CN105483275B - The new application of mir-1299 and its maturation miRNA - Google Patents

Abstract

The present invention relates to the new applications of mir-1299 and its maturation miRNA.Metastatic bone sarcoma patients and normal healthy controls crowd are sequenced in invention, obtain the expression data of its miRNA and mRNA, filter out candidate miR-1299 and its target gene C5AR1, ITPRIP.Further fluorescent quantitation experimental result shows that miR-1299 and its target gene are closely related with osteosarcoma, inhibits the expression of miR-1299 that can effectively inhibit the migration and invasion of osteosarcoma cell.The present invention provides good bone and flesh tumor metastasis diagnosis and treatment target spot for clinic, has good practical application value.

Description

The new application of mir-1299 and its maturation miRNA

Technical field

The present invention relates to molecular biology fields, are specifically related to the new application of mir-1299 and its maturation miRNA, more New application of the new application for being related to mir-1299 and its maturation miRNA of body in diagnosis and treatment bone and flesh tumor metastasis.

Background technique

MiRNA is a kind of endogenous non-coding small molecule, by specific binding target gene come the expression of controlling gene. There are two types of its major ways regulated and controled: one is the not fully complementary pairings of 3 ' UTR with target gene mRNA, block turning over for target gene It translates, to adjust gene expression；Another kind be it is similar with siRNA, when miRNA and mRNA complete complementary match clock synchronization, Ago2 albumen Its degradation is directly resulted in by cutting mRNA, realizes gene silencing.In short, be presently believed to miRNA in which way with purpose base Because effect and miRNA are related with the pairing degree of target gene.MiRNA and target gene pairing it is incomplete when, miRNA just with The expression of target gene is inhibited to play a role；When miRNA and target gene section sequence match complete, it is possible to cause purpose base Because leading to gene silencing in complementary region fracture.In addition, miRNAs sometimes also leads to the DNA of histidine modification and promoter region Methylation, to influence the expression of target gene.Research shows that miRNA participates in various adjusting approach, including development, disease Malicious defence, hematopoiesis, orga- nogenesis, cell Proliferation and apoptosis, fat metabolism etc., with important gene expression regulation work With.

Osteosarcoma (osteosarcoma, OSA) is common malignant bone tumor, is directly produced by the sarcoma cell of malignant proliferation Raw neoplastic osteoid or immature bone, histological characteristic be hyperplasia shuttle shape tumour cell directly generate osteoid matrix or Immature bone, nearly all bone and flesh tumor metastasis are transferred to lung through blood, and minority is transferred to the internal organs such as brain, kidney and through lymph It carries down shifting.The invasion and transfer of osteosarcoma seriously affect the quality of life and prognosis of patient.The treatment of gene level has become The research hotspot of numerous scholars, but still lack effective target of osteosarcoma diagnosing and treating at present, it is raw further to find molecule Substance markers, will be with important theory and clinical value.It, will be to bone simultaneously as can obtaining osteosarcoma serodiagnosis marker The diagnosing and treating of sarcoma brings far-reaching influence.

The present invention is based on high-flux sequence methods, survey to 5 metastatic bone sarcoma patients and 5 normal healthy controls crowds Sequence obtains the expression data of its miRNA and mRNA, and then carries out bioinformatic analysis, chooses standby miRNA and mRNA and carries out Molecular biology verifying and target checking, the results show that miR-1299 provided by the invention and its target gene and osteosarcoma are close Correlation can be used for the clinical diagnosis and prevention detection of osteosarcoma, have good practical application value.

Summary of the invention

The purpose of the present invention is to provide mir-1299 and its maturation miRNA in preparation diagnosis and/or prevention and treatment osteosarcoma examination Application in agent.The sequence of miR-1299 is shown in sequence table SEQ ID NO 1, and mir-1299 sequence is shown in sequence table SEQ ID NO 2.

Further, osteosarcoma is metastatic bone sarcoma.Preferably, diagnose and/or prevent and treat osteosarcoma reagent be diagnosis and/or The reagent for preventing and treating osteosarcoma cell invasion and transfer.

Further, diagnosis osteosarcoma reagent include based on high-flux sequence method and/or based on quantifying PCR method and/or The transcription of mir-1299 and its maturation miRNA in osteosarcoma sample are detected based on probing procedure or are based on immunologic detection method Detect the expression of the target gene of mir-1299 and its maturation miRNA regulation in osteosarcoma sample, it is preferred to use northern Hybridizing method, miRNA chip of expression spectrum, ribozyme protect analytical technology, RAKE method, in situ hybridization, the fluidic cell based on microballoon Art detects the transcription of mir-1299 and its maturation miRNA in osteosarcoma sample；It is detected using ELISA and/or colloidal gold strip The expression of the target gene of mir-1299 and its maturation miRNA regulation in osteosarcoma sample.It is preferred that the mir-1299 and its The target gene of mature miRNA regulation is C5AR1 and/or ITPRIP.

