CN105597109B

CN105597109B - The diagnosis and treatment molecular labeling of primary osteosarcoma

Info

Publication number: CN105597109B
Application number: CN201610003253.3A
Authority: CN
Inventors: 谢琳; 杨祚璋; 廖冶丹; 任明艳; 袁中琴; 余顺玲
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-01-04
Filing date: 2016-01-04
Publication date: 2019-07-23
Anticipated expiration: 2036-01-04
Also published as: CN105597109A

Abstract

The present invention relates to the diagnosis and treatment molecular labelings of primary osteosarcoma, more particularly relate to the new application of miR-3688-3p and its target gene in diagnosis and treatment osteosarcoma.Invention carries out high-flux sequence to primary Patients with Osteosarcoma and normal control, by bioinformatics confluence analysis, filters out candidate miR-3688-3p and its target gene CSRNP1, PFKFB3 and SLC1A3.Further experiment results show that miR-3688-3p and its target gene are closely related with osteosarcoma, can be used for the clinical diagnosis and prevention detection of osteosarcoma, have good practical application value.

Description

The diagnosis and treatment molecular labeling of primary osteosarcoma

Technical field

The present invention relates to molecular biology fields, are specifically related to the diagnosis and treatment molecular labeling of primary osteosarcoma, more specifically It is related to the new application of miR-3688-3p and its target gene in diagnosis and treatment osteosarcoma.

Background technique

Osteosarcoma (osteosarcoma) is the most common primary malignant tumor of teenager and the treatment of orthopaedics educational circles One of difficult point.It is shown according to China's Statistical data, osteosarcoma disease incidence account for the first in primary malignant tumor.Although bone in recent years Sarcoma the cause of disease, occurrence and development, in terms of have some progress, but very slow, the specific pathogenesis that is in progress It is still unclear.Next lacks effective target of osteosarcoma diagnosing and treating, further finds molecular biomarker, will have weight The theory and clinical value wanted.Simultaneously as can obtaining osteosarcoma serodiagnosis marker, by the diagnosis to osteosarcoma and control Treatment brings far-reaching influence.

MiRNA is a kind of endogenous non-coding small molecule, regulates and controls its expression by specific binding target gene.With target There are mainly two types of the typical effect modes of mRNA.In most cases, 3 ' of the single-stranded miRNA and said target mrna in compound The not fully complementary pairing of UTR, blocks the translation of target gene, to adjust gene expression.Another mode of action and siRNA class Seemingly, when miRNA and mRNA complete complementary match clock synchronization, Ago2 albumen directly results in its degradation by cutting mRNA, realizes that gene is heavy It is silent.In short, being presently believed to miRNA, and target gene acts on and miRNA is related with the pairing degree of target gene in which way. When miRNA and target gene match incomplete, miRNA is just to inhibit the expression of target gene to play a role；MiRNA and purpose base When complete because of certain section of sequence pairing, it is possible to cause target gene to be broken in complementary region and lead to gene silencing.In addition, miRNAs The DNA methylation for sometimes also leading to histidine modification and promoter region, to influence the expression of target gene.

Invention is based on high-flux sequence method, and 5 primary Patients with Osteosarcoma and 10 normal persons are sequenced, and obtains The expression data of its miRNA and mRNA, and then bioinformatic analysis is carried out, it chooses standby miRNA and mRNA and carries out molecule life Object verifying and target checking, the results show that miR-3688-3p provided by the invention and its target gene and the close phase of osteosarcoma It closes, can be used for the clinical diagnosis and prevention detection of osteosarcoma, there is good practical application value.

Summary of the invention

The purpose of the present invention is to provide a kind of osteosarcoma diagnostic reagent, the osteosarcoma diagnostic reagent is able to detect bone and flesh The transcription of miR-3688-3p or immunologic detection method detect the target base of miR-3688-3p regulation in osteosarcoma sample in tumor sample The expression of cause.

The sequence of miR-3688-3p is shown in sequence table SEQ ID NO 1.MiR-3688-3p precursor be mir-3688-1 and Mir-3688-2 sequence is shown in sequence table SEQ ID NO 2 and SEQ ID NO 3.

Further, the osteosarcoma is primary osteosarcoma.

