CN105734155A - Chondroblast-type osteosarcoma disease-causing gene and application thereof - Google Patents
Chondroblast-type osteosarcoma disease-causing gene and application thereof Download PDFInfo
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Abstract
The invention relates to a chondroblast-type osteosarcoma disease-causing gene and an application thereof, in particular to new applications of mir-4676, mature miRNA and a target gene DDX58 of the miRNA to treating chondroblast-type osteosarcoma. Chondroblast-type osteosarcoma patients and healthy crowds are subjected to high-throughput sequencing, the expression data of the miRNA and the expression data of mRNA are obtained, and a candidate gene is screened; then the candidate miRNA and the candidate mRNA are subjected to molecular biology verification and target verification. The result shows that the miRNA and the target gene DDX58 of the miRNA are closely related to chondroblast-type osteosarcoma and can be used for clinical diagnosis and preventing detection of chondroblast-type osteosarcoma.
Description
Technical field
The present invention relates to disease treatment field, be specifically related to chondroblastic osteosarcoma Disease-causing gene and answer
With, more particularly relate to mir-4676 and ripe miRNA and its target gene DDX58 thereof soft in diagnosis and treatment
New application in osteoblastic osteosarcoma.
Background technology
Osteosarcoma (osteosarcoma, OSA) is a kind of common primary malignant bone tumor, is apt to occur in blue or green few
Year, having multiple hypotype, chondroblastic osteosarcoma is one hypotype, inside has the obvious atypia of more tool
Cartilaginous element.Differentiate that the osteosarcomatous meaning of chondroblastic is to prevent it to be misdiagnosed as chondrosarcoma, because of
Treatment and prognosis for both have marked difference.Chondrosarcomatous Therapeutic Method is mainly excision, prognosis with
Histological classification and excision are the most relevant, and prognosis is preferable.And osteosarcoma is except excision external demand
It is aided with chemotherapy, and overall prognosis relatively chondrosarcoma is poor.Due to Therapeutic Method and the difference of prognosis, differentiate that cartilage is female
Cellular type osteosarcoma and chondrosarcoma are significant to the determination of clinical treatment, bone the most in recent years
The treatment of sarcoma emphasizes that staging tomography protects limb rate and survival rate to improve, and preoperative correct diagnosis is particularly important.
Chondroblastic osteosarcomatous histopathologic diagnosis standard is that tumor is mainly by the cartilage having obvious atypia
Cell forms, and around has spindle-type malignant hypertrophy, can directly produce osteoid tissue and immature bone.Cause
This biopsy exists the pathological diagnosis osteosarcomatous for chondroblastic of bone sample ingredient particularly significant.Although
Diseased region is all taken from all biopsies, but the inhomogeneity of tumor often results in biopsy sample error, and then directly affects
Correct diagnosis.Biopsy sample error be cause chondroblastic osteosarcoma mistaken diagnosis be chondrosarcomatous important because of
Element.
The present invention is based on high-flux sequence method, to 3 example chondroblastic Patients with Osteosarcomas and 10 health
Comparison crowd check order, it is thus achieved that the expression data of itself miRNA and mRNA, and then carries out bioinformatics
Analyzing, choose standby miRNA and mRNA and carry out molecular biology checking and target checking, result shows,
MiR-4676-5p and target gene DDX58 thereof that the present invention provides are closely related with chondroblastic osteosarcoma,
Can be used for the osteosarcomatous clinical diagnosis of chondroblastic and prevention detection.
Summary of the invention
The preparation that it is an object of the invention to provide a kind of mir-4676 of detection and ripe miRNA thereof is examined in preparation
Application in knochenbruch sarcoma reagent.
The sequence of mir-4676 is shown in that sequence table SEQ ID NO 1, miR-4676-5p sequence are shown in sequence table SEQ ID
NO 2, miR-4676-3p sequence is shown in sequence table SEQ ID NO 3.
Further, osteosarcoma is chondroblastic osteosarcoma.
Further, diagnosis osteosarcoma reagent includes based on high-flux sequence method and/or based on quantifying PCR method
And/or based in probing procedure detection osteosarcoma sample mir-4676 and ripe miRNA thereof transcribe or
Based on the target gene of mir-4676 and ripe miRNA regulation and control thereof in immunologic detection method detection osteosarcoma sample
Expression, it is preferred to use skill is analyzed in the protection of northern hybridizing method, miRNA chip of expression spectrum, ribozyme
Mir-4676 in art, RAKE method, in situ hybridization, Flow cytometry osteosarcoma sample based on microsphere
And the transcribing of ripe miRNA；Use in ELISA and/or colloidal gold strip detection osteosarcoma sample
The expression of the target gene of mir-4676 and ripe miRNA regulation and control thereof.The most described mir-4676 and one-tenth thereof
The target gene of ripe miRNA regulation and control is DDX58.
Preferably, osteosarcoma sample is peripheral blood.