It preferably, include the primer of specific amplification mir-1299 and its maturation miRNA based on quantifying PCR method, into one Preferably, the primer sequence of specific amplification miR-1299 is SEQ ID NO 3 to step；Include based on probing procedure and mir- The probe of the nucleic acid array hybridizing of 1299 and its maturation miRNA；Immunologic detection method includes and mir-1299 and its maturation miRNA Controlling gene expresses the antibody that protein-specific combines, further preferably in conjunction with C5AR1 and/or ITPRIP protein-specific Antibody.

Further, prevention and treatment osteosarcoma reagent includes transcription and/or the promotion mir- for raising mir-1299 and its maturation miRNA The active reagent of 1299 and its maturation miRNA.

Preferably, using the acquired technology of microRNA function and/or gene specific miR Mimics skill based on RNA Art raises the transcription of mir-1299 and its maturation miRNA and/or promotes the activity of mir-1299 and its maturation miRNA.It is preferred that people Work synthesizes the short hairpin RNA of miR-1299 or raises mir-1299 and its maturation miRNA by regulation promoter.

The purpose of the present invention is to provide the target genes of mir-1299 and its maturation miRNA in preparation diagnosis and/or prevention and treatment Application in osteosarcoma preparation.Preferably, target gene is C5AR1 or ITPRIP.It is furthermore preferred that target gene is ITPRIP.

Further, osteosarcoma is metastatic bone sarcoma.

Further, diagnosis osteosarcoma preparation detects bone and flesh using PCR kit for fluorescence quantitative, genetic chip, immunization method The expression of C5AR1 and/or ITPRIP3 gene in tumor peripheral blood in patients.Preferably, contain in the PCR kit for fluorescence quantitative There is the primer of specific amplification C5AR1 and/or ITPRIP gene；Include in the genetic chip and C5AR1 and/or ITPRIP The probe of the nucleic acid array hybridizing of gene.It is furthermore preferred that in PCR kit for fluorescence quantitative containing specific detection C5AR1 and/or The upstream primer and downstream primer of ITPRIP gene, the upstream primer sequence for expanding C5AR1 is SEQ ID NO 4, downstream primer Sequence is SEQ ID NO 5；The upstream primer sequence for expanding ITPRIP gene is SEQ ID NO 6, downstream primer sequence SEQ ID NO 7。

Further, the diagnostic preparation of osteosarcoma using immunization method detection osteosarcoma peripheral blood in C5AR1 and/or The expression product of ITPRIP gene.Preferably, immunization method is that ELISA is detected and/or colloidal gold detects.Further, it detects The ELISA method of C5AR1 and/or ITPRIP albumen is to use ELISA detection kit.Antibody in kit can be used commercially available C5AR1 and/or ITPRIP monoclonal antibody or polyclonal antibody.Further, kit includes: coating C5AR1 and/or ITPRIP The solid phase carrier of antibody, enzyme labelled antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction are whole Only liquid etc..

Further, the colloidal gold method for detecting C5AR1 and/or ITPRIP albumen is using detection kit, and antibody can be used Commercially available C5AR1 and/or ITPRIP monoclonal antibody or polyclonal antibody.Further, gold-immunochromatographyreagent reagent for assay box uses colloid Golden immunochromatography technique or colloidal gold percolation.Further, the detection zone (T) on gold-immunochromatographyreagent reagent for assay box nitrocellulose filter Specking has anti-C5AR1 and/or ITPRIP antibody, quality control region (C) specking to have Immunoglobulin IgG.

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

The purpose of the present invention is to provide a kind of preparations for preventing and treating osteosarcoma, in preparation containing inhibit C5AR1 and/or The reagent or compound of transcription or the expression of ITPRIP gene.Further, the table of C5AR1 and/or ITPRIP gene, gene The high expression in osteosarcoma transfer group up to product.

The expression of the known suppressor of those skilled in the art and its expression product usually can be using one of following methods And/or several: the suppressor for activating the gene, the albumen that the inhibition gene is added, addition inhibit the chemicals of the gene (such as SiRNA etc.), the molecule for promoting protein degradation or blocks protein maturation, albumen of the removal activation gene is added etc..

The purpose of the present invention is to provide a kind of prevention and treatment osteosarcoma reagents, and the reagent includes:

(a) it raises the transcription of mir-1299 and its maturation miRNA and/or promotes the work of mir-1299 and its maturation miRNA The reagent of property；

(b) receptible carrier in pharmacy.

Preferably, using the acquired technology of microRNA function and/or gene specific miR Mimics skill based on RNA Art raises the transcription of mir-1299 and its maturation miRNA and/or promotes the activity of mir-1299 and its maturation miRNA.It is preferred that people Work synthesizes the short hairpin RNA (short hairpin RNA, shRNA) of miR-1299 or raises mir- by regulation promoter The expression of 1299 and its maturation miRNA.

Further, the osteosarcoma is metastatic bone sarcoma.

The purpose of the present invention is to provide a kind of osteosarcoma diagnostic reagent, osteosarcoma diagnostic reagent is able to detect osteosarcoma sample In this transcription of mir-1299 and its maturation miRNA or immunologic detection method detect in osteosarcoma sample mir-1299 and its at The expression of the target gene of ripe miRNA regulation.

