CN105483275A

CN105483275A - New application of mir-1299 and mature miRNA thereof

Info

Publication number: CN105483275A
Application number: CN201610068656.6A
Authority: CN
Inventors: 李曙光; 杜玉梅; 杨承刚
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: GU'AN BOJIAN BIOTECHNOLOGY CO., LTD.
Priority date: 2016-02-01
Filing date: 2016-02-01
Publication date: 2016-04-13
Anticipated expiration: 2036-02-01
Also published as: CN105483275B

Abstract

The invention relates to new application of mir-1299 and mature miRNA thereof. According to the new application, patients with metastatic osteosarcoma and a healthy contrast crowd are sequenced to obtain expression data of miRNA and mRNA thereof; candidate miR-1299 genes and target genes C5AR1 and ITPRIP of the miR-1299 genes are screened out; further, a fluorescent quantitative experimental result shows that the miR-1299 genes and the target genes thereof are closely related to osteosarcoma, and migration and invasion of osteosarcoma cells can be effectively inhibited by inhibiting expression of the miR-1299 genes. The new application provides a good diagnosis and treatment target spot for osteosarcoma metastasis clinically and has high practical application value.

Description

The novelty teabag of mir-1299 and ripe miRNA thereof

Technical field

The present invention relates to biology field, be specifically related to the novelty teabag of mir-1299 and ripe miRNA thereof, relate to the novelty teabag of novelty teabag in the transfer of diagnosis and treatment osteosarcoma of mir-1299 and ripe miRNA thereof more specifically.

Background technology

MiRNA is a kind of Inner source property non-coding small molecules, is carried out the expression of regulatory gene by specific binding target gene.The major way of its regulation and control has two kinds: a kind of is the incomplete complementary pairing of 3 ' UTR with target gene mRNA, blocks the translation of target gene, thus regulatory gene is expressed; Another kind is similar with siRNA, and when miRNA and mRNA complete complementary matches, Ago2 albumen directly causes it to degrade by cutting mRNA, realizes gene silencing.In a word, currently think that miRNA is relevant with the pairing degree of goal gene effect and miRNA and goal gene in which way.When miRNA and goal gene match incomplete, miRNA is just to suppress the expression of goal gene to play a role; When miRNA and goal gene section sequence are matched complete, goal gene just may be caused to cause gene silencing in the fracture of complementary district.In addition, miRNAs sometimes also causes the DNA methylation of Histidine modification and promoter region, thus affects the expression of target gene.Research shows that miRNA participates in various adjustment approach, comprises growth, virus defense, hematopoiesis, orga-nogenesis, cell proliferation and apoptosis, metabolism of fat etc., has important gene expression regulation effect.

Osteosarcoma (osteosarcoma, OSA) be common malignant bone tumor, neoplastic osteoid or immature bone is directly produced by the sarcoma cell of malignant proliferation, its histological characteristic is that the fusiformis tumour cell of hyperplasia directly produces osteoid matrix or immature bone, the transfer of nearly all osteosarcoma is all transferred to lung through blood, and minority is transferred to the internal organs such as brain, kidney and through nodus lymphoideus transferring rate.Osteosarcomatous Infiltration and metastasis has a strong impact on quality of life and the prognosis of patient.The treatment of gene level has become the study hotspot of numerous scholars, but still lacks effective target of osteosarcoma Diagnosis and Treat at present, finds molecular biomarker further, will have important theory and clinical value.Simultaneously if obtain osteosarcoma serum diagnosis mark, far-reaching influence will be brought to osteosarcomatous Diagnosis and Treat.

The present invention is based on high-flux sequence method, 5 routine metastatic bone sarcoma patients and 5 normal healthy controls crowds are checked order, obtain the expression data of itself miRNA and mRNA, and then carry out bioinformatic analysis, choose standby miRNA and mRNA and carry out molecular biology checking and target checking, result shows, miR-1299 provided by the invention and target gene thereof and osteosarcoma closely related, can be used for osteosarcomatous clinical diagnosis and prevention detection, there is good actual application value.

Summary of the invention

The object of the present invention is to provide the application that mir-1299 and ripe miRNA thereof diagnoses in preparation and/or prevents and treats in osteosarcoma reagent.The sequence of miR-1299 is shown in sequence table SEQ IDNO1, and mir-1299 sequence is shown in sequence table SEQ IDNO2.

Further, osteosarcoma is metastatic bone sarcoma.Preferably, diagnose and/or prevent and treat the reagent that osteosarcoma reagent is diagnosis and/or control osteosarcoma cell Infiltration and metastasis.

Further, diagnosis osteosarcoma reagent comprises based on high-flux sequence method and/or based on quantifying PCR method and/or the expression transcribing or detect based on immunologic detection method the target gene that mir-1299 and ripe miRNA thereof regulates and controls in osteosarcoma sample detecting mir-1299 and ripe miRNA thereof in osteosarcoma sample based on probing procedure, preferred employing northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, based on transcribing of mir-1299 and ripe miRNA thereof in the Flow cytometry osteosarcoma sample of microballoon, ELISA and/or colloidal gold strip is adopted to detect the expression of the target gene that mir-1299 and ripe miRNA thereof regulates and controls in osteosarcoma sample.The target gene that preferred described mir-1299 and ripe miRNA thereof regulates and controls is C5AR1 and/or ITPRIP.

