The Effects And Mechanisms Of MiR-135a-5p And MiR-199a-5p On Myocardial Hypertrophy

Myocardial hypertrophy is a common complication of hypertension, pulmonaryarterial hypertension and other cardiovascular diseases, and it is the typical feature ofmyocardial remodeling and may causeheart failure, which eventually leads to death.Pulmonary arterial hypertensionleads to right ventricular hypertrophy, in which pulmonaryvascular remodeling and increased resistanceis the important reasons; left ventricularhypertrophy often occurs in patients with hypertension.It is caused widespread concern thatmyocardial hypertrophy becomes the independent risk factors of morbidity and mortality ofcardiovascular disease. The pathogenesis of cardiac hypertrophy has not been fullyelucidated, which causes the lack of effective clinical drugs for myocardial hypertrophy. Itis important that clarification of the pathogenesis of cardiac hypertrophyhelp to find newtargets on prevention of myocardial hypertrophy.microRNAs (miRNAs) is a non-coding RNA about22~25nucleotides, which involvedin post transcriptional regulation of gene andaffect cellular growth via binding with the3’non-coding regionof the target gene specifically.Although some researches show thatclosely relationship of miRNAs andoccurrence of myocardial hypertrophy, but that is stillnot enoughto clarifythe regulatory role of miRNAs in myocardial hypertrophy. Researcherspredicted amount of miRNAsassociated with incidence of myocardial hypertrophy bybioinformatics. Our preliminary studies found that the expression of miR-135a-5p andmiR-199a-5pincreased in the process of myocardial hypertrophysignificantly. According toprevious studies, we hypothesized that both of miRNAs have important functions onmyocardial hypertrophy. Therefore, this study aims to discuss the effects and mechanismsof miR-135a-5p and miR-199a-5p on right ventricular hypertrophy and left ventricularhypertrophy respectively, which promote to understandthe molecular mechanism and findout the effective intervention measures for myocardial hypertrophy. Methods1Methods about the actions and mechanisms of miR-135a-5p on right ventricularhypertrophy induced by PAH in rats.1.1Preparation of right ventricular hypertrophy model and animal groups: male SDrats were treated with monocrotaline (60mg/kg MCT) by subcutaneous injection, and thecontrol rats were administered withthe vehicle.The animals were sacrificed after1,2,3and4weeks respectively.1.2TheSigns of rat life was observed closely during the experiments, and mPAP andRVSP were detected at4th week; rat right ventricular hypertrophy index(RVHI), rightventricular mass index (RVMI) and wet lung index(LI)were detected and calculated. HEstaining morphological changes of lung tissue and right ventricular were observed by lightmicroscopy; the expression level of miRNAs in rat lung tissue was measured by RT-qPCR.1.3The expression level of miR-135a-5p was detected by RT-qPCR in the lung tissueof right ventricular hypertrophy modelswhich induced by monocrotaline (60mg/kg MCT) in1,2,3and4weeks respectively.1.4Primarypulmonary artery smooth muscle cells (PASMCs) were cultured, and thePASMCs proliferation model was induced by three percent oxygen, and the expression ofmiR-135a-5p was detected by RT-qPCRat6,12,18,24and48hours.1.5miR-135a-5p mimic or inhibitor was transfected into PASMCsusing LipofectamineRNAiMAX transfection reagent, and the effects of miR-135a-5p on cell proliferation weredetected with CCK-8,[3H]-leucine incorporation, cell cycleandexpression levels of mRNAof proliferating cell nuclear antigen (PCNA).2Methods about the actions and mechanisms of miR-199a-5p on left ventricularhypertrophy induced by transverse aortic coarctation (TAC) in rats.2.1Preparation of left ventricular hypertrophy model and animal groups: the model ofleft ventricular hypertrophy of male SD ratsinduced bytransverse aortic coarctation, and theratswere randomly divided into sham operation group, model group and losartan group.Losartan group were given10mg/kg/d of losartan potassium, sham operation