Further, osteosarcoma diagnostic reagent is based on high-flux sequence method and/or is based on quantifying PCR method and/or is based on Probing procedure detects the transcription of mir-1299 and its maturation miRNA in osteosarcoma sample or detects bone and flesh based on immunization method The expression of the target gene of mir-1299 and its maturation miRNA regulation in tumor sample, it is preferred to use northern hybridizing method, MiRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, bead-based flow-cytometry detect bone The transcription of mir-1299 and its maturation miRNA in sarcoma sample；Osteosarcoma sample is detected using ELISA and/or colloidal gold strip The expression of the target gene of mir-1299 and its maturation miRNA regulation in this.It is preferred that the mir-1299 and its maturation miRNA The target gene of regulation is C5AR1 and/or ITPRIP, in ELISA and/or colloidal gold strip containing with C5AR1 and/or The antibody that ITPRIP protein-specific combines.

The purpose of the present invention is to provide the preparations of above-mentioned prevention and treatment osteosarcoma in preparation clinical treatment of osteosarcoma drug or reagent Application.

Definition:

The method for detecting the expression of miRNA at this stage mainly includes based on high throughput sequencing technologies, based on nucleotide The miRNA detection method of hybridization and based on PCR.MiRNA detection method based on probe hybridization technique is a kind of direct Detection Method, It does not need to expand sample rna in advance, including northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analysis skill The technologies such as art, RAKE method, in situ hybridization, bead-based flow-cytometry.

(1) Northern hybridizes

Also known as Northern blot is most classic detection eucaryote RNA size, estimates the experimental method of its abundance.Base Present principles are as follows: fixing miRNA sample on carrier (such as silicon wafer, microballoon or film) first, then miscellaneous with the probe by label It hands over, carries out signal detection after washing extra hybridization probe；It can also be first fixed complementary with target miRNA sequence on carrier Then DNA probe hybridizes with the sample miRNA by label, then carries out signal detection.The method of signal label includes isotope Label, fluorescent marker and nano gold mark etc..

(2) miRNA chip of expression spectrum

Principle is equally using the target molecule on label probe detection solid support.Pass through miRNA in design chips Gene and internalcontrol sequence, can Accurate Analysis go out the expression of corresponding miRNA in sample.Genetic chip has the excellent of high throughput Point can once detect whole expression of several hundred a genes in same sample.The liquid-phase chip that Luminex company develops (Liquid chip) is also known as multifunctional suspending dot matrix (Multi analyte suspension array, MASA), is out new Generation biochip technology.Liquid-phase chip system is made of many spherulas for main matrix, is fixed with not on every kind of spherula With probe molecule, in order to distinguish different probes, each for label probe sphere matrix all have one it is unique Color numbers constitute liquid-phase chip system as soon as these spherulas are suspended in a liquid-phase system.The system can be to same Multiple and different molecules in one trace sample carry out quick qualitative and quantitative analysis simultaneously, and this detection technique is referred to as FMAP (Flexible multianalyte profiling) technology.Molecule hybridization carries out in aaerosol solution, detects speed pole Fastly.

(3) ribozyme protection analytical technology (RPA)

The detection of miRNA can also protect analytical technology using ribozyme, and the probe marked and RNA sample to be measured are mixed Close, hybridize after thermal denaturation, non-hybridized RNA and the single-chain nucleic acid enzymic digestion of extra probe, after heat inactivation nuclease purifying by Probe, colour developing is separated by electrophoresis finally by denaturation PAGE in the RNA molecule of protection.This new method based on solution hybridization is simple Quickly, high sensitivity, but be also only used for analyzing known miRNA.

(4) RAKE method

RAKE method (RNA primed array based Klenow emzyme) is the base in miRNA microarray The Klenow segment of DNA polymerase i, the method for hybridizing miRNA with fixed DNA probe are utilized on plinth.RAKE can be sensitive MiRNA is specifically detected, suitable for largely quickly screening all known miRNA.It can be in specific cell and tumour Detect miRNA express spectra situation.Moreover, RAKE method can also be from the tissue of the paraffin embedding secured by formalin In isolate miRNA and analyze it, for from the door for achieving analysis miRNA in sample and opening hope.

(5) in situ hybridization (in situ hybridization)

Hybridization in situ technique can intuitively understand miRNA expression way, be a kind of easier of observation miRNA spatial and temporal expression Method, normal mark mode include digoxin, biotin, fluorescent marker etc..In situ hybridization (Locked on the basis of locked nucleic acid Nucleic Acid (LNA) based in situ hybridization (LNA-ISH)) it is the more probe side of current application Formula.

(6) bead-based flow-cytometry (bead-based flow cytometry)

It is a kind of liquid-phase chip technology, FCM analysis is organically combined with chip technology, had concurrently by this method The features such as flux is big, detection speed is fast, high sensitivity and specificity are good.

