CN107254537A

CN107254537A - The application of miR 1912 and its target gene in diagnosis and treatment myocardial infarction

Info

Publication number: CN107254537A
Application number: CN201710591408.4A
Authority: CN
Inventors: 孙锦云; 李曙光
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2017-07-19
Filing date: 2017-07-19
Publication date: 2017-10-17

Abstract

The present invention relates to the application of miR 1912 and its target gene in diagnosis and treatment myocardial infarction.Invention is to the expression data by two generations sequencing acquisition myocardial infarction and the miRNA and mRNA of normal healthy controls, simultaneously, with reference to GEO database hub fleshes infarct correlation miRNA and mRNA data, carry out bioinformatic analysis checking and Molecular, as a result show, the miR 1912 and its target gene ZDHHC18 and myocardial infarction that the present invention is provided are closely related, are that the exploitation of clinically dependent diagnostic reagent or chip etc. lays the foundation available for the clinical diagnosis and prevention detection of myocardial infarction.

Description

The application of miR-1912 and its target gene in diagnosis and treatment myocardial infarction

Technical field

The present invention relates to medical diagnosis on disease therapy field, mir-1912 and its maturation miRNA new application are specifically related to, more It is specifically related to the new opplication of mir-1912 and its maturation miRNA in diagnosis and treatment myocardial infarction.

Background technology

MiRNA is a kind of endogenous non-coding small molecule, by specifically binding target gene come the expression of controlling gene. MiRNA plays regulation feedback and buffered in normal cell physiology, as accurate adjustment target gene protein level, prevents it from exceeding The key factor of normal physiological category.In addition, miRNA is also to adjust the switch between different molecular signal path.MiRNA expression quantity The normal physiological function of cell is very likely influenceed to cause the generation of disease with the exception of activity.

Oneself is verified for some results of animal in one's early years, and miRNA cardiac muscle in transcription or posttranscriptional modification Level tune Grow, fibrosis and reconstruct etc..By the dynamic monitoring to myocardial infarction animal blood serum, find some miRNA in cardiac muscle Dynamic change after infarct.Can be a large amount of early stage the miRNA of cardiac muscle growth, development and fibrosis is wherein affected after myocardial infarction Expression.Therefore, miRNA dynamic change is significant in patient's serum after myocardial infarction.Although have now been found that some and Myocardial infarction closely related miRNA, such as miR-1, miR-133, miR-24, miR-499, miR-208, but these are also remote Not nearly enough, clinical accurate diagnosis and treatment needs more candidate molecules marks.

The present invention is based on high-flux sequence method, to the blood tissues and normal healthy controls blood group of 5 myocardial infarction patients Progress two generations sequencing is knitted, miRNA and mRNA expression data are obtained, meanwhile, it is related with reference to GEO database hub fleshes infarct MiRNA and mRNA data, carry out bioinformatic analysis checking, several divided are picked out from the miRNA and mRNA of candidate Sub- biometric authentication, as a result shows, mir-1912 and its target gene ZDHHC18 and the close phase of myocardial infarction that the present invention is provided Close, be that the exploitation of clinically dependent diagnostic reagent or chip etc. is established available for the clinical diagnosis and prevention detection of myocardial infarction Basis.

The content of the invention

Detect application of mir-1912 or its ripe miRNA preparation in diagnosing myocardial infarction reagent is prepared.

The sequence of the mir-1912 is shown in that sequence table SEQ ID NO 1, mir-1912 maturations miRNA are miR-1912, sequence Row are shown in sequence table SEQ ID NO 2.

Further, diagnosing myocardial infarction reagent include based on high-flux sequence method and/or based on quantifying PCR method and/ Based on probing procedure detect myocardial infarction sample in mir-1912 and its maturation miRNA transcription or based on immune detection The expression of the target gene of mir-1912 and its maturation miRNA regulation and control in method detection myocardial infarction sample.

It is preferred to use northern hybridizing methods, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE methods, original position Mir-1912 and its maturation miRNA transcription in hybridization, bead-based flow-cytometry detection myocardial infarction sample；Using ELISA and/or colloidal gold strip detect the table of the target gene of mir-1912 and its maturation miRNA regulation and control in myocardial infarction sample Up to situation.