Preferably, drawing of specific amplification mir-4676 and ripe miRNA thereof is included based on quantifying PCR method
Thing, further preferably, the primer sequence of specific amplification miR-4676-5p is SEQ ID NO 4；Based on spy
Pin hybridizing method includes and mir-4676 and the probe of the nucleic acid array hybridizing of ripe miRNA thereof；Immune detection
Method includes the antibody specific binding with mir-4676 and ripe miRNA controlling gene expressing protein thereof, enters
The antibody that one step is preferably combined with DDX58 protein-specific.
The preparation that it is an object of the invention to provide mir-4676 and ripe miRNA thereof prevents and treats osteosarcoma in preparation
Application in reagent.
Further, prevent and treat osteosarcoma reagent and include lowering transcribing of mir-4676 or its ripe miRNA, or press down
The reagent of the activity of mir-4676 processed or its ripe miRNA.
Preferably, use antisense oligonucleotide, antagomiRs, miRNA sponge, miRNA Erasers,
The method of Target Masking and/or Mutiple Targets antisense oligonucleotide lowers mir-4676 and/or it is ripe
MiRNA transcribes and/or blocks mir-4676 and/or the activity of its ripe miRNA.
It is an object of the invention to provide the target gene of mir-4676 and ripe miRNA thereof in preparation diagnosis or anti-
Control the application in osteosarcoma preparation.Preferably, target gene is DDX58.
Further, osteosarcoma is chondroblastic osteosarcoma.
Further, diagnosis osteosarcoma preparation uses PCR kit for fluorescence quantitative, gene chip, immunization method
The expression of DDX58 gene in detection Patients with Osteosarcoma peripheral blood.Preferably, in PCR kit for fluorescence quantitative
Primer containing specific amplification DDX58 gene；Described gene chip includes and DDX58 gene
The probe of nucleic acid array hybridizing.It is furthermore preferred that PCR kit for fluorescence quantitative is contained within specific detection DDX58
The forward primer of gene and downstream primer, forward primer sequence is SEQ ID NO 5, and downstream primer sequence is
SEQ ID NO 6。
Further, DDX58 gene during osteosarcomatous diagnostic preparation uses immunization method detection osteosarcoma peripheral blood
Expression product.Preferably, immunization method is ELISA detection and/or gold colloidal detection.Further, detection
The ELISA method of DDX58 albumen is for using ELISA detection kit.Antibody in test kit can use commercially available
DDX58 monoclonal antibody or polyclonal antibody, the most commercially available Anti-DDX58 antibody (ab180675)
Antibody.Further, test kit includes: be coated the solid phase carrier of DDX58 antibody, enzyme labelled antibody, the end of enzyme
Thing, protein standard substance, negative controls, diluent, cleaning mixture, enzyme reaction stop buffer etc..
Further, the colloidal gold method of detection DDX58 albumen is for using detection kit, and antibody can use commercially available
DDX58 monoclonal antibody or polyclonal antibody.Further, gold-immunochromatographyreagent reagent for assay box uses gold colloidal to exempt from
Epidemic disease chromatographic technique or gold colloidal percolation.Further, the detection on gold-immunochromatographyreagent reagent for assay box nitrocellulose filter
District (T) specking has anti-DDX58 antibody, quality control region (C) specking to have immunoglobulin IgG.
It is an object of the invention to provide above-mentioned osteosarcoma diagnostic preparation answering in preparation osteosarcoma diagnostic tool
With.
It is an object of the invention to provide the osteosarcomatous preparation of a kind of preventing and treating, described reagent comprises:
A () inhibitor and/or inhibitor combination, described inhibitor and/or inhibitor combination lower mir-4676
And/or its ripe miRNA transcribe and/or block mir-4676 and/or the activity of its ripe miRNA；
Receptible carrier on (b) pharmaceutics.
Preferably, use antisense oligonucleotide, antagomiRs, miRNA sponge, miRNA Erasers,
The method of Target Masking and/or Mutiple Targets antisense oligonucleotide lowers mir-4676 and/or it is ripe
MiRNA transcribes and/or blocks mir-4676 and/or the activity of its ripe miRNA.
Further, described osteosarcoma is chondroblastic osteosarcoma.
It is an object of the invention to provide a kind of osteosarcoma diagnostic reagent, osteosarcoma diagnostic reagent can detect kindred
In tumor sample, mir-4676's and ripe miRNA thereof transcribes or in immunologic detection method detection osteosarcoma sample
The expression of the target gene of mir-4676 and ripe miRNA regulation and control thereof.