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

Fluorescence detection PCR instrument can be to the cumulative speed drafting dynamic changing curve of extension increasing sequence during entire PCR.Anti- Answer the initial concentration of target sequence in mixed system bigger, it is desirable that the PCR cycle number for obtaining amplified production specific output is (general Expressed with specific threshold recurring number Ct) it is fewer.Since miRNA length is only 22nt, traditional qRT-PCR is not suitable for amplification such as This short segment.There are several real time quantitative PCR methods for miRNA, such as tailing method, neck ring method now.Neck ring method is one The ideal miRNA of kind detects qRT-PCR method: special loop-stem structure primer is designed first, it is inverse as template using miRNA to be measured Transcription synthesis the first chain of cDNA, which is stem Loop primer, and stem loop structure, which is opened, substantially increases the length of cDNA Degree then carries out real-time quantitative PCR amplification by template design primer of the cDNA of synthesis.QRT-PCR has specificity high, sensitive It spends, a variety of advantages such as quickly and easily.

(8) PCR sequencing PCR

MiRNA known to major part is that discovery and identification is sequenced by cDNA clone.The method needs first to construct miRNA CDNA library, then carry out PCR amplification, amplified production is then cloned on expression vector and is sequenced.Takada develops one kind and changes Into amplification PCR cloning PCR (miRNA amplification profiling, mRAP), mRAP method first connects at the 3 ' ends of miRNA Connector, then with the reverse transcription primer reverse transcription complementary with connector.Because specific reverse transcriptase has end deoxynucleotide Transferase active, some nucleotide (mainly deoxycytidylic acid) can be connected to 3 ' ends of the cDNA chain that reverse transcription goes out.When 5 ' After the annealing of poly (C) cohesive end of end connector and cDNA chain, a pair of of general primer is added and can be realized, the PCR of cDNA is expanded Increase.It, can be directly with the expression quantity of miRNA in clone and a small amount of tissue of sequencing technologies detection due to mRAP High sensitivity.Label Sequence PCR cloning PCR is a kind of to have developed detection efficiency on the basis of serial analysis of gene expression (SAGE) technology higher MiRAGE (miRNA SAGE) PCR cloning PCR, the method can detect multiple by generating big sub-series by single sequencing reaction MiRNA, hence it is evident that improve detection efficiency.

High-flux sequence (High-throughput sequencing) is also known as next-generation sequencing technologies (next Generation sequencing) it is the change for tradition being sequenced revolution, once to hundreds of thousands to millions of DNA Molecule carries out sequencing, greatly improves sequencing efficiency.This kind of large scale sequencing technology greatly improves multiple species and loses The solution reading rate of communication breath, for the sequence information for obtaining all miRNA, decryption miRNA map provides guarantee.High pass simultaneously It measures sequence to carry out the analysis of careful overall picture to the transcript profile and genome of species, so the depth that is otherwise known as Degree sequencing (deep sequencing).The representative of high-flux sequence platform is 454 sequenator (Roch of Roche Holding Ag (Roche) GSFLX sequencer), the Solexa genome analysis instrument (Illumina Genome Analyzer) of Illumina company With the SOLiD sequenator (ABI SOLiD sequencer) of ABI.

Immunologic detection method is carried out to determinand quantitative or qualitative using a kind of antibody or Multiple Antibodies as analytical reagent The detection method of analysis.The basic principle is that the interaction between antibody and antigen.To improve the quick of antigen and antibody test Perception, the substance that will easily be shown in known antibodies or antigenic mark, by detecting marker, reflection whether there is or not antigen-antibody reaction, To measure micro antigen or antibody indirectly.Common marker has enzyme, fluorescein, radioactive isotope, colloidal gold and electricity Sub- dense matter etc..The specific reaction for showing that object is carried out on this antigen or antibody label is known as immunolabelling technique (immunolabelling technique).Immunoassay technology most widely used at present mainly has: enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA), colloidal gold immunity chromatography etc..

Enzyme-linked immunosorbent assay principle is to combine antigen or antibody and substrate (enzyme), it is made to keep immune response and enzyme Activity.The antigen or antibody of label and the ligand binding that is coated on solid phase carrier, then it is allowed to and corresponding colorless substrate It acts on and display color, determines result according to the range estimation of colour developing depth degree or with microplate reader measurement OD value.

Colloidal gold strip is generally made of sample pad, gold-labelled pad, chromatographic film, four part of water absorption pad.Chromatographic material has nitre Change tunica fibrosa (NC), polyester film, nylon membrane and pvdf membrane etc. need may be selected the film of different requirements, wherein NC film according to test It is the most commonly used, it can determine the need for activating according to test concrete condition before or handle, in most cases without processing, i.e., It can be used directly.By gold mark protein solution even application in gold-labelled pad, dry at room temperature spare.NC film can capture a certain amount of Coating (antibody) and secondary antibody as detection line and nature controlling line.Finally sample pad, gold-labelled pad, NC film and blotting paper are successively fixed In PVC board, test strips.

The acquired technology of microRNA function based on RNA be by precursor substance that exogenous supplement miRNAs is synthesized come Increase the level of miRNAs.For example, can the artificial synthesized and consistent short hair clip sample RNA (short of endogenous miRNA sequence Hairpin RNA, shRNA), promoter is done by polymerase II or III, is that carrier transfects cell with virus, by Dicer enzyme modification It is loaded into RISC afterwards to play a role, is equivalent to the level for increasing pre-miRNA, function and effect are stable and lasting.

