The Effect Of MicroRNA-214in H₂O₂-induced Rat Cardiac Myocytes Injury And Its Mechanism

Objectives: Diseases of the cardiovascular system represent the primarycause of human morbidity and mortality. Several studies have demonstratedthat miRNAs play important roles not only in cardiovascular development, butalso in cardiovascular disease, such as cardiac hypertrophy, heart failure,Ischemic heart disease.During cardiac infarction, reactive oxygen species （ROS） was increased inboth infarct and non-infarct area, which may induce cells apoptosis. ROS havebeen reported to be generated at an accelerated level in the postischemicmyocardium. Accumulating evidence suggests that ROS function as signaltransduction intermediates to induce transcription factor activation, geneexpression, cell growth, and apoptosis. H₂O₂is one of ROS that may causecell damage. In the experiments H₂O₂often used to simulate the myocardialischemia damage, however, the effects of H₂O₂on gene expression regulationat the translational level in heart cells are currently uncertain. The objective ofthe current study is to determine the effect of a ROS, H₂O₂, on miRNA-214expression in cultured cardiac myocytes and to determine whethermiRNA-214plays a role in ROS mediated gene expression regulation andcellular injury responses in heart cells.Studies about human ovarian cancerhave confirmed Phosphatase and tensin homolog deleted on chromosome10（PTEN） is target genes of the miRNA-214, so we choose PTEN meak furtherresearch to clarify it in myocardial cells associated with the miRNA-214.Methods:1Primary cultures of neonatal rat cardiac ventricular myocytes andidentification.In brief, the hearts of1-to3-day-old Sprague–Dawley rats（Hebei Medical University Laboratory Animal Center） were removed afterhypothermia-induced anesthesia. After repeated rinsing, the atria were removed, and the ventricles were minced with scissors. The minced tissue wasdispersed，filtrated, centrifugaed and resuspended the cells The cardiacmyocytes （0.5×106cells/ml） were cultured in Dulbecco’s modified Eagle’smedium/F12（DMEM/F12） supplemented with13%bovine serum,1%penicillin,1%streptomycin, and100μmol/L5-bromo-2-deoxyuridine torestrict fibroblast growth. Then, the cardiac myocytes were seeded onto theappropriate plates. The medium was replaced every48hours. After3days,observed with inverted microscope: cardiomyocytes were fusiform, star, orirregular shape and so on. They touched with each other by extentedprocesses, which linking together make a network, emerged into a pieceand beated synchronously at a frequency mostly60-120times/min. Cellvitality will keep good condition in3to7days, this cells suitable forexperimental studies.The expression of cardiac myocytes specific a-actin was detected byimmunocytochemical stain to identify the cardiac myocytes differentiation.The cardiac myocytes expressed a-actin, and the positive cells contain brownand filamentous structures in cytoplasm. The purity of neonatal rat cardiacmyocytes in the process of cultured for72hours reach up to75-85%.2Establish cardiac myocytes oxidate stress damage model.Briefly, ratcardiac myocytes cultured in DMEM/F12were treated with either the vehicle（normal control） or H2O2（10,30,50,100,200μmol/L） for6h,12h,24h.Afterwards, cell vitality was measured by MTT. Detect each group the cellvitality, grope for H2O2appropriate concentration and time of H2O2.The cells（>2â…¹106） that were exposed to H2O2（30,50,100,200μmol/L） for6h weredigested,centrifugation and stained with Annexin V-FITC and PI （BD） for15min. The apoptotic cells were identified by EPICS XL flow cytometry（Beckman Coulter, CA, USA）. Determine the concentration of H2O2inpreparation for the follow-up test.H2O2oxidation stress myocardial celldamage model is established.3The effect of H2O2on miRNA-214expression.Cultured rat cardiacmyocytes were treated with either vehicle （normal cells） or H2O2 （30,50,100μmol/L） for6h. RNA was then isolated from the cultured cellsusing an RNA isolation kit. miRNA-214expression was determined byquantitative real-time RT-PCR （qRT-PCR） to observe the miRNA-214expression in cardiac myocytes after H₂O₂stimulation.4AnnexinV/PI analysis the effect of miRNA-214on the H₂O₂-inducedapoptosis of cardiac myocytes.Briefly, the cells were incubated for48-72hafter seeding and then knock down miRNA-214, a miRNA-214inhibitor（anti-miR-214）（Ambion, Inc） was added to the culture media at a finaloligonucleotide concentration of30nM. To upregulate miR-214, pre-miR-214（Ambion, Inc.） was added directly to the complexes at a final oligonucleotideconcentration of30nM. The vehicle control, an oligo control for anti-miR-214（anti-scramble）, an oligo control for pre-miR-214（pre-scramble, Ambion,Inc.）, and a scramble control were also applied. Then cells were treated withH₂O₂（100μmol/L） for6h. The cells were fixed and stained with annexinV-FITC and PI （BD） for15min then apoptosis were identified by EPICS XLflow cytometry.5Western blot analysis.Proteins isolated from cultured normal cardiacmyocytes, H₂O₂-induced cardiac myocyte, the cells which transfected withpre-miR-214, anti-miR-214and also from the sham group at the same times.Proteins isolated from cultured cardiac myocytes were analyzed by westernblotting.Results:1H₂O₂could induce cultured cardiac myocytes apoptosis.The cardiacmyocyte vitality was decreased by high concentrations （30–200μM） of H₂O₂in a dose and time-dependent manner under our experimental conditions. Theapoptosis rate was measured by flow cytometry method （FCM）. Apoptosis ratewas significantly higher in the H₂O₂-treated cells than in the normal cells, andthis effect was dose dependent. We have chosen the relative lowerconcentration of H₂O₂（100μM）, for the further experiment.2In the H₂O₂-induced cardiac myocyte miRNA-214expression hasbeen sensitive changed.the short-term exposure （6h） of cardiac myocytes to H₂O₂（30-100μmol/L）resulted in the increased expression of miRNA-214,and the effect was dose dependent.3miRNA-214had a protective effect against the H₂O₂-inducedapoptosis of cardiac myocytes.Pre-miR-214decreased the rate ofH₂O₂-induced cardiac myocyte apoptosis，in contrast, the apoptosis rate incardiac myocytes increased after treatment with anti-miR-214as determinedby flow cytometry method （FCM）.Representative results for the cardiacmyocytes treated with the veh-icle control （normal cells,H₂O₂-induced）, thecontrol scramble oligo, pre-miR-214and anti-miR-214are shown.The resultsindicate that miR-214had a protective effect against the H₂O₂-inducedapoptosis of cardiac m-yocytes.4PTEN is a miRNA-214target gene in cardiac myocytes.Wedecreased the activity of PTEN using a PTEN inhibitor（VO-OHpic） andfound that the protective effect of pre-miR-214against H₂O₂-induced cardiacmyocyte apoptosis was stronger,but the effective of anti-miRNA-214incardiac myocyte apoptosis become inapparent. H₂O₂decrease the expressionof this protein in cardiac myocytes anti-miRNA-214increased andpre-miRNA-214decreased PTEN protein level in cultured cardiac myocytes.Conclusion:1miRNA-214expression is sensitive to H₂O₂stimulation in cardiacmyocytes.miRNA-214is sensitive to H₂O₂in cardiac myocyte. The short-termexposure （6h） of cardiac myocytes to H₂O₂resulted in the increasedexpression of miR-214, and the effect was dose-dependent.Indicates thatmiRNA-214plays a special role in the H₂O₂-induced H₂O₂-induced injury ofcardiac myocytes.2miRNA-214had a protective effect against the H₂O₂-inducedapoptosis of cardiac myocytes.Pre-miRNA-214decreased the rate ofH₂O₂-induced cardiac myocyte apoptosis，in contrast, the apoptosis rate incardiac myocytes increased after treatment with anti-miRNA-214asdetermined by flow cytometry method （FCM）.3The results indicate that miR-214had a protective effect against the H₂O₂-induced apoptosis of cardiac myocytes via its target gene PTEN.