Further, osteosarcoma diagnostic reagent based on high-flux sequence method and/or based on quantifying PCR method and/or
Based on the transcribing or based on exempting from of mir-4676 and ripe miRNA thereof in probing procedure detection osteosarcoma sample
The expression of the target gene of mir-4676 and ripe miRNA regulation and control thereof in epidemic disease method detection osteosarcoma sample,
Preferably employ northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE
Mir-4676 and maturation thereof in method, in situ hybridization, Flow cytometry osteosarcoma sample based on microsphere
MiRNA transcribes；Use ELISA and/or colloidal gold strip detection osteosarcoma sample in mir-4676 and
The expression of the target gene of its ripe miRNA regulation and control.The most described mir-4676 and ripe miRNA thereof
The target gene of regulation and control is containing special with DDX58 albumen in DDX58, ELISA and/or colloidal gold strip
Property combine antibody.
It is an object of the invention to provide the target gene of mir-4676 and ripe miRNA regulation and control thereof in preparation diagnosis
Or prevent and treat the application in osteosarcoma reagent.
Further, target gene is DDX58 gene.
Further, the preparation of the target gene of detection mir-4676 and ripe miRNA regulation and control thereof is at preparation diagnosis bone
Application in sarcoma reagent.
Further, this preparation of preparation raising DDX58 gene prevents and treats the application in osteosarcoma reagent.
Preferably, osteosarcoma is chondroblastic osteosarcoma.
It is an object of the invention to provide the osteosarcomatous preparation of above-mentioned preventing and treating in preparation clinical treatment of osteosarcoma medicine or examination
Application in agent.
Definition:
The method of expression of present stage detection miRNA mainly include based on high throughput sequencing technologies, based on
The miRNA detection method of nucleotide hybridization and PCR-based.MiRNA based on probe hybridization technique detects
Method is a kind of direct Detection Method, it is not necessary to sample rna is carried out pre-amplification, including northern hybridization side
Method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, based on microsphere
The technology such as flow cytometry.
(1) Northern hybridization
It is the most classical detection eukaryote RNA size also known as RNA engram technology, estimates the experiment of its abundance
Method.Ultimate principle is as follows: first at the upper fixing miRNA sample of carrier (such as silicon chip, microsphere or film etc.),
Hybridize with the probe through labelling again, after washing unnecessary hybridization probe, carry out signal detection；Can also be at carrier
The upper first fixing DNA probe complementary with target miRNA sequence, then miscellaneous with the sample miRNA through labelling
Hand over, then carry out signal detection.The method of signal labelling includes isotope labelling, fluorescent labeling and nano gold mark
Deng.
(2) miRNA chip of expression spectrum
Principle is to use the target molecule on label probe detection solid support equally.By in design chips
MiR-96 gene and internal reference sequence, Accurate Analysis can go out the expression of corresponding miRNA in sample.Gene core
Sheet has high-throughout advantage, once can detect whole expression of hundreds of gene in same sample.
The liquid-phase chip (Liquid chip) that Luminex company develops is also known as multifunctional suspending dot matrix (Multi analyte
Suspension array, MASA), it is the biochip technology of new generation.Liquid-phase chip system is little by many
Spheroid is that main matrix is constituted, and every kind of spherula is fixed with different probe molecules, in order to distinguish different spies
Pin, each for the sphere matrix of label probe all with a unique color numbers, by these spherulas
It is suspended in a liquid-phase system, just constitutes liquid-phase chip system.This system can be to same trace sample
In multiple different moleculars carry out quick qualitative and quantitative analysis simultaneously, this detection technique is referred to as
FMAP (Flexible multianalyte profiling) technology.Molecule hybridization is carried out in aaerosol solution, detection speed
Degree is exceedingly fast.
(3) ribozyme protection analytical technology (RPA)
The detection of miRNA can also use ribozyme to protect analytical technology, the probe that labelling is good and RNA to be measured
Sample mixes, and hybridizes, non-hybridized RNA and unnecessary probe single-chain nucleic acid enzymic digestion after thermal denaturation, heat
The shielded RNA molecule of purification after inactivation nuclease, is separated by electrophoresis probe finally by degeneration PAGE, aobvious
Color.This new method based on solution hybridization is simple and quick, highly sensitive, but is also only used for analyzing known
miRNA。
(4) RAKE method
RAKE method (RNA primed array based Klenow emzyme) is at miRNA microarray
On the basis of utilize the Klenow fragment of DNA polymerase i, make miRNA and the hybridization of fixing DNA probe
Method.RAKE sensitivity can detect miRNA specifically, it is adaptable to a large amount of quickly screenings are all, and oneself knows
MiRNA.MiRNA express spectra situation can be detected in specific cell and tumor.Moreover,
RAKE method can also isolate miRNA and to it from the paraffin-embedded tissue secured by formalin
It is analyzed, opens the door of hope for analyzing miRNA from archive specimen.
(5) in situ hybridization (in situ hybridization)
Hybridization in situ technique can intuitively understand miRNA expression way, is the one of observation miRNA spatial and temporal expression
Easier method, normal mark mode includes digoxin, biotin, fluorescent labeling etc..On the basis of locked nucleic acid
In situ hybridization (Locked Nucleic Acid (LNA) based in situ hybridization (LNA-ISH)) be
The probe mode that current application is more.