This technique avoids the nonspecific actions of miRNA and gene for gene specific miR Mimics technology.This people The specific oligonucleotide chain of the combination complementary with 3 ' UTR of target gene of work synthesis, after transcription identical with miRNA can be played Adjustment effect.

Include in the pharmacy of pharmaceutical composition of the invention the carrier permitted be the load usually utilized in preparation Body, which includes lactose (lactose), dextrose (dextrose), sucrose (sucrose), sorbierite (sorbitol), sweet Reveal alcohol (mannitol), starch, acacia gum, calcium phosphate, alginates (alginate), gel (gelatin), calcium silicates, Microcrystalline cellulose, polyvinylpyrrolidone (polyvinylpyrrolidone), cellulose (cellulose), water, syrup, first Base cellulose (methyl cellulose), methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxy benzene Propyl formate (propyl hydroxybenzoate), talcum, magnesium stearate (stearic acid magnesium) and mineral Oily (mineral oil) etc., but it is not limited to this.

Pharmaceutical composition of the invention can also include lubricant, wetting agent, sweetener, perfume (or spice) in addition to the above ingredients Taste agent, emulsifier, suspending agent, preservative etc..The suitable carrier and preparation permitted in pharmacy is recorded in Lei Mingdengshi in detail Pharmacy pandect.

Pharmaceutical composition of the invention can by it is oral or it is non-oral be administered, when as non-oral administration, can lead to Cross intravenous injection, intranasal injection, locally injecting, intraventricular injection, spinal cavity injection is subcutaneously injected, intraperitoneal injection, percutaneously The modes such as administration are administered.

The suitable dosage of pharmaceutical composition of the invention according to preparation ways, administration mode, patient year The factor of age, weight, gender, morbid state, food, administration time, administration route, drainage rate and draw property etc and can be with Carry out a variety of prescriptions, in general, skilled practitioner can be easily determined by and prescription to it is desired treatment or prevention effectively to Pharmaceutical quantities.

Person of an ordinary skill in the technical field can be easy to implement pharmaceutical composition of the invention according to the present invention Method, carried out using carrier receptible in pharmacy and/or excipient it is formulation, so as in the form of unit dose It is prepared by preparation or interior be mounted in multicapacity container.At this point, dosage form be solution in oiliness or aqueous medium, suspension or Emulsion form is either also possible to extract, powder agent, granule, tablet or capsule form, can also include dispersion Agent or stabilizer.

Detailed description of the invention

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

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

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

Specific embodiment

Present invention will be further explained below with reference to specific examples, for explaining only the invention, and should not be understood as to this The limitation of invention.It will be understood by those skilled in the art that: without departing from the principle and spirit of the present invention may be used To carry out a variety of change, modification, replacement and modification to these embodiments, the scope of the present invention is limited by claim and its equivalent It is fixed.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to item proposed by manufacturer Part examinations.

The collection of 1 sample of embodiment and Total RNAs extraction

5 metastatic bone sarcoma patients and 5 normal healthy controls.Metastatic bone sarcoma patients group 5 and 5 people of control group want Ask at least 12h on an empty stomach, at room temperature in next morning 7:00~8:00, it is anti-in ethylenediamine tetra-acetic acid (EDTA) to extract 10ml venous blood Solidifying pipe, extracts peripheral blood mononuclear cells PBMCs, is added 1ml Trizol reagent (Invitrogen company), mixes well ,- 80 DEG C of preservation samples, to be extracted for RNA.All blood samples and pathological examination are answered true and reliable, and research is through Ethics Committee batch Standard, patient's informed consent.

RNA extraction standard: RNA purity: OD260/280≤1.8,28S/18S≤1；RNA integrality: Zhi≤7.0 RIN. RNA integrality detection method: Agilent 2100 (6000 Nano kit of RNA), (Ago-Gel is dense for agarose gel electrophoresis Degree: 1% agarose gel；Voltage: 5V/cm；Time: 20min).

The sequencing of embodiment 2 and data analysis

Sequencing: with llumina Hiseq2500/Miseq second generation high throughput sequencing technologies to mRNA and miRNA into Row sequencing obtains final data by the processes such as removing connector, going low quality, depollute complete the processing of data.

T-test is carried out after carrying out background correction to miRNA and mRNA initial data by transcript profile Data Analysis Software P value is obtained, is then examined using Fisher and merges P value, screens differential expression miRNA and mRNA.P value < 0.01 is set, is sieved altogether The miRNA of 12 differential expressions is selected, wherein miRNA 11 of expression downward.P value < 0.05 is set in analytic process, The mRNA of 425 differential expressions is filtered out altogether, wherein gene 324 of expression up-regulation.Using include RNA22, These algorithm forecasted variances of miRanda, miRDB, miRWalk, PICTAR2 and Targetscan express the target gene of miRNA, Choose the miRNA's for the differential expression that >=4 algorithms predict the target gene come, and have verified that in miRWalk database lookup Then gene negatively correlated with miRNA expression in all target genes and mRNA sequencing result is carried out confluence analysis by target gene, I 105 miRNA- target gene relationships pair are obtained, pass through prediction obtain up-regulation miRNA (miR-1299) miRNA- target base Because of relationship pair, target gene C5AR1, ITPRIP etc..

