MiR-30 Affects Daunorubicin-induced Cardiomyocyte Apoptosis In Rats Via ERK1/2-p53 And NFATc4

Anthracycline represented by daunorubicin(Daunorubicin,DNR)is a broadspectrum antineoplastic drug commonly used in the clinic.Its cardiotoxicity is the most common and severe adverse reaction,which greatly limits its clinical application.The pathogenesis of anthracycline-induced cardiotoxicity(AIC)is complex and diverse,and cardiomyocyte apoptosis is one of the most important reasons.Many studies have shown that micro RNAs(miRNAs)play an important role in the regulation of cardiovascular function and disease.The potential target genes of miRNAs in organisms range from several to hundreds,and their expression and regulation functions are extraordinarily complex,with timing and tissue specificity.The expression of miR-30 is abundant in myocardial tissue,which is closely related to cardiomyocyte apoptosis.The miR-30 gene expression and its effects on AIC related cardiomyocyte apoptosis remain to be studied.In this study,1μM DNR was used to treat H9c2 rat cardiomyocytes to establish the AIC model.Detection: changes of miR-30 family gene expression profile in AIC myocardial injury;changes in proliferation and apoptosis by overexpressed miR-30 in H9c2 cell;changes in m RNA and protein expression of ERK1/2,p53,Bax,and Bcl-2 in AIC myocardial injury;changes in protein expression of p-ERK1/2,p53 and Bax/Bcl-2 by overexpressed miR-30 in H9c2 cell;changes in NFATc4 nuclear translocation by overexpressed miR-30 in H9c2 cell.The above experiments provide valuable experimental data for clarifying miR-30 effect on the cardiomyocyte apoptosis in AIC which may enrich the understanding of the AIC pathogenesis of.In addition,this study twould be a preliminary attempt for the prevention or treatment for AIC by miR-30.Objective: To observe the changes of miR-30 family expression in DNR treated H9c2 cardiomyocytes and then explore the effect of miR-30 on AIC cardiomyocyte apoptosis.Methods:(1)H9c2 rat cardiomyocytes were treated with 1μM DNR for 0,3,6,12,24 h to establish AIC myocardial injury model;(2)H9c2 cardiomyocytes overexpression of miR-30;(3)miRNA RT-q PCR detect the changes of miR-30 family gene expression;(4)RT-q PCR and Western Blot detect the changes of ERK1/2,p53,Bax and Bcl-2 m RNA and protein expression;(5)Ed U cell proliferation test detect cell survival rate;(6)Annexin V-FITC detect cell apoptosis rate;(7)Immunofluorescence technique detects NFATc4 nuclear translocation.Results:(1)In DNR treatment H9c2 rat cardiomyocytes,miR-30 expression increased at first and then decreased within 24 hours overall,in which miR-30a-5p and miR-30e-5p decreased more significantly after 24 hours treatment.(2)overexpression miR-30a-5p and miR-30e-5p in H9c2 cell improved the survival rate by 40.1% and 52.7%,respectively,in AIC model.Overexpression miR-30e-5p decreased the apoptosis rate,decreased by 55.9%.(3)In the model of AIC myocardial injury,the m RNA and protein content of ERK1/2,p53 and Bax increased significantly in DNR treated H9c2 cell,while the m RNA and protein content of Bcl-2 decreased,which yielded a marked increase in Bax/Bcl-2 ratio.(4)miR-30e-5p overexpression inhibited the protein expression of p-ERK1/2 and p53,depressed Bax/Bcl-2 ratio in AIC myocardial injury.(5)miR-30e-5p overexpression reduced the content of NFATc4 and nuclear translocation in DNR injured H9c2 cell.Conclusion: miR-30e-5p gene expression is significantly decreased in DNR-induced H9c2 rat cardiomyocyte injury.miR-30e-5p overexpression significantly improves the survival rate and lower the apoptosis rate in DNR treated H9c2 cell.miR-30e-5p overexpression also inhibits the activation of ERK1/2-p53 signal pathway and NFATc4 nuclear translocation.In summary,miR-30e-5p may regulate DNR-induced cardiomyocyte injury and apoptosis through ERK1/2-p53 and NFATc4,and then affect the occurrence and development of AIC.