(6) flow cytometry based on microsphere (bead-based flow cytometry)
Being a kind of liquid-phase chip technology, FCM analysis is organically combined by the method with chip technology,
The feature such as have that flux is big, detection speed is fast, highly sensitive concurrently and specificity is good.
(7) Real-Time Fluorescent Quantitative PCR Technique (Real-time PCR, RT-PCR)
The cumulative speed of extension increasing sequence during whole PCR can be drawn and dynamically change song by fluoroscopic examination PCR instrument
Line.In reaction mixture, the initial concentration of target sequence is the biggest, it is desirable to obtain amplified production specific output
PCR cycle number (typically expressing by specific threshold period Ct) is the fewest.Owing to miRNA length is only 22nt,
Traditional qRT-PCR is not suitable for expanding the shortest fragment.There is several real-time quantitative for miRNA now
PCR method, such as tailing method, neck ring method etc..Neck ring method is that a kind of preferably miRNA detects qRT-PCR
Method: first design special loop-stem structure primer, synthesizes cDNA with miRNA to be measured for template reverse transcription
First chain, this cDNA one end is stem Loop primer, and stem circulus is opened and substantially increases the length of cDNA
Degree, the cDNA with synthesis carries out real-time quantitative PCR amplification for template design primer subsequently.QRT-PCR has
Have that specificity is high, sensitivity is good, the multiple advantage such as quickly and easily.
(8) sequencing
The known miRNA of major part is found by cDNA clone order-checking and identifies.This method needs elder generation
Building the cDNA library of miRNA, then carry out PCR amplification, amplified production is cloned into expression vector subsequently
Upper order-checking.Takada develop a kind of improvement amplification cloning (miRNA amplification profiling,
MRAP), mRAP method first connects joint at the 3 ' of miRNA ends, then with the reverse transcription complementary with joint
Primed reverse transcription.Because specific reverse transcription has terminal deoxynucleotidyl transferase activity, some nucleotide
(mainly dCMP 1beta-Deoxyribofuranosylcytosine-5'-phosphate) can be connected to 3 ' ends of the cDNA chain that reverse transcription goes out.When 5 ' end connectors with
After poly (C) the sticky end annealing of cDNA chain, add a pair general primer and can realize the PCR to cDNA
Amplification.Due to mRAP High sensitivity, can be directly with miRNA in clone and a small amount of tissue of sequencing technologies detection
Expression.Sequence label cloning is a kind of on the basis of serial analysis of gene expression (SAGE) technology
Having developed the higher miRAGE of detection efficiency (miRNA SAGE) cloning, this method is by generating big series connection
Son, can detect multiple miRNA by single sequencing reaction, hence it is evident that improve detection efficiency.
High-flux sequence (High-throughput sequencing) is also known as sequencing technologies (next of future generation
Generation sequencing) it is the change to tradition order-checking revolution, once to hundreds of thousands to millions of
Bar DNA molecular carries out sequencing, greatly improves order-checking efficiency.This kind of large scale sequencing technology is great
Improve the solution reading rate of multiple species hereditary information, for obtaining the sequence information of all miRNA, deciphering
MiRNA collection of illustrative plates provides guarantee.High-flux sequence makes the transcript profile to species and genome enter simultaneously
The analysis of the careful overall picture of row is possibly realized, so degree of depth order-checking (deep sequencing) that is otherwise known as.High flux
The representative of order-checking platform is 454 sequenators (Roch GSFLX sequencer) of Roche Holding Ag (Roche),
The Solexa gene element analyzer (Illumina Genome Analyzer) of Illumina company and the SOLiD of ABI
Sequenator (ABI SOLiD sequencer).
Immunologic detection method is using a kind of antibody or Multiple Antibodies as analytical reagent, determinand is carried out quantitatively or
The detection method of qualitative analysis.Its ultimate principle is the interaction between antibody and antigen.For improve antigen and
The sensitivity of antibody test, by the material of easily display in known antibodies or antigenic mark, by detection label,
Reflection is with or without antigen antibody reaction, thus indirectly measures antigen or the antibody of trace.Conventional label have enzyme,
Fluorescein, radiosiotope, gold colloidal and electron dense substances etc..Show on this antigen or antibody labeling
The specific reaction that thing is carried out is referred to as immunolabelling technique (immunolabelling technique).Application at present
The widest immunoassay technology mainly has: elisa (enzyme-linked immunosorbent
Assay, ELISA), colloidal gold immunity chromatography etc..
Elisa principle is antigen or antibody to be combined with substrate (enzyme) so that it is keep immunity anti-
Should be with the activity of enzyme.Antigen or the antibody of labelling are combined with the part being coated on solid phase carrier, then be allowed to
Corresponding colorless substrate effect and Show Color, according to colour developing depth degree range estimation or measure OD value by microplate reader
Result of determination.
