The Effect Of Knockdown Of ANP32A On Doxorubicin-induced Cardiac Myocyte Apoptosis In Vitro

Objective:In this study, DOX-induced apoptosis in neonatal cardiac myocyte was used toinvestigate whether transient ANP32A gene down-expression could decreaseapoptosis in neonatal cardiac myocytes. And we will investigate whether ANP32A isa direct target of miR-21in neonatal cardiac myocytes.Methods：(1) ANP32A/GV115-RNAi-Lentivirus and rno-miR-21(Ubi)-Lentivirus wereconstructed, and were transiently transfected into neonatal cardiac myocytes.(2) Neonatal cardiac myocytes were divided into five groups:①control group:only neonatal cardiac myocytes;②GFP group: transfected with NC-GFP-LV;③knockdown of ANP32A group: transfected with ANP32A/GV115-RNAi,④DOXgroup: neonatal cardiac myocytes, were cultured with DOX；⑤knock down ofANP32A+DOX group: post transfecting with ANP32A/GV115-RNAi, neonatalcardiac myocytes were cultured with DOX.(3) Primary cultures of neonatal cardiac myocytes were prepared as described.ANP32A/GV115-RNAi-Lentivirus and the control lentivirus (NC-GFP-LV-Lentivirus) were transfected into neonatal cardiac myocytes, then cultured with DOX.The apoptosis and viability of neonatal cardiac myocytes were assessed by MTTassay and AnnexinV-FITC/PI. To test the possibility that ANP32A induces apoptosisby regulating expression of bcl-2and bax in neonatal cardiac myocytes,the level ofbcl-2and bax was examined by Western blot. Rno-miR-21(Ubi)-Lentivirus and thecontrol lentivirus (NC-GFP-LV-Lentivirus) were transfected into neonatal cardiacmyocytes,and the expression of ANP32A was analyzed with qRT-PCR and westernblot.(4) All results are expressed as mean values±standard deviation (SD). Datawere evaluated by one-way analysis of variance (ANOVA) using statistical analysissoftware SPSS17.0. P values <0.05consider statistically significant changes. Results：(1) Western blot identificationand DNA sequencing showed that theANP32A/GV115-RNAi-Lentivirus were constructed successfully.,and qRT-PCRidentificationand DNA sequencing showed that the rno-miR-21(Ubi)-Lentiviruswere constructed successfully.(2) These results suggest that ANP32A mediated regulation of bcl-2and baxinfluences apoptosis in DOX-induced neonatal cardiac myocyte apoptosis in vitro,transient ANP32A gene down-expression could decrease apoptosis in neonatalcardiac myocytes.(3) Overexpression of miR-21decreased ANP32A expression in neonatal cardiacmyocytes.Couclusions：(1) ANP32A could express in neonatal cardiac myocytes.(2) Knowdown of ANP32A could decrease apoptosis of DOX-treatedneonatal cardiac myocyte in vitro.(3) ANP32A may be a direct target of miR-21in neonatal cardiac myocytes.