Preferably, comprise the primer of specific amplification mir-1299 and ripe miRNA thereof based on quantifying PCR method, further preferably, the primer sequence of specific amplification miR-1299 is SEQIDNO3; The probe with the nucleic acid array hybridizing of mir-1299 and ripe miRNA thereof is comprised based on probing procedure; Immunologic detection method comprises the antibody be combined with mir-1299 and ripe miRNA regulate gene expression protein-specific thereof, the antibody be preferably combined with C5AR1 and/or ITPRIP protein-specific further.

Further, prevent and treat osteosarcoma reagent and comprise the reagent of activity that transcribing and/or promoting mir-1299 and ripe miRNA thereof raising mir-1299 and ripe miRNA thereof.

Preferably, the activity transcribing and/or promote mir-1299 and ripe miRNA thereof raising mir-1299 and ripe miRNA thereof based on the microRNA gain-of-function technology of RNA and/or gene specific miRMimics technology is adopted.The short hairpin RNA of preferred synthetic miR-1299 or raise mir-1299 and ripe miRNA thereof by regulation and control promotor.

The object of the present invention is to provide the application that the target gene of mir-1299 and ripe miRNA thereof is diagnosed in preparation and/or prevented and treated in osteosarcoma preparation.Preferably, target gene is C5AR1 or ITPRIP.Preferred, target gene is ITPRIP.

Further, osteosarcoma is metastatic bone sarcoma.

Further, osteosarcoma preparation is diagnosed to adopt PCR kit for fluorescence quantitative, gene chip, immunization method to detect the expression of C5AR1 and/or ITPRIP3 gene in Patients with Osteosarcoma peripheral blood.Preferably, the primer of specific amplification C5AR1 and/or ITPRIP gene is contained in described PCR kit for fluorescence quantitative; Described gene chip comprises the probe with the nucleic acid array hybridizing of C5AR1 and/or ITPRIP gene.Preferred, contain upstream primer and the downstream primer of specific detection C5AR1 and/or ITPRIP gene in PCR kit for fluorescence quantitative, the upstream primer sequence of amplification C5AR1 is SEQIDNO4, and downstream primer sequence is SEQIDNO5; The upstream primer sequence of amplification ITPRIP gene is SEQIDNO6, and downstream primer sequence is SEQIDNO7.

Further, osteosarcomatous diagnostic preparation adopts immunization method to detect the expression product of C5AR1 and/or ITPRIP gene in osteosarcoma peripheral blood.Preferably, immunization method is that ELISA detects and/or Radioactive colloidal gold detects.Further, the ELISA method of C5AR1 and/or ITPRIP albumen is detected for using ELISA detection kit.Antibody in test kit can adopt commercially available C5AR1 and/or ITPRIP monoclonal antibody or polyclonal antibody.Further, test kit comprises: wrap by the solid phase carrier of C5AR1 and/or ITPRIP antibody, enzyme labelled antibody, the substrate of enzyme, protein standard substance, negative controls, diluent, washings, enzyme reaction stop buffer etc.

Further, detect the colloidal gold method of C5AR1 and/or ITPRIP albumen for using detection kit, antibody can adopt commercially available C5AR1 and/or ITPRIP monoclonal antibody or polyclonal antibody.Further, gold-immunochromatographyreagent reagent for assay box adopts colloidal gold immunochromatographimethod technology or Radioactive colloidal gold percolation process.Further, detection zone (T) specking on gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-C5AR1 and/or ITPRIP antibody, quality control region (C) specking has immunoglobulin IgG.

The object of the present invention is to provide the application of above-mentioned osteosarcoma diagnostic preparation in preparation osteosarcoma diagnostic tool.

The object of the present invention is to provide the osteosarcomatous preparation of a kind of control, containing the reagent of transcribing or expressing or the compound that suppress C5AR1 and/or ITPRIP gene in preparation.Further, the expression product of C5AR1 and/or ITPRIP gene, gene high expression level in osteosarcoma transfer group.

The expression that those skilled in the art know suppressor gene and expression product thereof can adopt the one in following method and/or several usually: activate the suppressor gene of this gene, add suppress the albumen of this gene, add suppress the chemical (as siRNA etc.) of this gene, add promote proteolytic degradation or blocks protein maturation molecule, remove the albumen etc. activating this gene.

The object of the present invention is to provide one to prevent and treat osteosarcoma reagent, described pack contains:

A () is raised transcribing of mir-1299 and ripe miRNA thereof and/or is promoted the reagent of activity of mir-1299 and ripe miRNA thereof;

Receptible carrier in (b) pharmaceutics.

Preferably, the activity transcribing and/or promote mir-1299 and ripe miRNA thereof raising mir-1299 and ripe miRNA thereof based on the microRNA gain-of-function technology of RNA and/or gene specific miRMimics technology is adopted.The short hairpin RNA (shorthairpinRNA, shRNA) of preferred synthetic miR-1299 or the expression by regulation and control promotor rise mir-1299 and ripe miRNA thereof.

Further, described osteosarcoma is metastatic bone sarcoma.

