CN105505936A - Metastasis of osteosarcoma resistant biological agent and preparation method thereof - Google Patents

Metastasis of osteosarcoma resistant biological agent and preparation method thereof Download PDF

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CN105505936A
CN105505936A CN201610068863.1A CN201610068863A CN105505936A CN 105505936 A CN105505936 A CN 105505936A CN 201610068863 A CN201610068863 A CN 201610068863A CN 105505936 A CN105505936 A CN 105505936A
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osteosarcoma
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边洋
李瑜
杨承刚
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GU'AN BOJIAN BIOTECHNOLOGY CO., LTD.
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Abstract

The invention relates to a metastasis of osteosarcoma resistant biological agent and a preparation method thereof, in particular to application of a biological agent including mir-4520-1 and mature miRNA in preparation of metastasis of osteosarcoma resistant medicaments. Metastatic osteosarcoma patients and healthy contrast people are sequenced to obtain miRNA expression data, and candidate miR-4520-3p can be screened out. A verification experiment result shows that miR-4520-3p is closely related to osteosarcoma; promotion of expression of miR-4520-3p can effectively suppress migration and invasion of osteosarcoma cells. The metastasis of osteosarcoma resistant biological agent provides a new metastasis of osteosarcoma diagnosis and treatment target clinically, and has an extremely high application value.