It is preferred that, specific amplification mir-1912 and its maturation miRNA primer is included based on quantifying PCR method, enters one Step is preferred, and specific amplification miR-1912 primer sequence is SEQ ID NO 3；Included based on probing procedure and mir- The probe of 1912 and its maturation miRNA nucleic acid array hybridizing.

Preparing diagnosis it is an object of the invention to provide mir-1912 and its maturation miRNA target gene and/or preventing and treating Application in myocardial infarction preparation.

The target gene be DIANA-MICROT, MICRORNA.ORG, MIRDB, RNA22-HAS, TARGETMINER, The miR-1912 target genes that TARGETSCAN-VERT databases show.

It is preferred that, the target gene is ZDHHC18.

Further, diagnosing myocardial infarction reagent include based on high-flux sequence method and/or based on quantifying PCR method and/ Or the transcription of myocardial infarction sample mir-1912 and its maturation miRNA target genes is detected or based on being immunized based on probing procedure The expression of mir-1912 and its maturation miRNA target genes in detection method detection myocardial infarction sample.

It is preferred to use northern hybridizing methods, miRNA chip of expression spectrum, ribozyme protection analytical technology, RAKE methods, original position The transcription of mir-1912 and its maturation miRNA target genes in hybridization, bead-based flow-cytometry detection myocardial infarction sample； Using the expression of mir-1912 and its maturation miRNA target genes in ELISA and/or colloidal gold strip detection myocardial infarction sample Situation.

It is an object of the invention to provide up-regulation mir-1912 and its maturation miRNA transcription and/or promotion mir-1912 And its maturation miRNA active reagent is preparing the application in preventing and treating myocardial infarction preparation.

It is preferred that, using the microRNA gain-of-functions technology based on RNA and/or gene specific miRMimics skills Art up-regulation mir-1912 and its maturation miRNA transcription and/or the activity for promoting mir-1912 and its maturation miRNA.It is preferred that people Work synthesizes short hairpin RNA or raises mir-1912 and its maturation miRNA by regulating and controlling promoter.

The preventing and treating heart is being prepared it is an object of the invention to provide the inhibitor of mir-1912 and its maturation miRNA target gene Application in flesh infarct preparation.

It is an object of the invention to provide a kind of myocardial infarction diagnosis reagent, myocardial infarction diagnosis reagent can detect cardiac muscle Mir-1912 and its maturation miRNA transcription or immunologic detection method detect mir-1912 in myocardial infarction sample in infarct sample And its expression of the target gene of maturation miRNA regulation and control.

Further, myocardial infarction diagnosis reagent includes detecting that the specificity of the target gene ZDHHC18 expression of miRNA regulation and control is drawn Thing, it is preferred that primer sequence is SEQ ID NO 4 and SEQ ID NO 5.

Myocardial infarction treatment medicine or examination are being prepared it is an object of the invention to provide the above-mentioned preparation for preventing and treating myocardial infarction Application in agent.

Definition：

The method of detection miRNA expression is mainly included based on high throughput sequencing technologies, based on nucleotides at this stage Hybridization and the miRNA detection methods of PCR-based.MiRNA detection methods based on probe hybridization technique are a kind of direct Detection Methods, Sample rna need not in advance be expanded, including northern hybridizing methods, miRNA chip of expression spectrum, ribozyme protection analysis skill The technologies such as art, RAKE methods, in situ hybridization, bead-based flow-cytometry.

(1) Northern hybridizes

Also known as Northern blot is most classical detection eucaryote RNA sizes, estimates the experimental method of its abundance.Base Present principles are as follows：MiRNA samples are fixed on carrier (such as silicon chip, microballoon or film) first, then it is miscellaneous with the probe of process mark Hand over, signal detection is carried out after washing unnecessary hybridization probe；Can also the first fixed and target miRNA sequence complementation on carrier DNA probe, then with the sample miRNA hybridization by mark, then carries out signal detection.The method of signal mark includes isotope Mark, fluorescence labeling and nano gold mark etc..