The object of the present invention is to provide a kind of osteosarcoma diagnostic reagent, osteosarcoma diagnostic reagent can detect the expression that transcribing of mir-1299 and ripe miRNA thereof in osteosarcoma sample or immunologic detection method detect the target gene that mir-1299 and ripe miRNA thereof regulates and controls in osteosarcoma sample.

Further, osteosarcoma diagnostic reagent, based on high-flux sequence method and/or based on quantifying PCR method and/or the expression transcribing or detect based on immunization method the target gene that mir-1299 and ripe miRNA thereof regulates and controls in osteosarcoma sample detecting mir-1299 and ripe miRNA thereof in osteosarcoma sample based on probing procedure, preferably adopts northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, transcribing based on mir-1299 and ripe miRNA thereof in the Flow cytometry osteosarcoma sample of microballoon; ELISA and/or colloidal gold strip is adopted to detect the expression of the target gene that mir-1299 and ripe miRNA thereof regulates and controls in osteosarcoma sample.The target gene that preferred described mir-1299 and ripe miRNA thereof regulate and control is containing the antibody be combined with C5AR1 and/or ITPRIP protein-specific in C5AR1 and/or ITPRIP, ELISA and/or colloidal gold strip.

The object of the present invention is to provide the application of the osteosarcomatous preparation of above-mentioned control in preparation clinical treatment of osteosarcoma medicine or reagent.

Definition:

Present stage, the method for expression level that detects miRNA mainly comprised based on high throughput sequencing technologies, miRNA detection method based on nucleotide hybridization and PCR-based.MiRNA detection method based on probe hybridization technology is a kind of direct Detection Method; do not need to increase in advance to sample rna, comprise northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, the technology such as flow cytometry based on microballoon.

(1) Northern hybridization

Also known as the detection eukaryote RNA size that RNA engram technology is the most classical, estimate the experimental technique of its abundance.Ultimate principle is as follows: first at the upper fixing miRNA sample of carrier (as silicon chip, microballoon or film etc.), then with the probe hybridization through marking, carry out signal detection after washing unnecessary hybridization probe; Also can first fix the DNA probe with the complementation of target miRNA sequence on carrier, then hybridize with the sample miRNA through marking, then carry out signal detection.The method of signal mark comprises isotopic labeling, fluorescent mark and nano gold mark etc.

(2) miRNA chip of expression spectrum

Principle is the target molecule on applying marking probe in detecting solid support equally.By miR-96 gene in design chips and internal reference sequence, Accurate Analysis the expression level of corresponding miRNA in sample can be gone out.Gene chip has high-throughout advantage, once can detect whole expression of a hundreds of gene in same sample.The liquid-phase chip (Liquidchip) of Luminex company development, also known as multifunctional suspending dot matrix (Multianalytesuspensionarray, MASA), is the biochip technology of new generation.Liquid-phase chip system is that main matrix is formed by many microspheres, often kind of microsphere is fixed with different probe molecules, in order to distinguish different probes, each sphere matrix for label probe is all with a unique color numbers, these microspheres are suspended in a liquid-phase system, just constitute liquid-phase chip system.This system can carry out qualitative and quantitative analysis fast to the multiple differing moleculars in same trace sample simultaneously, and this detection technique is called as FMAP (Flexiblemultianalyteprofiling) technology.Molecular hybridization carries out in aaerosol solution, and detection speed is exceedingly fast.

(3) ribozyme protection analytical technology (RPA)

The detection of miRNA can also adopt ribozyme to protect analytical technology; the probe marked and RNA sample to be measured are mixed; hybridize after thermally denature; the RNA of not hybridizing and unnecessary probe single-chain nucleic acid enzymic digestion; the shielded RNA molecule of purifying after heat inactivation nuclease; finally by sex change PAGE electrophoretic separation probe, colour developing.This novel method based on solution hybridization is simple and quick, highly sensitive, but also can only be used for analyzing known miRNA.

(4) RAKE method

RAKE method (RNAprimedarraybasedKlenowemzyme) is the Klenow fragment utilizing DNA polymerase i on the basis of miRNAmicroarray, makes the method that miRNA is hybridized with fixing DNA probe.RAKE sensitivity can detect miRNA specifically, is applicable to screen all miRNA that oneself knows fast in a large number.MiRNA express spectra situation can be detected in specific cell and tumour.Moreover, RAKE method can also be isolated miRNA and analyze it from the paraffin-embedded tissue secured by formalin, for analyzing the door that miRNA opens hope from file sample.

(5) in situ hybridization (insituhybridization)

Hybridization in situ technique can intuitively understand miRNA phraseology, and be a kind of easier method of observation miRNA spatial and temporal expression, normal mark mode comprises digoxin, vitamin H, fluorescent mark etc.In situ hybridization (LockedNucleicAcid (LNA) basedinsituhybridization (LNA-ISH)) on locked nucleic acid basis is the more probe mode of current application.

(6) based on the flow cytometry (bead-basedflowcytometry) of microballoon

Be a kind of liquid-phase chip technology, FCM analysis organically combines with chip technology by the method, has that flux is large, detection speed is fast concurrently, a feature such as highly sensitive and specificity is good.