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

The acquisition of 1 sample:

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

2 Total RNAs extractions:

The processing for removing Rnase of related experiment article:

1. 180 DEG C of high temperature dry 2 by soaking, 120 DEG C of high pressure 20min is rinsed with DEPC before the application of all glasswares Hour or more.

2. will be needed before plastic ware (such as: EP pipe/pipette tips) use with 0.1%DEPC water enchroachment (invasion) bubble overnight, after drain liquid, 120 DEG C of high pressure 20min, oven is dried spare.

Leucocyte separation

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

(2) isometric sterile PB S is added to be sufficiently mixed in peripheral blood, forms cell suspension；

(3) 4m1 lymphocyte separation medium is added in another centrifuge tube；

(4) draw 4m1 cell suspension be gently added to along tube wall lymphocyte separation medium surface (pay attention to not with lymphocyte Separating liquid mixing).It is centrifuged 1500rpm 20min；

(5) boundary layer (tunica albuginea) is gently sucked out with suction pipe to enter in another centrifuge tube.Sterile cold PBS is washed 2 times, is washed for last 1 time Washing can move into cell suspension in EP pipe, and supernatant is removed in centrifugation, for extracting RNA.

RNA is extracted

(1) first add lml Trizol in EP pipe, if freeze-stored cell is directly added into Trizol, be not required to thaw, piping and druming cracking After be stored at room temperature 5-l0min；

(2) 0.2m1 chloroform is added, acutely shakes 15s, is stored at room temperature 2-3min, 12000 turns of centrifugation 15min at 4 DEG C；

(3) the supernatant water 600ul that makes an appointment carefully is sucked out and moves into another centrifuge tube (being careful not to be extracted into albumin layer), is added 500:1 isopropanol, is mixed by inversion, and is stored at room temperature 10min；

(4) 4 DEG C of 1 2000g are centrifuged l 0min, abandon supernatant, bottom visible white substance；

(5) the rotation washing of the cold ethyl alcohol of lml 75% is added, cleans isopropanol；

(6) 4 DEG C of 7500g are centrifuged 5min, and dry in the air 5-l0min after removal ethyl alcohol, translucent, with the dissolution of 20u1DEPC water RNA.3u1RNA sample is taken, the electrophoresis in 1.5% Ago-Gel；Lu1RNA sample in UV spectrophotometer measuring concentration, It is considered as RNA sample qualification in 1.8-2.0 with A260/280.

3 reverse transcriptions

MRNA reverse transcription:

It takes 1 μ g total serum IgE as template ribonucleic acid, usesIII Reverse Transcriptase (invitrogen, article No. 18080-044) carries out cDNA reverse transcription, and experimental implementation is carried out by product description.The cDNA of acquisition It is spare that -20 DEG C of refrigerators are put in preservation.

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；Aqua sterilisa filling-in is to 20 μ l.ABI After 37 DEG C of heat preservation 60min keep reverse transcription reaction complete in 9700 type PCR instruments, 95 DEG C of 5min terminate reaction.80 μ l are added Nuclease-free H₂O is diluted to 100 μ l, and to be stored in -20 DEG C of refrigerators spare.

4 quantitative fluorescent PCRs

Design of primers:

C5AR1 primer (NM_001736.3):

Forward primer: 5 '-GTAAGTAATGATACAGAGG-3 ' SEQ ID NO 4

Reverse primer: 5 '-GTCAATATCCTGGTTATG-3 ' SEQ ID NO 5

Amplified production length 102bp.

ITPRIP primer (NM_033397.3):

Forward primer: 5 '-CTATGATGTAGGTGCTATTCT-3 ' SEQ ID NO 6

Reverse primer: 5 '-ACAGTAGGTAAGTCAGTTG-3 ' SEQ ID NO 7

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 H2O	-	Filling-in is to 25 μ l

3 parallel tube reactions are arranged in the detection of expression of mRNAs every time, using actin as internal reference.

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

The preparation of the RT-PCR system of miRNA:

3 parallel tube reactions are arranged in the detection of expression of miRNAs every time, using snRNA U6 as internal reference.

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 inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the relative quantification formula of qRT-PCR: 2- Δ Ct × 100% compares expression water of miR-1299, C5AR1, ITPRIP gene in metastatic bone sarcoma group and normal group It is flat.As the result is shown: qRT-PCR stable amplification result, wherein miR-1299 is significantly lower than in metastatic bone sarcoma group expression Normal group is approximately 1/6th (being specifically shown in Fig. 1) of control, and the table of C5AR1, ITPRIP in metastatic bone sarcoma group It is above normal control up to level, C5AR1 expression in metastatic bone sarcoma group is about 1.7 times of control group and (is specifically shown in Fig. 2), ITPRIP expression in metastatic bone sarcoma group is about 2.6 times (being specifically shown in Fig. 3) of control group, and result above is tested The result of the confluence analysis of high-throughput transcript profile expression data is demonstrate,proved.