Further, the osteosarcoma diagnostic reagent be based on high-flux sequence method and/or based on quantifying PCR method and/or The transcription of miR-3688-3p in osteosarcoma sample is detected based on probing procedure or is based on immunization method detection osteosarcoma sample The expression of the target gene of middle miR-3688-3p regulation, it is preferred to use northern hybridizing method, miRNA chip of expression spectrum, Ribozyme protects analytical technology, RAKE method, in situ hybridization, bead-based flow-cytometry to detect miR- in osteosarcoma sample The transcription of 3688-3p；Using the target base of miR-3688-3p regulation in ELISA and/or colloidal gold strip detection osteosarcoma sample The expression of cause.It is preferred that the target gene of the miR-3688-3p regulation is CSRNP1 and/or PFKFB3 and/or SLC1A3 egg It is white, contain in conjunction with CSRNP1 and/or PFKFB3 and/or SLC1A3 protein-specific in ELISA and/or colloidal gold strip Antibody.

The purpose of the present invention is to provide application of the miR-3688-3p in preparation diagnosis osteosarcoma reagent.

Further, the osteosarcoma is primary osteosarcoma.

Further, the diagnosis osteosarcoma reagent includes based on high-flux sequence method and/or being based on quantifying PCR method And/or the transcription of miR-3688-3p in osteosarcoma sample is detected based on probing procedure or is based on immunologic detection method detection The expression for the target gene that miR-3688-3p regulates and controls in osteosarcoma 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 osteosarcoma sample The transcription of miR-3688-3p in this；Using miR-3688-3p tune in ELISA and/or colloidal gold strip detection osteosarcoma sample The expression of the target gene of control.It is preferred that the target gene of the miR-3688-3p and/or its precursor control be CSRNP1 and/or PFKFB3 and/or SLC1A3.

Preferably, it is described based on quantifying PCR method include specific amplification miR-3688-3p primer, it is further excellent Choosing, the primer sequence of specific amplification miR-3688-3p are SEQ ID NO 10；It is described based on probing procedure include with The probe of the nucleic acid array hybridizing of miR-3688-3p；The immunologic detection method includes expressing with miR-3688-3p controlling gene The antibody that protein-specific combines, further preferably in conjunction with CSRNP1 and/or PFKFB3 and/or SLC1A3 protein-specific Antibody.

The purpose of the present invention is to provide application of the target gene of miR-3688-3p in preparation diagnosis osteosarcoma preparation. Preferably, target gene is CSRNP1 and/or PFKFB3 and/or SLC1A3.

Further, the osteosarcoma is primary osteosarcoma.

Further, the diagnosis osteosarcoma preparation is using PCR kit for fluorescence quantitative, genetic chip, immunization method detection The expression of CSRNP1 and/or PFKFB3 and/or SLC1A3 gene in Patients with Osteosarcoma peripheral blood.Preferably, the fluorescence is fixed Measure the primer containing specific amplification CSRNP1 and/or PFKFB3 and/or SLC1A3 gene in PCR kit；The gene It include the probe of the nucleic acid array hybridizing with CSRNP1 and/or PFKFB3 and/or SLC1A3 gene in chip.It is furthermore preferred that glimmering The upstream primer containing specific detection CSRNP1 and/or PFKFB3 and/or SLC1A3 gene is under in Fluorescent Quantitative PCR kit Primer is swum, the upstream primer sequence of amplification CSRNP1 gene is SEQ ID NO 4, and downstream primer sequence is SEQ ID NO5；Expand The upstream primer sequence for increasing PFKFB3 gene is SEQ ID NO 6, and downstream primer sequence is SEQ ID NO 7；Expand SLC1A3 The upstream primer sequence of gene is SEQ ID NO 8, and downstream primer sequence is SEQ ID NO 9.

Further, the diagnostic preparation of the osteosarcoma using immunization method detection osteosarcoma peripheral blood in CSRNP1 and/ Or the expression product of PFKFB3 and/or SLC1A3 gene.Preferably, the immunization method is that ELISA is detected and/or colloidal gold is examined It surveys.Further, the ELISA method of the detection CSRNP1 and/or PFKFB3 and/or SLC1A3 albumen is to detect to try using ELISA Agent box.Commercially available CSRNP1, PFKFB3, SLC1A3 monoclonal antibody or Anti-TNF-α can be used in antibody in the kit Body.Further, the kit includes: the solid phase carrier for being coated with CSRNP1, PFKFB3, SLC1A3 antibody, enzyme labelled antibody, enzyme Substrate, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid etc..