(7) Real-Time Fluorescent Quantitative PCR Technique (Real-timePCR, RT-PCR)

Fluoroscopic examination PCR instrument can draw dynamic changing curve to the cumulative speed of extension increasing sequence in whole PCR process.In reaction mixture, the initial concentration of target sequence is larger, requires that the PCR cycle number (generally expressing with specific threshold cycle number Ct) obtaining amplified production specific output is fewer.Because miRNA length is only 22nt, traditional qRT-PCR is not suitable for increasing so short fragment.There is several real time quantitative PCR method for miRNA now, as tailing method, neck ring method etc.Neck ring method is that a kind of desirable miRNA detects qRT-PCR method: first design special loop-stem structure primer, with miRNA to be measured for template reverse transcription synthesis cDNA first chain, this cDNA one end is stem Loop primer, stem ring texture is opened and substantially increases the length of cDNA, carries out real-time quantitative PCR amplification for template design primer subsequently with the cDNA of synthesis.QRT-PCR has that specificity is high, sensitivity good, the multiple advantage such as simple fast.

(8) sequencing

The known miRNA of major part is found by cDNA cloning and sequencing and identifies.This method needs the cDNA library first building miRNA, then carries out pcr amplification, and amplified production is cloned into subsequently on expression vector and checks order.Takada develops a kind of amplification cloning (miRNAamplificationprofiling, mRAP) of improvement, and mRAP method first connects joint, then with the reverse transcription primer reverse transcription with joint complementation at 3 ' of miRNA end.Because specific ThermoScript II has terminal deoxynucleotidyl transferase activity, some Nucleotide (mainly deoxycytidylic acid(dCMP)) can be connected to 3 ' end of the cDNA chain that reverse transcription goes out.After poly (C) sticky end of 5 ' end connector and cDNA chain is annealed, add the pcr amplification that a pair general primer can realize cDNA.Due to mRAP High sensitivity, the expression amount of miRNA in can directly organizing on a small quantity by Cloning and sequencing technology for detection.Sequence label cloning is that one has developed the higher miRAGE of detection efficiency (miRNASAGE) cloning on the basis in serial analysis of gene expression (SAGE) technology, this method is by generating large sub-series, multiple miRNA can be detected by single sequencing reaction, significantly improve detection efficiency.

High-flux sequence (High-throughputsequencing) is the change to tradition order-checking revolution also known as sequencing technologies of future generation (nextgenerationsequencing), once to millions of DNA moleculars, sequencing is carried out to hundreds of thousands of, greatly improve order-checking efficiency.This kind of large scale sequencing technology greatly improves the solution read rate of multiple species genetic information, and for obtaining the sequence information of all miRNA, deciphering miRNA collection of illustrative plates provides guarantee.Simultaneously high-flux sequence makes the analysis transcript profile of species and genome being carried out to careful overall picture become possibility, so degree of depth order-checking (deepsequencing) that is otherwise known as.The representative of high-flux sequence platform is 454 sequenators (RochGSFLXsequencer) of Roche Holding Ag (Roche), the Solexa gene element analyzer (IlluminaGenomeAnalyzer) of Illumina company and the SOLiD sequenator (ABISOLiDsequencer) of ABI.

Immunologic detection method is using a kind of antibody or Multiple Antibodies as analytical reagent, carries out quantitatively or the detection method of qualitative analysis determinand.Its ultimate principle is the interaction between antibody and antigen.For improving the susceptibility of antigen and antibody test, by the material of easily display in known antibodies or antigenic mark, by certification mark thing, reflect with or without antigen antibody reaction, thus indirectly measure antigen or the antibody of trace.Conventional marker has enzyme, fluorescein, radio isotope, Radioactive colloidal gold and electron dense substances etc.The specific reaction that on this antigen or antibody labeling, display object is carried out is called immunolabelling technique (immunolabellingtechnique).Immunoassay technology most widely used at present mainly contains: enzyme linked immunosorbent assay (enzyme-linkedimmunosorbentassay, ELISA), colloidal gold immunity chromatography etc.

Enzyme linked immunosorbent assay principle is combined antigen or antibody and substrate (enzyme), makes it keep the activity of immune response and enzyme.The antigen of mark or antibody and the ligand binding that is coated on solid phase carrier, then make it and corresponding colorless substrate effect and Show Color, according to the range estimation of colour developing depth degree or by microplate reader mensuration OD value result of determination.

Colloidal gold strip is generally made up of sample pad, gold mark pad, chromatographic film, absorbent pad four part.Chromatographic material has nitrocellulose membrane (NC), polyester film, nylon membrane and pvdf membrane etc., need to select the different film required according to test, wherein NC film is the most conventional, can determine whether to need activation or process according to test particular case before using, in most cases without the need to process, can directly use.Gold is marked protein solution even application on gold mark pad, dry for subsequent use under room temperature.NC film can catch a certain amount of bag by (antibody) and two anti-as detection line and nature controlling line.Finally sample pad, gold mark pad, NC film and thieving paper are fixed on PVC board successively, test strip.

Namely microRNA gain-of-function technology based on RNA raises the level of miRNAs by the precursor substance of exogenous supplementary miRNAs synthesis.Such as, sample RNA (shorthairpinRNA can be pressed from both sides by the synthetic bob consistent with endogenous miRNA sequence, shRNA), promotor is done by polymerase II or III, take virus as vector-transfected cell, being loaded into RISC after Dicer enzyme modification plays a role, and is equivalent to raise the level of pre-miRNA, and action effect is stable and lasting.