(2) miRNA chip of expression spectrum

Principle is equally that the target molecule on solid support is detected using label probe.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 high-throughout excellent Point, can once detect whole expression of hundreds of genes in same sample.The liquid-phase chip that Luminex companies develop (Liquid chip) is also known as multifunctional suspending dot matrix (Multi analyte suspension array, MASA), is new Generation biochip technology.Liquid-phase chip system is made up of many spherulas for main matrix, is fixed with not on every kind of spherula With probe molecule, in order to distinguish different probes, sphere matrix that each is used for label probe is all unique with one Color numbers, these spherulas are suspended in a liquid-phase system, liquid-phase chip system is just constituted.The system can be to same Multiple different moleculars 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 is carried out in aaerosol solution, detection speed pole It hurry up.

(3) ribozyme protection analytical technology (RPA)

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

(4) RAKE methods

RAKE methods (RNA primed array based Klenow emzyme) are the bases in miRNA microarray The Klenow fragments of DNA polymerase i are utilized on plinth, make miRNA and the method for fixed DNA probe hybridization.RAKE can be sensitive Specifically detect miRNA, it is adaptable to largely quickly screen all miRNA that oneself knows.Can be in specific cell and tumour Detect miRNA express spectra situations.Moreover, RAKE methods can also be from the tissue of the FFPE secured by formalin Isolate miRNA and it is analyzed, be the door that analysis miRNA opens hope from sample is achieved.

(5) in situ hybridization (in situ hybridization)

Hybridization in situ technique can intuitively understand miRNA expression ways, be a kind of easier of observation miRNA spatial and temporal expressions Method, normal mark mode includes digoxin, biotin, fluorescence labeling 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, had concurrently by this method with chip technology Flux is big, detection speed is fast, sensitivity is high and it is specific good the features such as.

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

Fluoroscopic examination PCR instrument can draw dynamic changing curve to the cumulative speed of extension increasing sequence during whole PCR.Anti- Answer the initial concentration of target sequence in mixed system bigger, it is desirable to which the PCR cycle number for obtaining amplified production specific output is (general to use Specific threshold period Ct is expressed) it is fewer.Because miRNA length is only 22nt, it is such that traditional qRT-PCR is not suitable for amplification Short fragment.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 Preferable miRNA detections qRT-PCR methods：Special loop-stem structure primer is designed first, using miRNA to be measured as template reverse transcription The chains of cDNA first are synthesized, the cDNA one end is stem Loop primer, and stem loop structure, which is opened, substantially increases cDNA length, 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

Most of known miRNA is to be sequenced to find and identify by cDNA clone.The method needs first to build miRNA CDNA library, then enter performing PCR amplification, amplified production be then cloned on expression vector be sequenced.Takada develops one kind and changed The amplification PCR cloning PCR (miRNA amplification profiling, mRAP) entered, mRAP methods are first connected at miRNA 3 ' ends Joint, then with the reverse transcription primer reverse transcription complementary with joint.Because specific reverse transcriptase has end deoxynucleotide Transferase active, some nucleotides (mainly deoxycytidylic acid) can be connected to 3 ' ends of the cDNA chains that reverse transcription goes out.When 5 ' End connector is the achievable PCR expansions to cDNA with after poly (C) cohesive end annealing of cDNA chains, adding a pair of general primers Increase.Due to mRAP High sensitivities, the expression quantity of miRNA in a small amount of tissue can be directly detected with clone and sequencing technologies.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 sub-series big by generating can detect multiple by single sequencing reaction MiRNA, hence it is evident that improve detection efficiency.

High-flux sequence (High-throughput sequencing) is also known as sequencing technologies (next of future generation Generation sequencing) it is the change to tradition sequencing 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 lost The solution reading rate of information is passed, to obtain all miRNA sequence information, decryption miRNA collection of illustrative plates, which is provided, to be ensured.While high flux The analysis for make it that careful overall picture is carried out to the transcript profile and genome of a species is sequenced, 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 analysises instrument (Illumina Genome Analyzer) of Illumina companies and ABI SOLiD sequenators (ABI SOLiD sequencer).

MicroRNA gain-of-functions technology based on RNA be by exogenous supplement miRNAs synthesis precursor substance come Raise miRNAs level.For example, can be with the artificial synthesized bob folder sample RNA (short consistent with endogenous miRNA sequence Hairpin RNA, shRNA), promoter is done by polymerase II or III, with virus for carrier transfectional cell, by Dicer enzyme modifications RISC is loaded into afterwards to play a role, it is lasting equivalent to rise pre-miRNA level, action effect stabilization.