group andmodel group were giventhe vehicle for4weeks.2.2TheSigns of rat life was observed closely during the experiment, the rats weresacrificed at4th week. The morphological changes of rat right ventricle was detectedbyHEstaining; the cardiac hypertrophy index and left ventricular hypertrophy index were measured; the expression ofmiR-199a-5p in left ventricle of rats was detected by RT-qPCR.2.3Cultured neonatal rat cardiac myocytes were stimulated by Angiotensin Ⅱ (AngⅡ), and the expression level of miR-199a-5p was detected by RT-qPCR after12h.2.4miR-199a-5p mimic or inhibitor was transfected into cultured neonatal rat cardiacmyocytesstimulated by AngⅡ, Protein synthesis of myocytes were evaluated by [3H]-leucine incorporation.Results1Results about the actions and mechanisms of miR-135a-5p on right ventricularhypertrophy induced by PAH in rats.1.1Forty percent rats died in MCT model group, and the survivals of MCT model ratwere in poor health, which body weight decreased significantly(p<0.01). At4th week,mPAP and RVSP of rats increased significantly (p<0.01, p<0.01); RVHI, RVMI and LIincreasedsignificantly (p<0.01, p<0.01, p<0.01); pulmonary vascular remodeling and rightventricular hypertrophy were observed by HE staining; miR-135a-5p, miR-211-5p,miR-204-5p, miR-224-5p and miR-93-5p expressions were upregulated, and theexpressionsof miR-135a-5were upregulated965%significantly in lung tissue of ratsdetected byRT-qPCR.1.2The expression of miR-135a-5p was decreased in lung tissue of rats at1,2and3weeks, which was increased at4weeksignificantly (p<0.01).1.3Theexpression of miR-135a-5p had no change at6h,12h,18h,24hand48h inproliferation PASMCs stimulated by three percent oxygen.1.4miR-135a-5p mimic (30nM) could significantly inhibit the proliferation ofPASMCs induced by three percent oxygen,which decreased cell viability,[3H]-leucineincorporation,proliferation index and levels of PCNAmRNA in cultured PASMCs.2Results about the actions and mechanisms of miR-199a-5p on left ventricularhypertrophy induced by transverse aortic coarctation (TAC) in rats.2.1There are3,3and4rats died in sham operation group, model group and losartangroup respectively, which are no significant differences between groups. The healthconditions of model rats group were poorer than sham operation group, and losartan groupwere better than model group significantly.Cardiac index and left ventricular hypertrophyindex of model ratsgroup were increased significantly at4week; left ventricular hypertrophy were observed by HE staining; the expression level of miR-199a-5p wassignificantly increased in left ventricle hypertrophy induced by transverse aortic coarctation.However, AngII receptor antagonistlosartan could reverse these changes significantly.2.2In vitro study, losartan significantly inhibited the expression level of miR-199a-5pin neonatal rat cardiac myocytes induced by AngⅡ.2.3miR-199a-5p inhibitor significantly decreased [3H]-leucine incorporation inneonatal rat cardiac myocytes induced by AngⅡ, and miR-199a-5p mimic significantlyenhanced [3H]-leucine incorporation in neonatal rat cardiac myocytes.Conclusion1. The expression of miR-135a-5p was decreased in lung tissue of right ventricularhypertrophy model rats induced by PAH at1,2and3weeks, which was increased at4weeksignificantly (p<0.01). Meantime, miR-135a-5p could significantly inhibit the proliferationof cultured PASMCs induced by three percent oxygen. These results imply thatmiR-135a-5p maybe is a new target for improving pulmonary vascular remodeling andpreventing right ventricular hypertrophy.2. The expression level of miR-199a-5p was significantly increased in left ventriclehypertrophy induced by transverse aortic coarctation. AngII receptor antagonistlosartancould inhibitleft ventricle hypertrophy and the increasingof miR-199a-5p significantly.Meantime, downregulation of miR-199a-5p decreased cultured neonatal rat cardiacmyocytes hypertrophy induced by Ang Ⅱ in vitro study. These results suggest thatmiR-199a-5p maybe play an important role in myocyte hypertrophy induced by AngⅡ.