Gene specific miRMimics technology this technique avoids the nonspecific action of miRNA and gene.This synthetic with the specific oligonucleotide chain that combines of target gene 3 ' UTR complementation, can play and identical with miRNA transcribe rear regulating effect.

Being included in the carrier that the pharmaceutics of pharmaceutical composition of the present invention is permitted is the carrier usually utilized when preparation, this carrier comprises lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbyl alcohol (sorbitol), N.F,USP MANNITOL (mannitol), starch, gum Arabic, calcium phosphate, alginate (alginate), gel (gelatin), Calucium Silicate powder, Microcrystalline Cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone), Mierocrystalline cellulose (cellulose), water, syrup, methylcellulose gum (methylcellulose), methyl hydroxybenzoate (methylhydroxybenzoate), propyl hydroxy propyl benzoate (propylhydroxybenzoate), talcum, Magnesium Stearate (stearicacidmagnesium) and mineral oil (mineraloil) etc., but it is not limited thereto.

Pharmaceutical composition of the present invention can also comprise lubricant, wetting agent, sweeting agent, flavouring agent, emulsifying agent, suspension agent, sanitas etc. except mentioned component.The carrier be applicable to that pharmaceutics is permitted and preparation are recorded in Lei Mingdengshi pharmacy pandect in detail.

Pharmaceutical composition of the present invention by oral or parenterally carry out administration, during as non-oral administration, by intravenous injection, intranasal injection, local injection, intracerebral ventricle injection, spinal cavity is injected, subcutaneous injection, abdominal injection, the modes such as percutaneous dosing carry out administration.

The dosage be applicable to of pharmaceutical composition of the present invention can carry out multiple prescription according to the factor of age of preparation ways, administering mode, patient, body weight, sex, morbid state, food, administration time, route of administration, drainage rate and being quick on the draw property and so on, usually, skilled doctor can easily determine and prescription to desired treatment or prevent effective dosage.

The method that pharmaceutical composition of the present invention can easily be implemented according to general technical staff of the technical field of the invention, to utilize in pharmaceutics receptible carrier and/or vehicle formulation to carry out, thus can with the preparation of unit dose form or in be contained in multicapacity container and prepare.Now, formulation is solution, suspension or emulsion form in oiliness or aqueous medium, or also can be extractum, powder agent, granule, tablet or capsule form, can also comprise dispersion agent or stablizer.

Accompanying drawing explanation

Fig. 1 is that RT-PCR detects miR-1299 relative expression levels figure in metastatic bone sarcoma

Fig. 2 is that RT-PCR detects C5AR1 relative expression levels figure in metastatic bone sarcoma

Fig. 3 is that RT-PCR detects ITPRIP relative expression levels figure in metastatic bone sarcoma

Embodiment

Below in conjunction with specific embodiment, setting forth the present invention further, only for explaining the present invention, and can not limitation of the present invention be interpreted as.Those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.The experimental technique of unreceipted actual conditions in the following example, the usually conveniently conditioned disjunction condition examinations of advising according to manufacturer.

The collection of embodiment 1 sample and Total RNAs extraction

5 routine metastatic bone sarcoma patients and 5 normal healthy controls.Metastatic bone sarcoma patients group 5 example and control group 5 people require on an empty stomach at least 12h, under m seq 7:00 ~ 8:00 room temperature, extract 10ml venous blood in ethylenediamine tetraacetic acid (EDTA) (EDTA) anticoagulant tube, extract peripheral blood mononuclear cell PBMCs, add 1mlTrizol reagent (Invitrogen company), abundant mixing, preserves sample, extracts for RNA for-80 DEG C.All blood samples and pathological examination should be true and reliable, study through Ethics Committee's approval, patient's informed consent.

RNA extracts standard: RNA purity: OD260/280≤1.8,28S/18S≤1; RNA integrity: RIN Zhi≤7.0.RNA integrality detection method: Agilent2100 (RNA6000Nanokit), agarose gel electrophoresis (sepharose concentration: 1% agarose gel; Voltage: 5V/cm; Time: 20min).

Embodiment 2 checks order and data analysis

Order-checking: use lluminaHiseq2500/Miseq s-generation high throughput sequencing technologies to check order to mRNA and miRNA, by removing joint, go inferior quality, process that the process such as to depollute completes data, obtain final data.

Carry out t-test after background correction being carried out to miRNA and mRNA raw data by transcript profile data analysis software and obtain P value, then utilize Fisher to check and merge P value, screening differential expression miRNA and mRNA.Setting p value < 0.01, filters out the miRNA of 12 differential expressions altogether, and wherein the miRNA11 of expression level downward is individual.Set P value < 0.05 in analytic process, filter out the mRNA of 425 differential expressions altogether, the gene 324 of wherein expression level rise.Utilization comprises RNA22, miRanda, miRDB, miRWalk, the target gene of these algorithm predicts differential expressions of PICTAR2 and Targetscan miRNA, choose >=4 algorithm predicts target gene out, and the target gene of the miRNA of the differential expression verified at miRWalk database lookup, then the gene of expressing negative correlation in all target genes and mRNA sequencing result with miRNA is carried out confluence analysis, we obtain 105 miRNA-target gene relations pair altogether, the miRNA-target gene relation pair raising miRNA (miR-1299) is obtained by prediction, its target gene is C5AR1, ITPRIP etc.