This technique avoids the nonspecific action of miRNA and gene for gene specific miR Mimics technologies.This people The specific oligonucleotide chain combined with the UTR of target gene 3 ' complementations of work synthesis, can be played with being adjusted after the transcription of miRNA identicals Section is acted on.

The major way of miRNA regulation and control has two kinds：A kind of is the not fully complementary pairings of 3 ' UTR with target gene mRNA, resistance The translation of disconnected target gene, so that regulatory gene is expressed；It is another be it is similar with siRNA, when miRNA and mRNA complete complementaries are matched When, Ago2 albumen directly results in its degraded by cutting mRNA, realizes gene silencing.In a word, miRNA is presently believed to which kind of side Formula is acted on target gene and miRNA is relevant with the pairing degree of target gene.When miRNA matches incomplete with target gene, MiRNA is just played a role with the expression for suppressing target gene；When miRNA matches complete with target gene section sequence, it is possible to Target gene is caused to be broken in complementary region and cause gene silencing.

Brief description of the drawings

Fig. 1 is the ROC curve figure of miR-1912 diagnosing myocardial infarctions

Fig. 2 is miR-1912 relative expression's situation maps

Fig. 3 is ZDHHC18 gene relative expression's situation maps

Embodiment

With reference to specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and it is not intended that to this The limitation of invention.It will be understood by those skilled in the art that：Can in the case where not departing from the principle and objective of the present invention So that these embodiments are carried out with a variety of change, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent It is fixed.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition or according to the bar proposed by manufacturer Part examinations.

The collection of the sample of embodiment 1 and Total RNAs extraction

Hospital in March, 2015 is collected each 6 to the myocardial infarction patient of in September, 2016 peripheral bloods and normal healthy controls peripheral blood. Diagnosis of Acute Myocardial Infarction standard：According to third time in 2012《Myocardial infarction whole world unified definition》The diagnostic criteria system of recommendation It is fixed.Detect myocardial necrosis marker (mainly troponin) level to rise and/or decline, at least 1 time more than reference value 99%th percentile of the upper limit, and at least with the symptom of a following ischemic.

1) symptom of myocardial ischemia；

2) the ischemic ECG of kainogenesis changes；

3) there are pathologic Q ripples in electrocardiogram；

4) radiological evidence shows new myocardial activity and lost or new regional wall motion abnormality；

5) coronarography or postmortem find there is fresh thrombus in coronary artery.

Blood rna extraction standard：RNA purity：OD260/280≤1.8,28S/18S≤1；RNA integralities：RIN Zhi≤ 7.0.RNA integrality detection methods：(agarose coagulates for Agilent 2100 (RNA 6000Nano kit), agarose gel electrophoresis Gum concentration：1% agarose gel；Voltage：5V/cm；Time：20min).

Embodiment 2 is sequenced, data analysis and electronically validating

Sequencing：MiRNA and mRNA is carried out with llumina Hiseq2500/Miseq second generations high throughput sequencing technologies Sequencing, by go joint, go low quality, the process such as depollute completes the processing of data, obtains final data.Pass through transcript profile number Progress t-test after background correction is carried out to miRNA and mRNA sequencing initial data according to analysis software and obtains P values, is then utilized Fisher, which is examined, merges P values, screening differential expression miRNA and mRNA.Using including miRanda, miRDB, miRWalk and These algorithm forecasted varianceses of Targetscan express miRNA target gene, choose >=4 algorithms and predict the target gene come, and The miRNA for the differential expression being had verified that in miRWalk database lookups target gene.

It is final to be selected from the differential expression miRNA and mRNA of candidate to mir-1912 and its target gene ZDHHC18 progress Later experiments are verified.