Colloidal gold strip is typically made up of sample pad, gold mark pad, chromatographic film, adsorptive pads four part.Chromatography material
Material has nitrocellulose membrane (NC), polyester film, nylon membrane and pvdf membrane etc., needs to may select not according to test
With the film required, wherein NC film is the most commonly used, can determine the need for living according to test concrete condition before using
Change or process, in most cases without processing, i.e. can be used directly.Gold is marked protein solution even application at gold
On mark pad, dry standby at room temperature.NC film can capture a certain amount of be coated (antibody) and two anti-as inspection
Survey line and nature controlling line.Finally sample pad, gold mark pad, NC film and absorbent paper are in turn secured to PVC board, i.e.
Become test strips.
MicroRNA gain-of-function technology based on RNA is i.e. by exogenous supplementary miRNAs synthesis
Precursor substance raises the level of miRNAs.For example, it is possible to synthetic and endogenous miRNA sequence one
Bob folder sample RNA (short hairpin RNA, shRNA) caused, is done promoter by polymerase II or III,
With virus as vector-transfected cell, played a role by being loaded into RISC after Dicer enzyme modification, be equivalent to raise
The level of pre-miRNA, action effect is stably lasting.
Gene specific miR Mimics technology this technique avoids the nonspecific action of miRNA and gene.
The specific oligonucleotide chain being combined with target gene 3 ' UTR complementation of this synthetic, it is possible to play with
Regulation effect after what miRNA was identical transcribe.
On the pharmaceutics of the pharmaceutical composition being included in the present invention, the carrier of license is for generally to utilize when preparation
Carrier, this carrier comprises lactose (lactose), dextrose (dextrose), sucrose (sucrose), Pyrusussuriensis
Alcohol (sorbitol), mannitol (mannitol), starch, acacia gum, calcium phosphate, alginate (alginate),
Gel (gelatin), calcium silicates, microcrystalline Cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone),
Cellulose (cellulose), water, syrup, methylcellulose (methyl cellulose), hydroxy benzoic acid first
Ester (methyl hydroxybenzoate), propyl hydroxy propyl benzoate (propyl hydroxybenzoate),
Talcum, magnesium stearate (stearic acid magnesium) and mineral oil (mineral oil) etc., but not office
It is limited to this.
The pharmaceutical composition of the present invention can also comprise lubricant, wetting agent, sweet taste in addition to mentioned component
Agent, flavouring agent, emulsifying agent, suspending agent, preservative etc..On pharmaceutics, the carrier being suitable for and the preparation of license are detailed
Carefully it is recorded in Lei Mingdengshi pharmacy pandect.
The pharmaceutical composition of the present invention can be by oral or parenteral be administered, during as non-oral administration,
Can be by intravenous injection, intranasal injection, local injection, intracerebral ventricle injection, spinal cavity injection, subcutaneous note
Penetrating, lumbar injection, the mode such as percutaneous dosing is administered.
The dosage being suitable for of the pharmaceutical composition of the present invention is according to preparation ways, administering mode, patient
Age, body weight, sex, morbid state, food, administration time, route of administration, drainage rate and be quick on the draw
The factor of property etc and multiple prescription can be carried out, generally, skilled practitioner can be easily determined by and prescription pair
Desired treatment or prevent effective dosage.
The pharmaceutical composition of the present invention can be easy according to general technical staff of the technical field of the invention
The method implemented, utilizes receptible carrier and/or excipient on pharmaceutics formulation to carry out such that it is able to
With unit dose form preparation or in be contained in multicapacity container in prepare.Now, dosage form is oiliness or water
Solution, suspension or emulsion form in property medium, or can also be extractum, powder agent, granule,
Tablet or capsule form, it is also possible to include dispersant or stabilizer.
Accompanying drawing explanation
Fig. 1 is miR-4676-5p relative expression levels figure in RT-PCR detection chondroblastic osteosarcoma
Fig. 2 is DDX58 relative expression levels figure in RT-PCR detection chondroblastic osteosarcoma
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and is not understood that
For limitation of the present invention.It will be understood by those skilled in the art that: without departing from the present invention principle and
These embodiments can be carried out multiple change in the case of objective, revise, replace and modification, the model of the present invention
Enclose and limited by claim and equivalent thereof.The experimental technique of unreceipted actual conditions in the following example, generally
According to normal condition or according to the condition examinations proposed by manufacturer.
The collection of embodiment 1 sample and Total RNAs extraction
3 example chondroblastic Patients with Osteosarcomas, clinical information is shown in Table 1,10 normal healthy controls.Cartilage is female thin
Born of the same parents' type Patients with Osteosarcoma group 3 example and matched group 10 people require at least 12h on an empty stomach, in m seq 7:00～8:
Under 00 room temperature, extraction 10ml venous blood, in ethylenediaminetetraacetic acid (EDTA) anticoagulant tube, extracts peripheral blood single
Nucleus PBMCs, adds 1ml Trizol reagent (Invitrogen company), fully mixes, and-80 DEG C preserve mark
This, extract for RNA.All of blood sample and pathological examination should be true and reliable, and research is through Ethics Committee
Approval, patient's informed consent.