Embodiment 3Real-timePCR detects the expression of miR-1299 and C5AR1 and ITPRIP gene in osteosarcoma tissue

1 sample collecting:

The peripheral blood of 49 routine metastatic bone sarcoma tumor patients and 63 routine normal healthy controls is all from hospital's (acquisition time in August ,-2015 in April, 2014).

2 Total RNAs extraction:

The process of removing Rnase of related experiment article:

1. invade bubble, 120 DEG C of high pressure 20min by all rinsing with DEPC before all glassware application, 180 DEG C of high temperature dry more than 2 hours.

2. need before being used by plastic ware (as: EP pipe/rifle head) to spend the night with 0.1%DEPC water enchroachment (invasion) bubble, rear control dry liquids, 120 DEG C of high pressure 20min, baking box is dried for subsequent use.

White corpuscle is separated

(1) 2m1 anticoagulation cirumferential blood (blood sampling time is no more than 3h) is got;

(2) add the aseptic PBS of equal-volume fully to mix 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 (noting not mixing with lymphocyte separation medium) gently along tube wall.Centrifugal 1500rpm20min;

(5) with suction pipe gently sucking-off interfacial layer (tunica albuginea) enter in another centrifuge tube.Aseptic cold PBS washes 2 times, and cell suspension can move in EP pipe by last 1 washing, centrifugally removes supernatant, for extracting RNA.

RNA extracts

(1) first add lmlTrizol in EP pipe, if freeze-stored cell directly adds Trizol, do not need to thaw, after piping and druming cracking, room temperature leaves standstill 5-l0min;

(2) add 0.2m1 chloroform, concuss 15s, room temperature leaves standstill 2-3min, and at 4 DEG C, 12000 leave heart 15min;

(3) the careful sucking-off supernatant water 600ul that makes an appointment moves into another centrifuge tube (noting not being extracted into egg white layer), and add 500:1 Virahol, put upside down mixing, room temperature leaves standstill 10min;

(4) 4 DEG C of centrifugal l0min of 12000g, abandon supernatant liquor, bottom visible white material;

(5) add the cold ethanol of lml75% and rotate washing, cleaning Virahol;

(6) 4 DEG C of centrifugal 5min of 7500g, dry in the air after removing ethanol 5-l0min, translucent, with 20u1DEPC water dissolution RNA.Get 3u1RNA sample, electrophoresis in 1.5% sepharose; Lu1RNA sample, in UV spectrophotometer measuring concentration, is considered as RNA sample with A260/280 at 1.8-2.0 qualified.

3 reverse transcriptions

MRNA reverse transcription:

Get 1 μ g total serum IgE as template ribonucleic acid, adopt iIIReverseTranscriptase (invitrogen, article No. 18080-044) carries out cDNA reverse transcription, and experimental implementation is undertaken by product description.It is for subsequent use 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 × miScriptHiSpecBuffer4 μ l; 10 × NucleicsMix2 μ l; MiScriptReverseTranscriptaseMix2 μ l; Aqua sterilisa fills to 20 μ l.After in ABI9700 type PCR instrument, 37 DEG C of insulation 60min make reverse transcription reaction completely, 95 DEG C of 5min termination reactions.Add 80 μ lNuclease-freeH ₂o is diluted to 100 μ l, and to be stored in-20 DEG C of refrigerators for subsequent use.

4 quantitative fluorescent PCRs

Design of primers:

C5AR1 primer (NM_001736.3):

Forward primer: 5 '-GTAAGTAATGATACAGAGG-3 ' SEQIDNO4

Reverse primer: 5 '-GTCAATATCCTGGTTATG-3 ' SEQIDNO5

Amplified production length 102bp.

ITPRIP primer (NM_033397.3):

Forward primer: 5 '-CTATGATGTAGGTGCTATTCT-3 ' SEQIDNO6

Reverse primer: 5 '-ACAGTAGGTAAGTCAGTTG-3 ' SEQIDNO7

Amplified production length 83bp.

The preparation of the RT-PCR system of mRNA:

Reactive component	Concentration	Volume (μ l)
			mix	2×	10
Upstream primer	10uM	0.5
			Downstream primer	10uM	0.5
cDNA	-	2
			Nuclease-free H ₂O	-	Fill to 25 μ l

The detection of expression of mRNAs arranges 3 parallel pipe reactions, using actin as internal reference at every turn.

Amplification program is: 95 ° of 10min, 45 circulations (95 DEG C of 15s, 52 DEG C of 60s).

The preparation of the RT-PCR system of miRNA:

The detection of expression of miRNAs arranges 3 parallel pipe reactions, using snRNAU6 as internal reference at every turn.

Amplification program: 95 DEG C of 10min; 40 circulations (95 DEG C of 10s, 60 DEG C of 30s).

