, sterile water solution, non-aqueous solution, suspension, emulsion, lyophilized formulations and suppository.
As used in the present invention, " effective dose " refers to that its dosage is enough to treat disease, with the conjunction suitable for any therapeutic treatment
Interests/risk-ratio of reason.The effective dose level of composition can according to the type of subject, the order of severity of disease, by
The age of examination person and sex, pharmaceutical activity, the sensitiveness to medicine, administration time, method of administration, excretion rate, treatment time, with
Other known facts determine in medicine associated with composition and medical field.The present invention pharmaceutical composition can be used alone or
It is administered in combination, and can be sequentially or simultaneously administered with the therapeutic agent of routine with other therapeutic agents.One or more agent can be used
Composition is applied in type.All above-mentioned factors are considered, in the case where minimum of the maximum efficiency without causing side effect can be shown
Most important using composition, the minimum can be readily determined by those skilled in the art.
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.Such as can be directly by FIBCD1 inhibitor by such as noting
The methods of penetrating delivers medicine to subject；Or can by certain approach will carry FIBCD1 inhibitor ceneme (such as
Expression vector or virus etc., or siRNA or shRNA) it is delivered on target spot, and the FIBCD1 inhibitor of expression activity is allowed to, have
Body situation need to be depending on the type 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 RNA sample 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 differential expression is presented in FIBCD1 in Patients with Osteosarcoma cancerous tissue and cancer beside organism, poor
It is different that there is statistical significance (P<0.05).
The differential expression of the QPCR sequence verification FIBCD1 genes of embodiment 2
1st, large sample QPCR checkings are carried out to FIBCD1 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 × gDNA Buffer 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 FIBCD1 genes in Genebank and house-keeping gene GAPDH genes to draw
Thing, synthesized by Bo Maide companies.
The primer sequence of FIBCD1 genes：
Forward primer sequence is 5 '-GACCATTCAGAGAACAACT-3 ' (SEQ ID NO.1)；
Reverse primer sequences are 5 '-GTACTGCCCATTGAGGTT-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 of 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, pass through melt curve analysis analysis and electrophoresis
Purpose band is determined, Δ Δ CT methods carry out relative quantification.
5th, result
As a result as shown in figure 1, compared with cancer beside organism, FIBCD1 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 FIBCD1 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 compared with cancer beside organism, expression of the FIBCD1 albumen in osteosarcoma tissue significantly rises
Height, difference have statistical significance.
The silence of the FIBCD1 genes of embodiment 4
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 '-UUUAUUGUUUUUUAAGCACAA-3 ' (SEQ ID NO.7)
Antisense strand is 5 '-GUGCUUAAAAAACAAUAAAUU-3 ' (SEQ ID NO.8)
siRNA2：
Positive-sense strand is 5 '-UUUUAAGCACAAACUUCAGUG-3 ' (SEQ ID NO.9)
Antisense strand is 5 '-CUGAAGUUUGUGCUUAAAAAA-3 ' (SEQ ID NO.10)
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 FIBCD1 genes is without homology.
3) transfect
Transfected using the liposome Lipofectamine 3000 of Invitrogen companies, by specification is grasped
Make.
5th, QPCR detects the transcriptional level of FIBCD1 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 FIBCD1 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), therefore selection siRNA2 carries out subsequent experimental.
The influence of the FIBCD1 gene pairs human osteosarcoma cell proliferations of embodiment 5
FIBCD1 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 FIBCD1 and osteosarcoma cell is closely related, drop can be passed through
Low FIBCD1 expression suppresses the propagation of osteosarcoma cell.
The influence of the FIBCD1 gene pairs apoptosis in osteosarcoma cells of embodiment 6
Use the influence of flow cytomery FIBCD1 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 significantly raises, and transfects the cell of siRNA2 groups
Apoptosis rate is (20.17 ± 0.021) %, and the apoptosis rate of transfection siRNA-NC groups is (4.89 ± 0.23) %, above-mentioned difference
With statistical significance (P<0.05), the above results show, FIBCD1 is relevant with the apoptosis of osteosarcoma cell, and the gene crosses table
Up to the apoptosis for suppressing osteosarcoma cell.
The influence of the FIBCD1 cell migrations of embodiment 7
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
According to, without significant difference, illustrating that FIBCD1 is relevant with the migration of osteosarcoma cell between group.
Influences of the FIBCD1 of embodiment 8 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 U-2OS and transfection siRNA-NC, explanation
FIBCD1 overexpression promotes the invasion and attack of osteosarcoma cell, and prompting its expression of silence, can suppress bone by targetting FIBCD1 genes
The invasion and attack of sarcoma.
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 (10)
1. the reagent for detecting biomarker is preparing the application in being used to diagnose the product of osteosarcoma, it is characterised in that including：
A) biomarker in the sample of subject is identified, wherein the biomarker is FIBCD1；And
B) by the biomarker compared with reference, wherein being used for the difference of the biomarker with reference to compared with
Detect osteosarcoma.
2. application according to claim 1, it is characterised in that the sample is tissue.
3. application according to claim 1, it is characterised in that with reference to compared with, biomarker expression level raises.
4. a kind of product, it is characterised in that the product includes the horizontal reagents of detection FIBCD1.
5. product according to claim 4, it is characterised in that the reagent is selected from：
The probe of specific recognition FIBCD1 genes；Or
The primer of specific amplification FIBCD1 genes；Or
Specifically bind the antibody or part of the albumen of FIBCD1 codings.
6. product according to claim 5, it is characterised in that the primer sequence such as SEQ of specific amplification FIBCD1 genes
Shown in ID NO.1~2.
A kind of 7. pharmaceutical composition, it is characterised in that described pharmaceutical composition includes FIBCD1 inhibitor, and/or with it is described
Other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material of inhibitor compatibility.
8. pharmaceutical composition according to claim 7, it is characterised in that the inhibitor is selected from：Nucleic acid inhibitor, albumen
Inhibitor, proteolytic enzyme, protein binding molecule.
9. pharmaceutical composition according to claim 8, 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.
10. the application described in following any one：
1) application of the product described in any one of claim 4-6 in the instrument for preparing diagnosis osteosarcoma.
2) applications of the FIBCD1 in the medicine for preparing treatment tumour；
3) applications of the FIBCD1 in the pharmaceutical composition for preparing treatment bone and flesh tumor metastasis；
4) applications of the FIBCD1 in the drug candidate of screening treatment osteosarcoma.
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| CN109655608A (en) * | 2018-12-13 | 2019-04-19 | 杭州多泰科技有限公司 | A kind of excretion body protein for osteosarcoma diagnosis and its instant detection method |
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| CN106834486A (en) * | 2017-02-28 | 2017-06-13 | 北京泱深生物信息技术有限公司 | Osteosarcoma molecule diagnosis and treatment mark and its application |
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| WO2008119356A1 (en) * | 2007-04-03 | 2008-10-09 | Syddansk Universitet | The use of a type ii transmembrane protein designated fibcd1 as an endocytosis receptor of acetylated compounds |
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