The Role Of MiR-375-3p/YAP1 In The Cardiotoxicity Of Doxorubicin

Objective:To investigate the effect of miR-375-3p/YAP1 on myocardial injury induced by doxorubicin(DOX)in rats,and explore underlying mechanisms of miR-375-3p/YAP1 pathway on sodium phosphocreatine relieving cardiotoxicity induced by DOX.Methods:(1)In vivo experiment:Forty male SD rats were randomly divided into 3 groups,normal control group(5 m L�kg^-1of normal saline by ip injection),DOX group(DOX 2 mg�kg^-1by ip injection for 7 times),CP+DOX group(CP 200 mg�kg^-1by ip injection for the first 3 times,and then CP 200 mg�kg^-1by ip injection for 30 min,and DOX 2 mg�kg^-1by ip injection for 7 times in total,finally CP 200 mg�kg^-1by ip injection for 3 times again),each group was given medicine once every other day,and kept feed for 3 weeks after the last dose.The expression level of miR-375-3p in heart tissue of rats s detected by qRT-RCR.The expression profiles of non-coding RNA related to cardiotoxicity of DOX was searched in the GEO database,and the data were downloaded to analyze the expression difference of miR-375-3p in cardiotoxicity induced by DOX.The target genes of human miR-375-3p were predicted by Target Scan 7.2 and miRTarbase online database,and the intersection genes of the target genes of the two databases were obtained.KEGG pathway enrichment analysis was performed by DIVID database to search for enrichment pathways and regulatory targets related to cardiac pathophysiology.The expressions of yes-associated protein 1(YAP1),cleaved-caspase 3 and cycle-dependent protein kinase inhibitor 1A(P21)in heart tissue of rats were detected by Western-blot.(2)In vitro experiment:(1)H9c2 cells were treated with DOX 1?mol�L^-1alone or combined with CP 1 mmol�L^-1and n-acetyl-L-cysteine(NAC)0.5 m mol�L^-1for 24 h.The expression of miR-375-3p was detected by qRT-RCR.The expressions of YAP1,cleaved-caspase 3 and P21 were detected by Western-blot.CCK8 kit was used to detect H9c2 cells viability,EdU stain was used to detect the proliferation of H9c2 cells.(2)H9c2 cells were transfected with miR-375-3p mimics or inhibitor for 24 h,and then treated with DOX 1?mol�L^-1 for 24 h.The expression of YAP1 and cleaved-caspase 3 were detected by Wes tern-blot analysis.The viability of H9c2 cells was detected by CCK8 kit.Results:(1)In vivo experiment:qRT-RCR results showed that compared with the normal control group,the expression of miR-375-3p in the DOX group was up-regulated,and the expression level of miR-375-3p was significantly down-regulated after the intervention of CP compared with the DOX group(P<0.05).The expression profile analysis of non-coding RNA(GSE36239?GSE121275)showed that the expression level of miR-375-3p in DOX-induced cardiotoxicity was significantly higher than that in the normal group.The software analyzed the intersection of human miR-375-3p target genes,a total of 49 genes,and the KEGG pathway enrichment analysis of the above genes,and the results only showed that the Hippo signaling pathway was enriched.Western-blot results showed that compared with the normal control group,the expression of YAP1 in the DOX group was down-regulated,and the expression of cleaved-caspase 3 and P21 were up-regulated;after the intervention of CP,the expression of YAP1 was significantly up-regulated,and the expression of cleaved-caspas e 3 and P21 were significantly down-regulated compared with the DOX group(P<0.05).(2)In vitro experiment:(1)The expression of miR-375-3p,YAP1,cleaved-caspase 3,and P21 in H9c2 cells in CON group vs DOX group and DOX group vs CP+DOX group were consistent with the results of in vivo experiments(P<0.05).In addition,compared with DOX group,expression levels of miR-375-3p,cleaved-caspase 3,and P21 were significantly down-regulated account for NAC intervention,and YAP1 expression level was significantly up-regulated(P<0.05),which was similar to the results of CP intervention.CCK8 test showed that the H9c2 cells viability was significantly lowered in DOX group than in normal control group,and NAC intervention significantly alleviated the myocardial cell viability decrease induced by DOX(P<0.05).Ed U test showed that the proliferation cells in DOX group were decreased compared with normal control group,while the proliferation cells were increased compared with DOX group after the intervention of CP.(2)H9c2 cells transfected with miR-375-3p mimics or inhibitor showed that YAP1 expression was down-regulated,cleaved-caspase 3 expression was up-regulated,and myocardial cell viability was decreased in the miR-375-3p mimics group compared with the normal group.Compared with the DOX group,YAP1 expression was down-regulated and cleaved-caspase 3 expression was up-regulated in the miR-375-3p mimics+DOX gr oup,and the myocardial cell viability decreased more significantly.However,YAP1 expression was up-regulated and cleaved-caspase 3 expression was down-regulated in miR-375-3p inhibitor+DOX group compared with DOX group,and myocardial cell viability was increased(P<0.05).Conclusion:DOX up-regulated the expression of miR-375-3p and inhibited YAP1,and promoted cleaved caspase 3 and P21 expression in the myocardial injury induced by DOX.The intervention of CP reduced the up-regulated expression of miR-375-3p induced by DOX,and alleviated up-regulated expression of cleaved caspase 3 and P21 and improved myocardial injury induced by DOX.