Table 1 chondroblastic Patients with Osteosarcoma clinical information
RNA extracts standard: RNA purity: OD260/280 1.8,28S/18S 1；RNA integrity:
RIN value 7.0.RNA integrality detection method: Agilent 2100 (RNA 6000 Nano kit), agar
Sugar gel electrophoresis (agarose gel concentration: 1% agarose gel；Voltage: 5V/cm；Time: 20min).
Embodiment 2 library construction, order-checking and data analysis
MRNA library construction: ' end has the structure of ployA tail, utilizes band for eukaryote mRNA 3
There is the enrichment with magnetic bead mRNA of Oligo (dT), after high temperature fragmentation, with it as template, synthesize cDNA.Warp
Cross magnetic beads for purifying, end reparation, 3 ' ends add base A, add sequence measuring joints after, carry out PCR amplification, structure
Build mRNA library.According to RNA pattern detection result, to chondroblastic Patients with Osteosarcoma with the most right
MRNA library construction is carried out according to group.
SRNA library construction: experiment uses Illumina TruSeq Small RNA test kit to build library,
Total serum IgE reclaims length 18-30nt RNA, becomes singly-bound cDNA, cDNA by RT-PCR reverse transcription
After amplification, reclaim cDNA product, build up tiny RNA s library.According to RNA pattern detection result, to soft
Osteoblastic Patients with Osteosarcoma and Normal group carry out sRNA library construction.
Order-checking: use llumina Hiseq2500/Miseq second filial generation high throughput sequencing technologies to mRNA and
MiRNA checks order, by removing joint, going low quality, the process such as depollute to complete the process of data,
To final data.
Carry out after miRNA and mRNA initial data being carried out background correction by transcript profile data analysis software
T-test obtains P value, then utilizes Fisher inspection to merge P value, screens differential expression miRNA and mRNA.
Set p value ＜ 0.01 and log2(Fold_change) absolute value >=3 of normalized are as significance threshold value,
Filter out the miRNA of 11 differential expressions, the miRNA 5 that wherein expression raises altogether, express water
The flat miRNA 6 lowered.Set P value ＜ 0.05 during analysis, filter out 929 differential expressions altogether
MRNA, wherein expression raise gene 455, expression lower gene 474.Its
In, the obvious miR-4676-5p of up-regulated includes our research range in.
Embodiment 3 Real-time PCR detection chondroblastic osteosarcoma tissue in miR-4676-5p and
The expression of DDX58 gene
1 sample collecting:
The peripheral blood of 19 example chondroblastic osteosarcoma tumor patients and 36 example normal healthy controls is all from hospital and (adopts
In March, 2014 collection time in December ,-2015).
2 Total RNAs extraction:
The process removing Rnase of related experiment article:
1. all rinsing with DEPC before being applied by all glass drying ovens and invade bubble, 120 DEG C of high pressure 20min, 180 DEG C high
Temperature dries more than 2 hours.
2. will plastic ware (such as: EP pipe/rifle head) use before need with 0.1%DEPC water enchroachment (invasion) steep overnight, after drain
Liquid, 120 DEG C of high pressure 20min, baking box is dried standby.
Leukocyte separates
(1) 2m1 anticoagulation cirumferential blood (blood sampling time is less than 3h) is taken；
(2) add equal-volume aseptic PB S to be sufficiently mixed in peripheral blood, form cell suspension；
(3) 4m1 lymphocyte separation medium is added in another centrifuge tube；
(4) draw 4m1 cell suspension and join lymphocyte separation medium surface along tube wall gently.Centrifugal 1500rpm
20min；
(5) with suction pipe during sucking-off boundary layer enters another centrifuge tube gently.Aseptic cold PBS washes 2 times, last 1 time
Cell suspension can be moved in EP pipe by washing, is centrifuged and removes supernatant, is used for extracting RNA.
RNA extracts
(1) first add lml Trizol in EP pipe, if freeze-stored cell is directly added into Trizol, is not required to thaw, blows
Breaking solution, rear chamber is gentle and quiet puts 5-l0min；
(2) adding 0.2m1 chloroform, acutely shake 15s, room temperature stands 2-3min, at 4 DEG C 1 2000 turns
Centrifugal 15min；
(3) the careful sucking-off supernatant water 600ul that makes an appointment moves into another centrifuge tube, adds 500:1 isopropanol, reverse
Mixing, room temperature stands 10min；
(4) 4 DEG C of 1 2000g are centrifuged l0min, abandon supernatant, bottom visible white material；
(5) add the cold ethanol of lml 75% and rotate washing, clean isopropanol；
(6) 4 DEG C of 7500g are centrifuged 5min, and dry in the air after removing ethanol 5-l0min, translucent, with 20u1DEPC
Water dissolution RNA.Take 3u1RNA sample, electrophoresis in 1.5% agarose gel；Lu1RNA sample is in purple
Outer spectrophotometer detectable concentration, is considered as RNA sample with A260/280 at 1.8-2.0 qualified.