Embodiment 4miR-1299 and C5AR1 and ITPRIP target gene relationship are verified

One, material prepares:

(1) Specimen origin

Human osteosarcoma cell line MG-63 is purchased from Chinese Academy of Sciences Shanghai cell research institute.

(2) main agents

LipofectamineTM2000 Transfection Reagent(Invitrogen)。

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

MiR-1299 sequence issues Synesis Company, asks its chemical synthesis miR-1299mimics and non specific control.

(3) main solution

1, cell culture fluid

+ 10% standard fetal calf serum of DMEM culture medium.

2, PBS (balanced salt solution)

8g NaCl is dissolved in 800m1 distilled water, 0.25g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 are used HCl adjusts the pH value of solution to 7.4, and water is added to be settled to 1L, high pressure sterilization, room temperature preservation.

3,0.25% tryptic digestive juice

0.25g trypsase is added in 100m1 deionized water, filter filtration sterilization, dispenses spare.

Two, experimental method

1, cell passes on

(1) culture solution original in the culture bottle for covering with cell is discarded, 0.25% trypsin solution 1m1, covering is added Cellular layer, bottleneck disinfection, covers；

(2) cellular change is observed under inverted microscope, over time, former adherent cell gradually tends to be round, Cytoplasm retraction, space between cells increase, discard pancreatin in also non-levitating, and the culture that 5ml contains 10% fetal calf serum is added Liquid terminates digestion；

(3) cell count: taking above-mentioned cell suspension 0.5mI, instills in blood cell counting plate after appropriate dilution, by leucocyte Counting method number quadrangle four big lattice inner cell sum, when counting, only count nucleus and the complete cell of cytoplasm, cell in heaps It is calculated by a cell, by the total number of cells in 4 block plaids by following formula scales at the cell number in every milliliter of cell suspension: Big lattice total number of cells/4 × 10 total number of cells/ml=4⁴× extension rate；

(4) according to cell counts, every milliliter is further diluted to containing 3 × 10 with DMEM complete culture solution⁵A cell Concentration is sub-packed in culture bottle (every bottle of 8m1/), is placed in 37 DEG C, 5%CO₂It is cultivated in incubator.2.miRNA is transiently transfected

It is transiently transfected, is operated according to Lipofectamin using cationic-liposome method^TM2000 reagent specifications into Row.For 24 hours by the good cell inoculation of growth conditions into 12 orifice plates before transfection, cell count about 2 × 10⁴, routine culture to turn The dye same day, cell fusion degree are tested when being 50-60%.20nM/40nM/80nM miRNA mimic is added to 100u1 It is soft to mix in DMEM culture medium；Separately 2u1 Lipofectamin is diluted with 100u1DMEM culture medium^TM2000 liposomes, softly It mixes, is incubated at room temperature 5min；DMEM- liposome and DMEM-miRNAs are mixed, 20min is incubated at room temperature, it is compound to form transfection Object；Then said mixture is added in cell culture medium, is mixed gently, replace complete medium after cultivating 6h.Wherein, non-spy Anisotropic sequence is as negative control.Cell total rna progress next step experiment is extracted after cultivating 48h.

3. experimental result:

MiR-1299mimics is transfected into cell line of human osteosarcoma MG-63, cell total rna, blank are extracted after 48h Control is the cell line of human osteosarcoma cell for not being transferred to miRNA, and non-specific sequences are as negative control.Quantitative PCR detection target The horizontal variation of the mRNA of gene C 5AR1, ITPRIP.The expression of 2 genes has the downward of different level as the result is shown, shows C5AR1, ITPRIP are the target target genes of miR-1299, and wherein ITPRIP gene deregulation becomes apparent.

Embodiment 5 transfects influence of the miR-1299 to human osteosarcoma cell proliferation

One, experimental material

Human osteosarcoma cell line MG-63 is purchased from Chinese Academy of Sciences Shanghai cell research institute.

Sequent synthesis: miR-1299 sequence is issued into Shanghai Ji Ma company, miR-1299mimics, anti-are synthesized by it miR-1299.Nucleotide sequence 5 ' end modified with FAM, can fluoresced green, can be in fluorescence microscope after transfecting into cell Lower observation.

MiR-1299mimics sequence:

5’-UUCUGGAAUUCUGUGUGAGGGA/CCUCACACAGAAUUCCAGAAUU-3’ (SEQ ID NO 8)

Anti-miR-1299 sequence:

5’-UCCCUCACACAGAAUUCCAGAA-3’(SEQ ID NO 9)

Two, experimental method

1.Transwell migration experiment detection cell migration ability

1) using 24 orifice plate of the cell Transwell of the poly- carbon ester film in the aperture containing 8um, in the lower room of the cell Transwell It is previously added DMEM culture solution of the 600ul containing 10%FBS, 37 DEG C balance 1 hour.

2) each group logarithmic phase cell is collected, is rinsed 3 times with PBS (pH7.4), 1640 culture solution of RPMI containing 10%FBS Group of cells is resuspended, it is 5 105/ml that cell density is adjusted after counting, takes the hole 100ul/ that upper chamber is added.It is placed in 5%CO2,37 It is incubated for 48 hours in DEG C constant incubator.