5 statistical analysis

Real-time quantitative PCR amplification curve flex point is clear, and the overall collimation of amplification curve is good, shows that the amplification efficiency of each reaction tubes is close, and the limit is flat and without raising up now, exponent phase slope is comparatively large, illustrates that amplification efficiency is higher; Sample amplified production solubility curve is all unimodal, illustrates that amplified production only has one, is specific amplification; Relative quantification formula according to qRT-PCR: 2-Δ Ct × 100%, compares the expression level of miR-1299, C5AR1, ITPRIP gene in metastatic bone sarcoma group and normal group.Result shows: qRT-PCR stable amplification result, wherein miR-1299 is starkly lower than Normal group at metastatic bone sarcoma group expression level, about 1/6th (specifically seeing Fig. 1) of contrast, and C5AR1, the expression level of ITPRIP in metastatic bone sarcoma group is all higher than normal control, C5AR1 expression level in metastatic bone sarcoma group is about 1.7 times (specifically seeing Fig. 2) of control group, ITPRIP expression level in metastatic bone sarcoma group is about 2.6 times (specifically seeing Fig. 3) of control group, the result of the confluence analysis of above result verification high-throughput transcript profile expression data.

Embodiment 4miR-1299 and C5AR1 and the checking of ITPRIP target gene relation

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

LipofectamineTM2000TransfectionReagent(Invitrogen)。

C5AR1, ITPRIP design of primers (see embodiment 3):

MiR-1299 sequence issues Synesis Company, please its chemosynthesis miR-1299mimics and non specific control.

(3) main solution

1, cell culture fluid

DMEM substratum+10% standard foetal calf serum.

2, PBS (balanced salt solution)

In 800m1 distilled water, dissolve 8gNaCl, 0.25gKCl, 1.44gNa2HPO4 and the 0.24gKH2PO4 pH value to 7.4 of HCl regulator solution, adds water and is settled to 1L, autoclaving, room temperature preservation.

3,0.25% tryptic digestive juice

0.25g trypsinase adds in 100m1 deionized water, and frit sterilizing, packing is for subsequent use.

Two, experimental technique

1, passage

(1) discarding covering with nutrient solution original in the culturing bottle of cell, adding 0.25% trypsin solution 1m1, cover cellular layer, bottleneck is sterilized, and adds a cover;

(2) observation of cell change under inverted microscope, As time goes on, former adherent cell is tending towards circular gradually, intercellular substance bounces back, intercellular substance is strengthened, and is discarded by pancreatin when also non-levitating, adds 5ml and stops digestion containing the nutrient solution of 10% foetal calf serum;

(3) cell counting: get above-mentioned cell suspension 0.5mI, instill after suitable dilution in blood cell counting plate, by the large lattice inner cell sum in white blood cell count(WBC) method number corner four, only count fine karyon and cytoplasm complete cell during counting, cell in heaps calculates by a cell, the total cellular score in 4 block plaid is become the cell count in every ml cells suspension by following formula scales: large lattice total cellular score/4 × 10 of total cellular score/ml=4 ⁴× extension rate;

(4) according to cell counts, dilute further for every milliliter containing 3 × 10 with DMEM complete culture solution ⁵individual cell concn, is sub-packed in (8m1/ every bottle) in culturing bottle, is positioned over 37 DEG C, 5%CO ₂cultivate in incubator.2.miRNA transient transfection

Adopt cationic-liposome method to carry out transient transfection, operate according to Lipofectamin ^tM2000 reagent specification sheetss carry out.Before transfection, cell good for growth conditions is inoculated in 12 orifice plates by 24h, cell counting about 2 × 10 ⁴, cellar culture, to transfection same day, is tested when cytogamy degree is 50-60%.20nM/40nM/80nMmiRNAmimic is joined in 100u1DMEM substratum, softly mix; Another 100u1DMEM substratum dilution 2u1Lipofectamin ^tM2000 liposomes, 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 perfect 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 miR-1299mimics transfection in cell line of human osteosarcoma MG-63, extract cell total rna after 48h, blank is the cell line of human osteosarcoma cell not proceeding to miRNA, and non-specific sequences is as negative control.The level change of the mRNA of quantitative PCR detection target gene C5AR1, ITPRIP.The expression that result shows 2 genes all has the downward of different levels, and show that C5AR1, ITPRIP are the target genes of miR-1299, wherein ITPRIP gene deregulation is more obvious.

MiR-1299 is on the impact of human osteosarcoma cell proliferation in embodiment 5 transfection

One, experiment material

Sequent synthesis: miR-1299 sequence is issued Shanghai Ji Ma company, by its synthesis miR-1299mimics, anti-miR-1299.5 ' end FAM of nucleotide sequence modifies, can fluoresced green, can observe after cell is entered in transfection under fluorescent microscope.

MiR-1299mimics sequence:

5’-UUCUGGAAUUCUGUGUGAGGGA/CCUCACACAGAAUUCCAGAAUU-3’(SEQIDNO8)

Anti-miR-1299 sequence:

5’-UCCCUCACACAGAAUUCCAGAA-3’(SEQIDNO9)

Two, experimental technique

1.Transwell migration experiment detects cell migration ability

1) adopt Transwell cell 24 orifice plate gathering carbon ester film containing 8um aperture, add the DMEM nutrient solution of 600ul containing 10%FBS in the lower room of Transwell cell in advance, 37 DEG C balance 1 hour.