Description

A kind of anti-osteosarcoma transfer biotechnological formulation and application thereof
Technical field
The present invention relates to biology field, be specifically related to a kind of anti-osteosarcoma transfer biotechnological formulation and application thereof, relate to the application of biotechnological formulation in the anti-osteosarcoma transfer of preparation comprising mir-4520-1 and ripe miRNA thereof more specifically.
Background technology
MicroRNA (miRNA) is that a newfound class length is the non-coding microRNA of 22-23 Nucleotide in recent years, extensively exists in eukaryote.MiRNA is combined by or incomplete complementary pairing complete with target gene mRNA3'-UTR, causes target miRNA degraded or Translational repression to regulate and control the expression of target gene, thus affects the biological behaviours such as cell proliferation, invasion and attack, differentiation and apoptosis.Research shows that miRNA plays an important role in the developing of tumour, and this Diagnosis and Treat being found to be tumour provides new thinking.But up to the present, the report both at home and abroad about the effect of miRNA in osteosarcoma and mechanism of action is still rare.
Osteosarcoma (osteosarcoma, OSA) is a kind of high malignancy noumenal tumour being primary in medullary space.In primary bone tumor, osteosarcomatous sickness rate is in the 2nd, is only second to plasma cell myeloma.Osteosarcoma develops from mesenchymal cell system, and the osteoid tissue produced with the fusiformis stroma cell and tumour cell that can produce osteoid tissue or immature bone are for feature.Osteosarcoma is modal Primary Malignant Bone Tumor clinically, is generally apt to occur in the Children and teenager phase.Osteosarcoma accounts for the 3-4% of malignant bone tumor 30% and all pediatric tumors, and prognosis is poor, and within 5 years, survival rate is lower.Osteosarcoma clinical cure difficult point is, even if treated by amputation, many patients also can die from Lung metastases in 1 year.Osteosarcoma transfer becomes one of most significant problems faced in clinical treatment of osteosarcoma.
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 its miRNA, and then carry out bioinformatic analysis, choose standby miRNA and carry out molecular biology checking, result shows, and miR-4520-3p provided by the invention and osteosarcoma shift 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 a kind of anti-osteosarcoma transfer biotechnological formulation, comprise:
A () is raised transcribing of mir-4520-1 and ripe miRNA thereof and/or is promoted the reagent of activity of mir-4520-1 and ripe miRNA thereof;
Receptible carrier in (b) pharmaceutics.
Mir-4520-1 sequence is shown in sequence table SEQ IDNO1, and the ripe miRNA of mir-4520-1 is that the sequence of miR-4520-3p and miR-4520-5p, miR-4520-3p is shown in sequence table SEQ IDNO2, and the sequence of miR-4520-5p is shown in sequence table SEQ IDNO3.
Preferably, the activity transcribing and/or promote mir-4520-1 and ripe miRNA thereof raising mir-4520-1 and ripe miRNA thereof based on the microRNA gain-of-function technology of RNA and/or gene specific miRMimics technology is adopted.The Mimics of preferred synthetic miR-4520-3p or short hairpin RNA (shorthairpinRNA, shRNA) or raised the expression of mir-4520-1 and ripe miRNA thereof by regulation and control promotor.
The object of the present invention is to provide the application of above-mentioned anti-osteosarcoma transfer biotechnological formulation in preparation treatment osteosarcoma diversion medicaments or reagent.
The object of the present invention is to provide a kind of osteosarcoma to shift diagnostic reagent, osteosarcoma diagnostic reagent can detect the expression that transcribing of mir-4520-1 and ripe miRNA thereof in osteosarcoma sample or immunologic detection method detect mir-4520-1 in osteosarcoma sample.
Further; osteosarcoma transfer diagnostic reagent based on high-flux sequence method and/or based on quantifying PCR method and/or detect mir-4520-1 and ripe miRNA thereof in osteosarcoma sample based on probing procedure transcribe situation, preferably adopt northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, transcribing based on mir-4520-1 and ripe miRNA thereof in the Flow cytometry osteosarcoma sample of microballoon.
Further, it is peripheral blood or osteosarcoma tissue that diagnosis osteosarcoma transfering reagent detects sample, preferred peripheral blood.
Above-mentioned osteosarcoma is the object of the present invention is to provide to shift the application of diagnostic reagent in preparation osteosarcoma diagnostic preparation.
The object of the present invention is to provide the application that mir-4520-1 and ripe miRNA thereof diagnoses in preparation and/or prevents and treats in osteosarcoma transfering reagent.
Preferably, diagnose and/or prevent and treat the reagent that osteosarcoma transfering reagent is diagnosis and/or control osteosarcoma cell Infiltration and metastasis.
Further; diagnosis osteosarcoma transfering reagent comprise based on high-flux sequence method and/or based on quantifying PCR method and/or detect mir-4520-1 and ripe miRNA thereof in osteosarcoma sample based on probing procedure transcribe situation, preferably adopt northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, transcribing based on mir-4520-1 and ripe miRNA thereof in the Flow cytometry osteosarcoma sample of microballoon.
Further, it is peripheral blood or osteosarcoma tissue that diagnosis osteosarcoma transfering reagent detects sample, preferred peripheral blood.
Preferably, comprise the primer of specific amplification mir-4520-1 and ripe miRNA thereof based on quantifying PCR method, further preferably, the primer sequence of specific amplification miR-4520-3p is SEQIDNO4; The probe with the nucleic acid array hybridizing of mir-4520-1 and ripe miRNA thereof is comprised based on probing procedure.
Further, prevent and treat osteosarcoma transfering reagent and comprise the reagent of activity that transcribing and/or promoting mir-4520-1 and ripe miRNA thereof raising mir-4520-1 and ripe miRNA thereof.
Preferably, the activity transcribing and/or promote mir-4520-1 and ripe miRNA thereof raising mir-4520-1 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-4520-3p or raise mir-4520-1 and ripe miRNA thereof by regulation and control promotor.
The target gene of mir-4520-1 and ripe miRNA thereof is the object of the present invention is to provide to diagnose in preparation and/or prevent and treat the application in osteosarcoma transfer preparation.
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-4520-3p 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 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 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.Setting p value < 0.01, filters out the miRNA of 12 differential expressions altogether, and wherein the miRNA11 of expression level downward is individual.The miR-4520-3p wherein lowered enters our research range.
Embodiment 3Real-timePCR detects the expression of miR-4520-3p 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
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
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; 35 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-4520-3p in metastatic bone sarcoma group and normal group.Result shows: qRT-PCR stable amplification result, wherein miR-4520-3p is starkly lower than Normal group at metastatic bone sarcoma group expression level, it is about the result of 1/4th (specifically seeing Fig. 1) of contrast, the confluence analysis of above result verification high-throughput transcript profile expression data.
MiR-4520-3p is on the impact of human osteosarcoma cell proliferation in embodiment 4 transfection
One, experiment material
Human osteosarcoma cell line MG-63 is purchased from Shanghai cell research institute of the Chinese Academy of Sciences.
Sequent synthesis: miR-4520-3p sequence is issued Shanghai Ji Ma company, by its synthesis miR-4520-3pmimics, anti-miR-4520-3p.5 ' end FAM of nucleotide sequence modifies, can fluoresced green, can observe after cell is entered in transfection under fluorescent microscope.
MiR-4520-3pmimics sequence:
5’-UUGGACAGAAAACACGCAGGAA/CCUGCGUGUUUUCUGUCCAAUU-3’(SEQIDNO5)
Anti-miR-4520-3p sequence:
5’-UUCCUGCGUGUUUUCUGUCCAA-3’(SEQIDNO6)
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, and intercellular substance bounces back, and 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/80nM composition sequence is joined in 100u1DMEM substratum, softly mixes; 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.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 5 × 10 5individual/ml, gets 100ul/ hole and adds room.Be placed in 5%CO 2, hatch 48 hours in 37 DEG C of constant incubators.
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.
4.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, by 50ul/cm 2ratio add the upper surface of the poly-carbon ester film of Transwell cell, 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 5 × 10 5individual/ml, gets 100ul/ hole and adds room.Be placed in 5%CO 2, 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-4520-3pmimics, anti-miR-4520-3p are all successfully transfected in osteosarcoma cell MG-63, and the cell with fluorescent grain accounts for about total cellular score 80%-85%, display transfection success.
The impact of miR-4520-3p 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-4520-3pmimics 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-4520-3p 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 (10)