3 reverse transcriptions
MRNA reverse transcription:
Take 1 μ g total serum IgE as template ribonucleic acid, employingIII Reverse Transcriptase
(invitrogen, article No. 18080-044) carries out cDNA reverse transcription, and experimental implementation is carried out by product description.
It is standby that-20 DEG C of refrigerators are put in the cDNA preservation obtained.
MiRNA reverse transcription:
The preparation of RT system: 1 μ g total serum IgE is as template ribonucleic acid；5×miScript HiSpec Buffer 4
μl；10×Nucleics Mix 2μl；miScript Reverse Transcriptase Mix 2μl；
Aquesterilisa filling-in is to 20 μ l.In ABI 9700 type PCR instrument, 37 DEG C of insulation 60min make reverse transcription reaction complete
Quan Hou, 95 DEG C of 5min terminate reaction.Add 80 μ l Nuclease-free H2O is diluted to 100 μ l storages
There are-20 DEG C of refrigerators standby.
4 quantitative fluorescent PCRs
Design of primers:
DDX58 primer (NM_014314.3):
Forward primer: 5 '-GGATAGATGAATGAATGG-3 ' SEQ ID NO 5
Reverse primer: 5 '-AATAGAAGGTTAAGAAGTG-3 ' SEQ ID NO 6
Amplified production length 162bp.
The preparation of the RT-PCR system of mRNA:
| Reactive component | Concentration | Volume (μ l) |
| mix | 2× | 10 |
| Forward primer | 10uM | 0.5 |
| Downstream primer | 10uM | 0.5 |
| cDNA | - | 2 |
| Nuclease-free H2O | - | Filling-in is to 25 μ l |
The detection of expression of mRNAs arranges 3 parallel pipe reactions every time, using actin as internal reference.
Amplification program is: 95 ° of 10min, 45 circulations (95 DEG C of 15s, 55 DEG C of 60s).
The preparation of the RT-PCR system of miRNA:
The detection of expression of miRNAs arranges 3 parallel pipe reactions every time, using snRNA U6 as internal reference.
Amplification program: 95 DEG C of 10min；40 circulations (95 DEG C of 10s, 55 DEG C of 30s).
5 statistical analysis
Real-time quantitative PCR amplification curve flex point understands, amplification curve entirety collimation is good, shows each reaction tube
Amplification efficiency is close, and the limit is flat and nothing raises up now, and exponent phase slope is relatively big, illustrates that amplification efficiency is higher；
Sample amplified production solubility curve is all unimodal, illustrates that amplified production only has one, for specific amplification；According to
The relative quantification formula of qRT-PCR: 2-Δ Ct × 100%, compares miR-4676-5p, DDX58 gene soft
Expression in osteoblastic osteosarcoma group and normal group.Result shows: qRT-PCR stable amplification result,
Wherein miR-4676-5p is at chondroblastic osteosarcoma group expression apparently higher than Normal group, is approximately
The octuple many (being specifically shown in Fig. 1) of comparison, and the expression that DDX58 is in chondroblastic osteosarcoma group
Less than 1/4th (being specifically shown in Fig. 2) of normal control, about matched group, result above demonstrates high flux
Transcript profile expresses the result of the confluence analysis of data.
Embodiment 4 miR-4676-5p Yu DDX58 target gene relation is verified
One, material prepares:
(1) Specimen origin
Human osteosarcoma cell line MG-63 is purchased from Shanghai cell research institute of the Chinese Academy of Sciences.
(2) main agents
LipofectamineTM2000 Transfection Reagent(Invitrogen)。
DDX58 design of primers (see embodiment 3):
MiR-4676-5p sequence issues Synesis Company, please its chemosynthesis miR-4676-5p mimics and non-spy
Specific control.
(3) main solution
1, cell culture fluid
DMEM culture medium+10% standard hyclone.
2, PBS (balanced salt solution)
8g NaCl, 0.25g KCl, 1.44g Na is dissolved in 800m1 distilled water2HPO4 and 0.24g KH2PO4
With the pH value of HCl regulation solution to 7.4, add water and be settled to 1L, autoclaving, room temperature preservation.
3,0.25% tryptic digestive juice
0.25g trypsin adds in 100m1 deionized water, and filter filtration sterilization, subpackage is standby.