2) collect each group of logarithmic phase cell, with PBS (pH7.4) rinsing 3 times, the resuspended each group of cell of the RPMI1640 nutrient solution containing 10%FBS, after counting, adjustment cell density is 5105/ml, gets 100ul/ hole and adds room.Be placed in 5%CO2,37 DEG C of constant incubators hatch 48 hours.

3) take out Transwell cell, strike off the cell in face, room on filter membrane with cotton swab.

4) fix with 4% paraformaldehyde after PBS rinsing, violet staining.

5) under 200 power microscopes, count the cell count in face, room under filter membrane, each hole counts the cell number in 5 visuals field immediately.Get its mean value, represent the transfer ability of each group of cell with the number of migrating cell.

2.Transwell Matrigel detects cell invasion ability

1) carry thawed on ice Matrigel the day before yesterday, prepare Tip, liquid-transfering gun, the Transwell cell of precooling.

2) by extracellular matrix Matrigel, add the upper surface of the poly-carbon ester film of Transwell cell in the ratio of 50ul/cm2,37 DEG C of placements make its plastic in 30 minutes.

3) in the lower room of Transwell cell, add the DMEM nutrient solution of 600ul containing 10%FBS in advance, 37 DEG C balance 1 hour.

4) collect each group of logarithmic phase cell, with PBS (pH7.4) rinsing 3 times, the resuspended each group of cell of the RPMI1640 nutrient solution containing 10%FBS, after counting, adjustment cell density is 5105/ml, gets 100ul/ hole and adds room.Be placed in 5%CO2, in 37 DEG C of constant incubators, hatch 48 hours.

5) take out Transwell cell, strike off the cell in face, room on filter membrane with cotton swab.

6) fix with 4% paraformaldehyde after PBS rinsing, violet staining.

7) under 200 power microscopes, count the cell count in face, room under filter membrane, each hole counts the cell number in 5 visuals field immediately.Get its mean value, represent the invasive ability of each group of cell with the number of migrating cell.

Three, experimental result

Fluorescence microscope transfection efficiency.After 24 hours, fluorescence microscopy Microscopic observation, miR-1299mimics, anti-miR-1299 are all successfully transfected in osteosarcoma cell MG-63, and the cell with fluorescent grain accounts for about total cellular score 85%-90%, display transfection success.

The impact of miR-1299 on cell MG-63 migration and invasion ability is analyzed with the experiment of Transwell cell migration and invasion, result shows, the MG-63 cell migration of transfection miR-1299mimics and the ability of invasion and attack comparatively control group all significantly decrease, meanwhile, the ability of the MG-63 cell migration of transfection anti-miR-1299 and invasion and attack comparatively control group be all significantly improved.

Although describe the present invention with reference to various preferred embodiment, it will be appreciated by those skilled in the art that and can carry out various change, and available equivalents substitutes its assembly and do not deviate from base region of the present invention.In addition, many changes can be carried out and not deviate from its base region to make particular case or material be suitable for instruction of the present invention.

Claims

The application that 1.mir-1299 and ripe miRNA thereof diagnoses in preparation and/or prevents and treats in osteosarcoma reagent.
2. application according to claim 1, is characterized in that, osteosarcoma is metastatic bone sarcoma.
3. application according to claim 1, is characterized in that, prevents and treats osteosarcoma reagent and comprises the reagent of activity that transcribing and/or promoting mir-1299 and ripe miRNA thereof raising mir-1299 and ripe miRNA thereof.
4. application according to claim 3, it is characterized in that, based on the microRNA gain-of-function technology of RNA and/or the activity transcribing and/or promote mir-1299 and ripe miRNA thereof of gene specific miRMimics technology rise mir-1299 and ripe miRNA thereof, the short hairpin RNA of preferred synthetic miR-1299 or the expression by regulation and control promotor rise mir-1299 and ripe miRNA thereof.
5. application according to claim 1, it is characterized in that, diagnosis osteosarcoma reagent comprises based on high-flux sequence method and/or based on quantifying PCR method and/or the expression transcribing or detect based on immunologic detection method the target gene that mir-1299 and ripe miRNA thereof regulates and controls in osteosarcoma sample detecting mir-1299 and ripe miRNA thereof in osteosarcoma sample based on probing procedure, preferred employing northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, based on transcribing of mir-1299 and ripe miRNA thereof in the Flow cytometry osteosarcoma sample of microballoon, ELISA and/or colloidal gold strip is adopted to detect the expression of the target gene that mir-1299 and ripe miRNA thereof regulates and controls in osteosarcoma sample.
6. application according to claim 5, is characterized in that, the target gene of regulation and control is C5AR1 and/or ITPRIP gene, preferred ITPRIP gene.
The target gene of 7.mir-1299 and ripe miRNA thereof is in the application preparing diagnosis and/or prevent and treat in osteosarcoma preparation.
8. application according to claim 7, is characterized in that, target gene is C5AR1 and/or ITPRIP gene.
9. application according to claim 7, is characterized in that, diagnosis osteosarcoma preparation adopts PCR kit for fluorescence quantitative or gene chip or immunization method to detect the expression of C5AR1 and/or ITPRIP gene in peripheral blood.
10. application according to claim 7, is characterized in that, prevents and treats in osteosarcomatous preparation containing the reagent of transcribing or expressing or the compound that suppress C5AR1 and/or ITPRIP gene.