The electronically validating of embodiment 3

Electronically validating：3 sets of mRNA data sets are obtained from the screening of GEO (Gene Expression Omnibus) database (GSE48060, GSE34198 and GSE61145-GPL6884) and 2 sets of miRNA data sets (GSE61741, GSE31568), 3 sets MRNA data sets (GSE48060, GSE34198 and GSE61145-GPL6884) have gene 13680, we by using MetaMA bags, which are calculated and merged using merging P value methods, obtains 612 (FDR after effect value<0.05) difference expression gene, wherein on 352 are adjusted, 260 are lowered；The common miRNA numbers 848 of 2 sets of data collection (GSE24371, GSE17498).Use R bags MetaMA merges the analysis of P values to two sets of miRNA data sets, finds 15 differential expression miRNA (FDR<0.001&&| Combined.ES|>1).As a result show that electronically validating result is consistent with sequencing result expression trend.

It is to set up Receiver Operating Characteristics for single miRNA molecule or the method for the efficiency evaluation of diagnostic model (receiver operating characteristic, ROC) curve, passes through (the Area Under of area under calculated curve Curve) come judge diagnosis ability.Area value under ROC curve is between 1.0 and 0.5, in AUC>In the case of 0.5, AUC Closer to 1, illustrate that diagnosis effect is better, AUC has relatively low accuracy in 0.5-0.7, AUC is fixed in 0.7-0.9 True property, has high accuracy when AUC is more than 0.9.We will utilize GSE31568 data sets (including 70 control groups and 20 Example case group), and then analyzed, as a result show, the AUC of miR-1912 diagnosing myocardial infarctions is 0.809 (see Fig. 1), is shown MiR-1912 can as diagnosing myocardial infarction molecular marker.

The collection of the sample of embodiment 4 and the extraction of total serum IgE

1 sample collection：

36 myocardial infarction patients and 22 normal healthy controls crowd peripheral bloods (acquisition time in January, 2014-2015 year 8 Month).

2 Total RNAs extractions：

The processing for removing Rnase of related experiment article：

1. it will be rinsed before the application of all glasswares with DEPC and invade bubble, 120 DEG C of high pressure 20min, 180 DEG C of high temperature dry 2 More than hour.

2. by plastic ware (such as：EP pipes/pipette tips) need before use to be stayed overnight with 0.1%DEPC water enchroachment (invasion)s bubble, after drain liquid, 120 DEG C of high pressure 20min, baking box is dried standby.

Leucocyte is separated

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

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

(3) 4m1 lymphocyte separation mediums are added in another centrifuge tube；

(4) draw 4m1 cell suspensions be gently added to along tube wall lymphocyte separation medium surface (note not with lymphocyte Separating liquid is mixed).Centrifuge 1500rpm 20min；

(5) boundary layer (tunica albuginea) is gently suctioned 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 pipes, and supernatant is removed in centrifugation, for extracting RNA.

RNA is extracted

(1) sample is taken out in -80 DEG C of refrigerators, is shredded, Trizol is added in EP pipes in 1ml/50-100mg ratio, Homogenized is carried out, 5-l0min is stored at room temperature；

(2) every milliliter of Trizol adds 0.2m1 chloroforms, acutely shakes 15s, is stored at room temperature 2-3min, 12000 at 4 DEG C Leave heart 15min；

(3) the careful supernatant water 600ul that makes an appointment that suctions out moves into another centrifuge tube (being careful not to be extracted into albumin layer), adds equivalent Isopropanol, overturns and mixes, be stored at room temperature 10min；

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

(5) the rotation washing of the cold ethanol of lml 75% is added, isopropanol is cleaned；

(6) 4 DEG C of 12000g centrifuge 5min, remove the 5-l0min that dried in the air after ethanol, translucent, are dissolved with 20u1DEPC water RNA.3u1RNA samples are taken, the electrophoresis in 1.5% Ago-Gel；Lu1RNA samples in UV spectrophotometer measuring concentration, RNA samples in 1.8-2.0 are considered as with A260/280 qualified.

The RT-PCR of embodiment 5 detects miR-1912 and ZDHHC18 expression

1 RT-PCR detects miR-1912 expression

Reverse transcription

The preparation of RT systems：1 μ g total serum IgEs are used 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 insulation 60min make reverse transcription reaction complete in 9700 type PCR instruments, 95 DEG C of 5min terminating reactions.Add 80 μ l Nuclease-free H₂O is diluted to 100 μ l, and to be stored in -20 DEG C of refrigerators standby.