Further, the colloidal gold method of the detection CSRNP1 and/or PFKFB3 and/or SLC1A3 albumen is using detection examination Commercially available CSRNP1, PFKFB3, SLC1A3 monoclonal antibody or polyclonal antibody can be used in agent box, the antibody.Further, The gold-immunochromatographyreagent reagent for assay box uses colloidal gold immunochromatographimethod technology or colloidal gold percolation.Further, the colloid Detection zone (T) specking on golden detection kit nitrocellulose filter has anti-CSRNP1 and/or PFKFB3 and/or SLC1A3 anti- Body, quality control region (C) specking 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.

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 It isolates miRNA and analyzes it, to analyze the door that miRNA opens hope from archive sample.

(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 to use Specific threshold recurring number Ct is expressed) it is fewer.Since miRNA length is only 22nt, it is such that traditional qRT-PCR is not suitable for amplification Short segment.There are several real time quantitative PCR methods for miRNA, such as tailing method, neck ring method now.Neck ring method is a kind of Ideal miRNA detects qRT-PCR method: special loop-stem structure primer is designed first, using miRNA to be measured as template reverse transcription The first chain of cDNA is synthesized, which is stem Loop primer, and stem loop structure, which is opened, substantially increases the length of cDNA, then Real-time quantitative PCR amplification is carried out by template design primer of the cDNA of synthesis.QRT-PCR has specificity height, sensitivity good, fast A variety of advantages such as fast simple.

(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.It is high-throughput simultaneously Sequencing 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 It is sequenced (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 and 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, is adjusted after capable of playing transcription identical with miRNA Section 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 RT-PCR detects miR-3688-3p expression in primary osteosarcoma

Fig. 2 RT-PCR detects CSRNP1 expression in primary osteosarcoma

Fig. 3 RT-PCR detects PFKFB3 expression in primary osteosarcoma

Fig. 4 RT-PCR detects SLC1A3 expression in primary osteosarcoma

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 primary Patients with Osteosarcoma and 10 normal persons.Primary Patients with Osteosarcoma group (5, A1-A5, clinical information It is detailed in Table 1) and control group (10 people) requirement empty stomach at least 12h, at room temperature in next morning 7:00~8:00, extraction 10ml Venous blood extracts peripheral blood mononuclear cells PBMCs in ethylenediamine tetra-acetic acid (EDTA) anticoagulant tube, and 1ml Trizol reagent is added (Invitrogen company), mixes well, -80 DEG C of preservation samples, to extract for RNA.All blood samples and pathological examination are answered True and reliable, research is ratified through Ethics Committee, 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: mRNA and miRNA is carried out with llumina Hiseq2500/Miseq second generation high throughput sequencing technologies Sequencing obtains final data by the processes such as removing connector, going low quality, depollute complete the processing of data.

Progress t-test is obtained after carrying out background correction to miRNA and mRNA initial data by transcript profile Data Analysis Software It to P value, is then examined using Fisher and merges P value, screen differential expression miRNA and mRNA.P value < 0.01 is set, is screened altogether The miRNA of 12 differential expressions out, wherein the miRNA1 of expression up-regulation is a (miR-3688-3p), what expression was lowered MiRNA 11.P value < 0.05 is set in analytic process, the mRNA of 425 differential expressions is filtered out altogether, wherein on expression The gene of tune 324, gene 101 of expression downward.Using include RNA22, miRanda, miRDB, miRWalk, These algorithm forecasted variances of PICTAR2 and Targetscan express the target gene of miRNA, and >=4 algorithms of selection predict next Target gene, and the target gene of the miRNA for the differential expression having verified that in miRWalk database lookup, then by all target genes Confluence analysis is carried out with gene negatively correlated with miRNA expression in mRNA sequencing result, 105 miRNA- target bases are obtained in we Because of relationship pair, the miRNA- target gene relationship pair of up-regulation miRNA (miR-3688-3p) is obtained by predicting, target gene is CSRNP1、PFKFB3、SLC1A3。

3 Real-time PCR of embodiment detect osteosarcoma tissue in miR-3688-3p and CSRNP1, PFKFB3, The expression of SLC1A3 gene

The acquisition of 1 sample:

The peripheral blood of 78 primary osteosarcoma tumor patients and 53 normal healthy controls is all from hospital's (acquisition time 2013 2 months -2015 years January of year).