3) cell Transwell is taken out, the cell in filter membrane upper chamber face is struck off with cotton swab.

4) it is fixed after PBS rinsing with 4% paraformaldehyde, violet staining.

5) cell number in room face under filter membrane is counted under 200 power microscopes, each hole counts the cell in 5 visuals field immediately Number.Its average value is taken, the transfer ability of group of cells is indicated with the number of migrating cell.

2.Transwell Matrigel detects cell invasion ability

1) melt Matrigel on ice on the day before mentioning, prepare Tip, the liquid-transfering gun, the cell Transwell of pre-cooling.

2) by extracellular matrix Matrigel, the poly- carbon ester film of the cell Transwell is added in the ratio of 50ul/cm2 Upper surface, placing 30 minutes for 37 DEG C makes its plastic.

3) DMEM culture solution of the 600ul containing 10%FBS, 37 DEG C of balances are previously added in the lower room of the cell Transwell 1 hour.

4) each group logarithmic phase cell is collected, is rinsed 3 times with PBS (pH7.4), 1640 culture solution of RPMI containing 10%FBS Group of cells is resuspended, it is 5 105/ml that cell density is adjusted after counting, takes the hole 100ul/ that upper chamber is added.It is placed in 5%CO2,37 It is incubated for 48 hours in DEG C constant incubator.

5) cell Transwell is taken out, the cell in filter membrane upper chamber face is struck off with cotton swab.

6) it is fixed after PBS rinsing with 4% paraformaldehyde, violet staining.

7) cell number in room face under filter membrane is counted under 200 power microscopes, each hole counts the cell in 5 visuals field immediately Number.Its average value is taken, the invasive ability of group of cells is indicated 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 is successfully transfected into osteosarcoma cell MG-63, and the cell with fluorescent grain accounts for total number of cells 85%-90% Left and right, display transfect successfully.

MiR-1299 is analyzed with the migration of the cell Transwell and Matrigel to cell MG-63 migration and invasive ability Influence, the results show that the ability of MG-63 cell migration and invasion of transfection miR-1299mimics has obviously compared with control group Decline, meanwhile, the ability for transfecting MG-63 cell migration and the invasion of anti-miR-1299 has compared with control group significantly to be mentioned It is high.

Although having referred to various preferred embodiments describes the present invention, it will be appreciated by those skilled in the art that can carry out each Kind variation, and available equivalents substitute its component without departing from base region of the invention.Come in addition, many changes can be carried out Specific condition or material is set to be suitable for the teachings of the present invention without departing from its base region.

Claims (11)

1. Application of the 1.mir-1299 and its maturation miRNA in preparation diagnosis and/or prevention and treatment osteosarcoma reagent.
2. 2. application according to claim 1, which is characterized in that osteosarcoma is metastatic bone sarcoma.
3. 3. application according to claim 1, which is characterized in that prevention and treatment osteosarcoma reagent include up-regulation mir-1299 and its at The transcription of ripe miRNA and/or the active reagent for promoting mir-1299 and its maturation miRNA.
4. 4. application according to claim 3, which is characterized in that prevention and treatment osteosarcoma reagent includes the microRNA based on RNA The acquired technology of function and/or gene specific miR Mimics technology up-regulation mir-1299 and its maturation miRNA transcription with/ Or promote the activity of mir-1299 and its maturation miRNA.
5. 5. application according to claim 4, which is characterized in that use the short hairpin RNA or logical of artificial synthesized miR-1299 Cross the expression of regulation promoter up-regulation mir-1299 and its maturation miRNA.
6. 6. application according to claim 1, which is characterized in that diagnosis osteosarcoma reagent includes being based on high-flux sequence method And/or based on quantifying PCR method and/or based on mir-1299 and its maturation in probing procedure detection osteosarcoma sample The transcription of miRNA or the target base regulated and controled based on mir-1299 and its maturation miRNA in immunologic detection method detection osteosarcoma sample The expression of cause.
7. 7. application according to claim 6, which is characterized in that use northern hybridizing method, miRNA express spectra core Piece, ribozyme protection analytical technology, RAKE method, in situ hybridization, bead-based flow-cytometry detect mir- in osteosarcoma sample The transcription of 1299 and its maturation miRNA；Using ELISA and/or colloidal gold strip detection osteosarcoma sample in mir-1299 and The expression of the target gene of its maturation miRNA regulation.
8. 8. application according to claim 6, which is characterized in that the target gene of regulation is C5AR1 and/or ITPRIP gene.
9. Application of the target gene of 9.mir-1299 and its maturation miRNA in preparation diagnosis osteosarcoma preparation.
10. 10. application according to claim 9, which is characterized in that target gene is C5AR1 and/or ITPRIP gene.
11. 11. application according to claim 9, which is characterized in that diagnosis osteosarcoma preparation uses PCR kit for fluorescence quantitative Or genetic chip or immunization method detect the expression of C5AR1 and/or ITPRIP gene in peripheral blood.