Quantitative fluorescent PCR

The preparation of miRNA RT-PCR systems：

Reaction system：MiRNAs detection of expression sets 3 parallel tube reactions every time, and internal reference is used as using snRNA U6.Expand Increasing program：95℃10min；40 circulations (95 DEG C of 10s, 60 DEG C of 30s).

2 RT-PCR detect ZDHHC18 expression

Reverse transcription

UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) enter Row cDNA reverse transcriptions.Using Reverse Transcriptase kit, converse record synthesis cDNA is carried out to l μ g total serum IgEs with RT Buffer.RT bodies The preparation of system：5 × RT Buffer, 5 μ l, 10mmol/l dNTP, 1.25 μ l, 0.1mmol/l DTT 2.5 μ l, 30 μm of mol/ 2 μ l, 200U/ μ l MMLV of l OligodT 1.25 μ l, the μ g of template ribonucleic acid 1, add aqua sterilisa to the μ l of total system 25.42 DEG C of incubations 1 hour, 72 DEG C 10 minutes, of short duration centrifugation.CDNA is preserved and is put the standby RNA of -20 DEG C of refrigerators.

Quantitative fluorescent PCR

Sequence (NM_032283.2) the design primer provided according to GenBank, send company to be synthesized.

The preparation of mRNA RT-PCR systems：

Component	Addition
		2×mix	10μl
Sense primer 10uM (SEQ ID NO 4)	0.5μl
		Anti-sense primer 10uM (SEQ ID NO 5)	0.5μl
Template	2μl
		Add sterile purified water	To 25 μ l

Reaction system：Use PowerGreen PCR Master Mix (invitrogen, article No. 4367659) enter Row amplification, interior participation in the election GAPDH, experimental implementation is carried out by product description.Amplification program is：95 DEG C of 10min, (95 DEG C of 15sec, 60 DEG C of 60sec) × 35 circulations.

3 statistical analysis

Real-time quantitative PCR sample amplified production solubility curve is all unimodal, illustrates that amplified production only has one, is specificity Amplification；Amplification curve flex point understands that amplification curve entirety collimation is good, shows that the amplification efficiency of each reaction tube is close, and the limit is put down And without raising up now, exponent phase slope is larger, illustrates that amplification efficiency is higher；According to qRT-PCR relative quantification formula meter Experimental group and the multiple proportion of control group destination gene expression are calculated, compares tables of the miR-1912 in myocardial infarction group and control group Up to level.As a result show：MiR-1912 is in about 0.4 times that the expression quantity of illness group is control group, tables of the ZDHHC18 in illness group It it is 3.5 times of control group up to amount, result above demonstrates the result of high-flux sequence data analysis.

The culture and transient transfection of the cardiac muscle cell of embodiment 6

First, material prepares：

People's primary cardiomyocytes (Human Cardiac Myocytes, HCM) are purchased from ScienCell companies of the U.S., culture In special myocardial cells culture base.

Lipofectamine^TM2000Transfection Reagent(Invitrogen)。

MiR-1912 sequences issue Synesis Company, ask its chemical synthesis miR-1912mimics, miR-1912inhibitor And its non specific control.

2nd, experimental method

1 miRNA is transiently transfected

Lipofectamine is pressed in operation^TM2000 reagent specifications are carried out.24h is thin by the good HCM of growth conditions before transfection Born of the same parents are inoculated into 6 orifice plates, and cell count is about 4 × 10⁵/ L, cellar culture to transfect the same day, cell fusion degree be 70-80% when Tested.100nM miR-1912mimics/miR1912inhibitor are added to 250 μ l opti-MEM culture mediums In, it is soft to mix；It is another to dilute 5 μ l Lipofectamine with 250 μ l opti-MEM culture mediums^TM2000 liposomes, it is soft mixed It is even, it is incubated at room temperature 5min；Opti-MEM- liposomes and Opti-MEM-miRNAs are mixed, 20min is incubated at room temperature, to form transfection Compound：Then said mixture is added in cell culture medium, gently mixed, complete medium is changed after culture 6h.Wherein, Nonspecific mimics Negative Control (mimics NC) and inhibitor Negative Control (inhibitor NC) sequence is used as control.Cell total rna is extracted after culture 24-48h, and reverse transcription is into cDNA, real-time quantitative PCR The change that miR-1912 is expressed after detection is transiently transfected.