Two, experimental technique
1, passage
(1) discard covering with culture fluid original in the culture bottle of cell, add 0.25% trypsin solution 1m1,
Covering cellular layer, bottleneck is sterilized, and adds a cover；
(2) observation of cell change under inverted microscope, As time goes on, former adherent cell gradually tends to round
Shape, intercellular substance bounces back, and intercellular substance strengthens, and is discarded by pancreatin when the most non-levitating, adds 5ml containing 10%
The culture fluid of hyclone terminates digestion；
(3) instill in blood cell counting plate after cell counting: take above-mentioned cell suspension 0.5mI, suitably dilution, press
Numeration of leukocyte method number corner four big lattice inner cell sum, only counts nucleus and cytoplasm complete thin during counting
Born of the same parents, cell in heaps is calculated by a cell, and by following formula scales, the total cellular score in 4 block plaid is become every
Cell number in milliliter cell suspension: total cellular score/ml=4 big lattice total cellular score/4 × 104× extension rate；
(4) according to cell counts, every milliliter it is diluted to further containing 3 × 10 with DMEM complete culture solution5
Individual cell concentration, is sub-packed in culture bottle (8m1/ every bottle), is positioned over 37 DEG C, 5%CO2Incubator is cultivated.
2.miRNA transient transfection
Using cationic-liposome method to carry out transient transfection, operation is according to LipofectaminTM2000 reagent explanations
Book is carried out.Before transfection, cell good for growth conditions is inoculated in 12 orifice plates by 24h, cell counting about 2 × 104,
Cellar culture is to transfecting the same day, and cell degrees of fusion is to test during 50-60%.By 20nM/40nM/80nM
MiRNA mimic joins in 100u1 DMEM culture medium, softly mixes；Another with 100u1 DMEM training
Support base dilution 2u1 LipofectaminTM2000 liposomees, softly mix, incubated at room 5min；Mixing
DMEM-liposome and DMEM-miRNAs, incubated at room 20min, to form transfection composite；Then
Said mixture is added in cell culture medium, mixes gently, after cultivating 6h, change complete medium.Wherein,
Non-specific sequences is as negative control.Extract cell total rna after cultivating 48h and carry out next step experiment.
3. experimental result:
By in miR-4676-5p mimics transfection to cell line of human osteosarcoma MG-63, after 48h, extract cell
Total serum IgE, blank is the cell line of human osteosarcoma cell not proceeding to miRNA, non-specific sequences conduct
Negative control.The level change of the mRNA of quantitative PCR detection target gene DDX58.Result display DDX58
Expression is lowered, and shows that DDX58 is the target gene of miR-4676-5p.
Although describing the present invention with reference to various preferred embodiments, it will be appreciated by those skilled in the art that can enter
The various changes of row, and available equivalents substitutes its assembly elemental range without departing from the present invention.Additionally, can
Carrying out many change makes particular case or material be suitable for the teachings of the present invention without departing from its elemental range.
Claims (10)
1. the preparation of detection mir-4676 and ripe miRNA thereof application in preparation diagnosis osteosarcoma reagent.
Application the most according to claim 1, it is characterised in that osteosarcoma is chondroblastic osteosarcoma.
Application the most according to claim 1, it is characterised in that diagnosis osteosarcoma reagent includes based on high flux
Sequence measurement and/or based on quantifying PCR method and/or based on probing procedure detection osteosarcoma sample in
Mir-4676 and ripe miRNA thereof transcribes or based in immunologic detection method detection osteosarcoma sample
The expression of the target gene of mir-4676 and ripe miRNA regulation and control thereof, it is preferred to use northern is miscellaneous
Friendship method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, based on
In the Flow cytometry osteosarcoma sample of microsphere, mir-4676's and ripe miRNA thereof transcribes；Use
Mir-4676 and ripe miRNA regulation and control thereof in ELISA and/or colloidal gold strip detection osteosarcoma sample
The expression of target gene.
Application the most according to claim 3, it is characterised in that the target gene of regulation and control is gene DDX58.
5. preventing and treating an osteosarcomatous preparation, described reagent comprises:
A () inhibitor and/or inhibitor combination, described inhibitor and/or inhibitor combination lower mir-4676
And/or its ripe miRNA transcribe and/or block mir-4676 and/or the activity of its ripe miRNA；
Receptible carrier on (b) pharmaceutics.
Preparation the most according to claim 5, it is characterised in that osteosarcoma is chondroblastic osteosarcoma.
Preparation the most according to claim 5, it is characterised in that use antisense oligonucleotide, antagomiRs,
MiRNA sponge, miRNA Erasers, Target Masking and/or Mutiple Targets antisense oligonucleotide
Method downward mir-4676 and/or its ripe miRNA transcribes and/or blocks mir-4676 and/or its one-tenth
The activity of ripe miRNA.
8. the preparation described in claim 5-7 prevents and treats the application in osteosarcoma reagent in preparation.
The target gene of 9.mir-4676 and ripe miRNA regulation and control thereof is in preparation diagnosis or prevents and treats answering in osteosarcoma reagent
With.
Application the most according to claim 9, it is characterised in that the target gene of regulation and control is gene DDX58.
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