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, 1 2000 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 4μl；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:

CSRNP1 primer (NM_033027.3):

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

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

Amplified production length 78bp.

PFKFB3 primer (NM_004566.3):

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

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

Amplified production length 118bp.

SLC1A3 primer (NM_004172.4):

Forward primer: 5 '-CATAATCATTGTCATCATC-3 ' SEQ ID NO 8

Reverse primer: 5 '-CTTCTCTTCTCATAGTTG-3 ' SEQ ID NO 9

Amplified production length 180bp.

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, 54 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 table of miR-3688-3p, CSRNP1, PFKFB3, SLC1A3 gene in osteosarcoma group and normal group Up to level.As the result is shown: qRT-PCR stable amplification result, wherein miR-3688-3p is bright in primary osteosarcoma group expression It is aobvious to be lower than Normal group, it is approximately the one third (being specifically shown in Fig. 1) of control, and CSRNP1, PFKFB3, SLC1A3 are in primary Expression in osteosarcoma group is above normal control, and CSRNP1 expression in primary osteosarcoma group is about control group 3 times (being specifically shown in Fig. 2), PFKFB3 expression in primary osteosarcoma group is about 4.5 times of control group and (is specifically shown in figure 3), SLC1A3 expression in primary osteosarcoma group is about 8 times (being specifically shown in Fig. 4) of control group, and result above demonstrates The result of the confluence analysis of high-throughput transcript profile expression data.

4 miR-3688 of embodiment and CSRNP1, PFKFB3, SLC1A3 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)。

CSRNP1, PFKFB3, SLC1A3 design of primers (see embodiment 3):

MiR-3688-3p sequence issues Synesis Company, asks its chemical synthesis miR-3688-3p mimics and non-specificity Control.

(3) main solution

1, cell culture fluid

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

2, PBS (balanced salt solution)

8g NaCl, 0.25g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 HCl are dissolved in 800m1 distilled water The pH value of solution is adjusted to 7.4, 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 It is soft to mix in 100u1DMEM culture medium；Separately 2u1Lipofectamin is diluted with 100u1DMEM culture medium^TM2000 liposomes, It is soft to mix, it is incubated at room temperature 5min；DMEM- liposome and DMEM-miRNAs are mixed, 20min is incubated at room temperature, it is multiple to form transfection Close object；Then said mixture is added in cell culture medium, is mixed gently, replace complete medium after cultivating 6h.Wherein, non- Specific sequence is as negative control.Cell total rna progress next step experiment is extracted after cultivating 48h.

3. experimental result:

MiR-3688-3p mimics is transfected into cell line of human osteosarcoma MG-63, cell total rna is extracted after 48h, it is empty White 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 The horizontal variation of the mRNA of target gene CSRNP1, PFKFB3, SLC1A3.The expression of three genes has different level as the result is shown Downward, showing CSRNP1, PFKFB3, SLC1A3 all is the target target gene of miR-3688-3p, wherein SLC1A3 gene deregulation It is the most obvious.

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

Application of the 1.miR-3688-3p in preparation diagnosis osteosarcoma reagent, which is characterized in that the sequence of miR-3688-3p is shown in Sequence table SEQ ID NO 1.
2. 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 probing procedure detection osteosarcoma sample in miR-3688-3p transcription or Expression based on the target gene of miR-3688-3p regulation in immunologic detection method detection osteosarcoma sample.
3. application according to claim 2, 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 3688-3p；Using the target base of miR-3688-3p regulation in ELISA and/or colloidal gold strip detection osteosarcoma sample The expression of cause.
4. application according to claim 2, which is characterized in that the target gene of regulation be CSRNP1 and/or PFKFB3 and/or SLC1A3 gene.