MicroRNA-214Effect on Fibrosis after Acute Myocardial Infarction in Rats
|School||Hebei Medical University|
|Keywords||acute myocardial infarction miRNA-214 myocardialfibrosis|
Objectives:Cardiovascular diseases is one of the common causesresulting in death in middle aged and aged people and cardiac failure is themajor cause.Recent research has shown the critical role ofmicroRNAs(miRNA) in cardiovascular diseases. Lots of studies haverevealed that miRNA-214relative levels were different in acute myocardialinfarction(AMI).We have researched the function that miRNA-214inhibitsleft ventricular remodeling in an acute myocardial infarction rat model viathe phosphatase and tension homolog. This experiment will continue in-depthstudy miR-214specific effects on fibrosis after myocardial infarction inrats,explore its effect on the collagens.Ligating the anterior descending of leftcoronary artery to product the model of acute myocardial infarction.Theprecursor and inhibitor of miR-214injected into the heart to regulate theexpression of miR-214level. Observing the collagen expression under thechang of miR-214.Methods:1The regulation of miRNA-214in a rat model of AMIAnesthetized the rats, inserted endotracheal intubation and opened thechest,completed aortic clamp while the intracardiac injection of reagent[ad-miRNA-214,anti-miRNA-214,phosphate buffer saline(PBS)],thenligated the left anterior descending coronary artery. Confirm the success ofsurgery by the means of injection of evans blue into the inferior venacava:Myocardial tissue is pale in infarcted zone, blue in non-infarctedzone.Eighty rats that underwent ligation were randomly divided into fourgroups:(1) the sham group (the normal group),(2)the control groups (ligatethe left anterior descending coronary artery after injection of PBS into theintracardiac,AMI+PBS,(3) the miR-214precursor group (ligate the left anterior descending coronary artery after injectionof ad-miRNA-214into theintracardiac, AMI+pre),(4)the miR-214inhibitor group (ligate the left anteriordescending coronary artery after injectionof anti-miRNA-214into theintracardiac, AMI+inh).Two weeks and six weeks after surgery, each groupwas divided into two batches and killed. To compare the each index changeof these two time periods.2Heart-to-body weight ratio and Histological analysisRats weighed and killed by an intraperitoneal injection of chloralhydrate,The heart were excised,weighed,and fixed in10%formalin. Sampleswere embedded in paraffin and cut into5μm thick sections.They weremounted on normal glass slides and stained with Masson trichrome forhistological examination.3Quantitative real-time PCR and Western blot analysisTake a small organization (1mm*1mm) of the infarct border zone into theEP tube to detect the gene expression of miR-214and mmp1and the proteinexpression of the col-1,col-3,MMP-1and TGF-β1.Results:1Heart-to-body weight ratioThe body weight(g) and heart weight(mg) of each rat were measured inthe terminal experiment. Heart-to-body weight ratio were significantlydifference between the control group and the miR-214precursorgroup, themiR-214inhibitor group and the miR-214precursorgroup in two weeksand six weeks(Table1). And the former was significantly greater than thelatter. These findings indirect indicate that the up-regulation of miRNA-214can suppress AMI-induced myocardial fibrosis. At the same time, it is advancethe myocardial fibrosis if the miRNA-214down-regulated.2Masson’s trichrome stainingMasson’s trichrome staining choosed the left atrium of thesix-week-rats.It showed that more fibre tissue hyperplasia could be seen inAMI than AD, and the most collagen expression in the AN.3RT-PCR The quantitative real-time PCR is a verification of the miRNA-214relative levels in different groups. At different times, the AMI group hadsignificantly decreased expression of miRNA-214when compared with theAD group.Within the AMI group and the AD group,the AN group appearedto have the lowest level of miRNA-214.And there were significantlydifferences among the three groups.With the level change of miRNA-214,theexpression of mmp-1had a opposite trend.There were differences between theAMI group and the AD group.The expression level of the AN group wassignificantly higher than the AD group.4Western blotWestern blot results showed every indicator there was a differentexpressions in each group..That the colla1,col3a1,MMP-1and TGF-β1proteinexpression were lower in high miRNA-214expression group(the ADgroup).In two week and six week, the NG,AMI,AN groups’expression wereobviously different with the AD group. There were not difference aboutcol1/col3in two week.It had a significantly expression of col1/col3in sixweek.Summary,in miRNA-214up-regulate group,the indicators expression ofpromote and reactive fibrosis were lower than the group of ligating the leftanterior descending coronary artery and PBS(AMI) and down-regulategroup.The normal group had a lowest expression.These showed thatmiRNA-214can influnce fibrosis-related protein expression and reducte themyocardial fibrosis.Conclusion1MiR-214has a protective effect about remodeling caused by myocardialfibrosis following the acute myocardial infarction. With its expression levelincreases,the heart-to-body weight ratio and collagen production havesignificantly reduced.2MiR-214could act the heart on the collagen formation and inhibit itsremodeling by affecting gene and protein expression of colla1,col3a1,MMP-1and TGF-β1.