2 experimental results

Transiently transfected using cationic-liposome method, respectively by miR-1912mimics or miR- 1912inhibitor and corresponding control sequence Negative Control (NC) transfection cardiac muscle cell's strain HCM.Transfect after 48h, take out Carry cell total rna.Using U6 as internal reference, real-time quantitative PCR detects miR-1912 expression.As a result show：Compared with control group, After HCM transfections miR-1912mimics, miR-1912 expression increases about 4.3 times；Transfect after miR-1912inhibitor, Expression have dropped nearly 63%.Result above shows, can by transiently transfecting miR-1912mimics and miR-1912inhibitor Effectively up-regulation or downward miR-1912 expression, reliable results can carry out subsequent experimental.

Embodiment 6 transfects influences of the miR-1912 to Human Cardiomyocytes ZDHHC18 gene expressions

MiR-1912mimics or miR-1912inhibitor and corresponding control sequence Negative will be transiently transfected HCM cells after Control (NC) are inoculated into 96 orifice plates, after transfection 48h, extract cell total rna.Using GAPDH as internal reference, Real-time quantitative PCR detects ZDHHC18 expression.

As a result show：Compared with control group, after HCM transfections miR-1912mimics, ZDHHC18 expression quantity reduction is about After 13%, transfection miR-1912inhibitor, ZDHHC18 expression quantity rises about 19%, preliminary proof miR-1912 and ZDHHC18 targeting relation.

Although describing the present invention with reference to various preferred embodiments, it will be appreciated by those skilled in the art that can carry out each Change is planted, and available equivalents substitute base region of its component without departing from the present invention.Come in addition, many changes can be carried out Particular case or material is set to be suitable for the teachings of the present invention without departing from its base region.

SEQUENCE LISTING

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<120>The application of miR-1912 and its target gene in diagnosis and treatment myocardial infarction

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Claims

1. detect application of the mir-1912 or miR-1912 preparation in diagnosing myocardial infarction reagent is prepared.

2. application according to claim 1, it is characterised in that mir-1912 sequence is shown in sequence table SEQ ID NO 1, MiR-1912 sequences are shown in sequence table SEQ ID NO 2.

3. application according to claim 1, it is characterised in that diagnosing myocardial infarction reagent includes being based on high-flux sequence side Method and/or based on quantifying PCR method and/or based on probing procedure detection myocardial infarction sample in mir-1912 and miR- 1912 transcription.

4. application according to claim 3, it is characterised in that using northern hybridizing methods, miRNA express spectra cores In piece, ribozyme protection analytical technology, RAKE methods, in situ hybridization, bead-based flow-cytometry detection myocardial infarction sample Mir-1912 and miR-1912 transcription.

Application of 5.mir-1912 and its maturation the miRNA target gene in diagnosing myocardial infarction preparation is prepared.

6. application according to claim 5, it is characterised in that diagnosing myocardial infarction reagent includes being based on high-flux sequence side Method and/or detect myocardial infarction sample mir-1912 and its maturation based on quantifying PCR method and/or based on probing procedure The transcription of miRNA target genes detects mir-1912 and its maturation miRNA targets in myocardial infarction sample based on immunologic detection method The expression of gene.

7. application according to claim 5, it is characterised in that using northern hybridizing methods, miRNA express spectra cores In piece, ribozyme protection analytical technology, RAKE methods, in situ hybridization, bead-based flow-cytometry detection myocardial infarction sample Mir-1912 and its maturation miRNA target genes transcription, or using ELISA and/or colloidal gold strip detection myocardial infarction sample The expression of mir-1912 and its maturation miRNA target genes in this.

8. the application according to any one of claim 3,4,6,7, it is characterised in that sample is peripheral blood.

9. the transcription for raising mir-1912 or miR-1912 and/or the active reagent for promoting mir-1912 or miR-1912 are being made The standby application prevented and treated in myocardial infarction preparation.

The inhibitor of 10.miR-1912 target gene is preparing the application in preventing and treating myocardial infarction preparation.