1. an anti-osteosarcoma transfer biotechnological formulation, comprises:
A () is raised transcribing of mir-4520-1 and ripe miRNA thereof and/or is promoted the reagent of activity of mir-4520-1 and ripe miRNA thereof;
Receptible carrier in (b) pharmaceutics.
2. biotechnological formulation according to claim 1, it is characterized in that, the microRNA gain-of-function technology of employing based on RNA and/or the activity transcribing and/or promote mir-4520-1 and ripe miRNA thereof of gene specific miRMimics technology rise mir-4520-1 and ripe miRNA thereof, the Mimics of preferred synthetic miR-4520-3p or the expression by regulation and control promotor rise mir-4520-1 and ripe miRNA thereof.
3. the application of the biotechnological formulation described in claim 1-2 in preparation treatment osteosarcoma diversion medicaments or reagent.
4. an osteosarcoma transfer diagnostic reagent, osteosarcoma diagnostic reagent can detect the expression that transcribing of mir-4520-1 and ripe miRNA thereof in osteosarcoma sample or immunologic detection method detect mir-4520-1 in osteosarcoma sample.
5. diagnostic reagent according to claim 4; it is characterized in that; diagnostic reagent based on high-flux sequence method and/or based on quantifying PCR method and/or detect mir-4520-1 and ripe miRNA thereof in osteosarcoma sample based on probing procedure transcribe situation, preferably adopt northern hybridizing method, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE method, in situ hybridization, transcribing based on mir-4520-1 and ripe miRNA thereof in the Flow cytometry osteosarcoma sample of microballoon.
6. diagnostic reagent according to claim 5, is characterized in that, adopt primer sequence be SEQIDNO4 carry out quantifying PCR method detect transcribe situation.
7. diagnostic reagent according to claim 5, is characterized in that, detecting osteosarcoma sample is peripheral blood or osteosarcoma tissue.
8. the application of the diagnostic reagent described in claim 4-7 in preparation osteosarcoma diagnostic preparation.
The application that 9.mir-4520-1 and ripe miRNA thereof diagnoses in preparation and/or prevents and treats in osteosarcoma reagent.
10. application according to claim 9, is characterized in that, osteosarcoma is metastatic bone sarcoma.
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CN106191264A (en) * 2016-07-21 2016-12-07 杨祚璋 Osteosarcomatous miRNA diagnosis marker

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ANGE´LIQUE GOUGELET ET AL.: "Micro-RNA profiles in osteosarcoma as a predictive tool for ifosfamide response", 《INTERNATIONAL JOURNAL OF CANCER》 *
EISUKE KOBAYASHI ET AL.: "MicroRNA Involvement in Osteosarcoma", 《SARCOMA》 *
GEORGES MAIRE ET AL.: "Analysis of miRNA-gene expression-genomic profiles reveals complex mechanisms of microRNA deregulation in osteosarcoma", 《CANCER GENETICS》 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105734155A (en) * 2016-04-27 2016-07-06 固安博健生物技术有限公司 Chondroblast-type osteosarcoma disease-causing gene and application thereof
CN105734155B (en) * 2016-04-27 2018-03-23 固安博健生物技术有限公司 Chondroblastic osteosarcoma Disease-causing gene and its application
CN106191264A (en) * 2016-07-21 2016-12-07 杨祚璋 Osteosarcomatous miRNA diagnosis marker
CN106191264B (en) * 2016-07-21 2019-08-20 杨祚璋 The miRNA diagnosis marker of osteosarcoma

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