| Objective Apoptosis is the major cause of cardiomyocyte death in myocardial ischemia/reperfusion injury(MI/RI).The major aim of the present study was performed to test whether mi R-327 participates in cardiomyocytes apoptosis both in vitro and in vivo,and reveal the potential molecular mechanism of mi R-327 regulated MI/RI through targeting apoptosis repressor with caspase recruitment domain(ARC).Methods In vitro: The rat H9C2 cardiomyocyte cell was cultured in high glucose Dulbecco's modified Eagle's medium(DMEM)and randomly divided into five groups,named Control group,hypoxia/reoxygenation(H/R)group,Ad?EGFP?NC + H/R(Ad?NC)group,Ad?mi R?327 + H/R(Ad?mi R?327)group and Ad?mi R?327?i + H/R(Ad?mi R?327i)group.The H9C2 cells were exposed to hypoxia for 4 h and reoxygenation for 12 h to mimic I/R injury before transfecting with mi R?327 recombinant adenovirus vectors.The transfected cells were observed under the fluorescence microscope and transfection efficiency was evaluated by flow cytometry.The cell viability was assessed by the cell counting kit?8(CCK?8)assay.The lactate dehydrogenase(LDH)were detected by the automatic biochemical analyzer.The apoptosis of H9C2 cells was determined by Annexin V?APC/propidium iodide dual staining.The level of mi R-327 and ARC m RNA were detected using Quantitative Real-time PCR(q PCR).The protein expression of ARC,cleaved Caspase-3,cleaved Caspase-9,Caspase-8,Cyt-c,Bax,Bcl-2,Fas and Fas L were also tested by Western blot.The target relationship between mi R?327 and ARC was verified by luciferase reporter assay.In vivo: A total of seventy male Sprague–Dawley rats were randomly allocated into five groups,including the Normal nonischemic group(sham),the myocardial I/R group(I/R),the myocardial I/R with Ad?EGFP?NC group(Ad?NC),the myocardial I/R with Ad?rno?mi R?327 group(Ad-mi R-327)and the myocardial I/R with Ad?rno?mi R?327?inhibition group(Ad-mi R-327i).After adenovirus or normal saline delivery following by 72 h,all rats were subjected to MI/RI by left anterior descending coronary artery occlusion for 30 min and reperfusion for 3 h with the exception of sham group.Then,the expression of Enhanced Green Fluorescent Protein(EGFP)was observed under fluorescence microscopy.The level of LDH was detected by the automatic biochemical analyzer.The infarcted area of myocardium was measured by 1.5% 2,3,5?triphenyltetrazolium chloride(TTC)staining,and apoptosis of cardiomyocytes was determined by terminal deoxynucleotide transferase-mediated d UTP nick-end labeling(TUNEL)staining.The levels of mi R-327 and ARC m RNA,as well as protein levels of ARC,cleaved Caspase-3,cleaved Caspase-9,Caspase-8,Cyt-c,Bax,Bcl-2,Fas and Fas L were evaluated by q PCR and Western blot,respectively.Results 1.In vitro:(1)Expression of mi R-327 and ARC in H9c2 cells after H/R treatment.Compared with the control group,mi R-327 was significantly upregulated,while ARC was downregulated both on m RNA and protein level in response to H/R treatment(P<0.05).(2)Adenovirus vectors were successfully transfected into H9C2 cell.After 48 h,green fluorescence protein(GFP)was observed under fluorescence microscope and the expression of GFP increased with the rise of MOI.The MOI of 50 was determined as the optimal multiplicities of infection.(3)Inhibition of mi R-327 improved H/R-induced cardiomyocyte injury and apoptosis.Compared with Ad-NC group,Ad?mi R?327i remarkably decreased mi R-327 expression.Meanwhile,cell viability was obviously increased,while the LDH activity,rate of apoptosis and the level of cleaved Caspase-3 was obviously decreased.Whereas the Ad?mi R?327 performed an opposite effect(All P<0.05)..(4)Inhibition of mi R-327 suppressed the both extrinsic and intrinsic apoptosis?associated molecules expression in vitro.Compared with the control group,the level of cleaved Caspase-9,Caspase-8,Cyt-c,Bax,Fas and Fas L were obviously increased and Bcl-2 was markedly decreased after H/R.Compared with the H/R group,the expression levels of these proapoptotic molecules were obviously reduced and the antiapoptotic molecule was elevated by mi R?327 downregulated,whereas the mi R?327 overexpression displayed an opposite effect(All P<0.05).(5)ARC was a downstream target gene of mi R?327.The ARC protein expression was obviously reduced by mi R?327 overexpression,but increased by mi R?327 knockdown(All P<0.05).However,there was no obvious change of ARC m RNA both in Ad?mi R?327 and Ad?mi R?327i group.The results of luciferase reporter assay indicated that mi R?327 was specifically bound to ARC 3? UTR region.2.In vivo:(1)MI/RI rat model was successfully established.Compared with the sham group,mi R?327 was significantly upregulated after MI/RI,accompanied with decrease of ARC both at m RNA and protein levels(P<0.05).(2)Adenovirus vectors were successfully transfected into myocardia tissue.High expression of green fluorescence was observed in the Ad-NC,Ad?mi R?327 and Ad?mi R?327i group.(3)mi R-327 regulated the expression of ARC in myocardium after MI/RI.Compared with Ad-NC group,Ad?mi R?327i remarkably decreased mi R-327 and increased ARC protein expression,while Ad?mi R?327 displayed an opposite effect(P<0.05).(4)Inhibition of mi R-327 attenuated MI/RI and apoptosis response.Compared with Ad-NC group,Ad?mi R?327i obviously decreased infarct size,LDH level,apoptotic index and cleaved Caspase-3 expression,whereas the Ad?mi R?327 performed an opposite effect(All P<0.05).(5)Inhibition of mi R-327 suppressed both extrinsic and intrinsic apoptosis?associated molecules in vivo.Downregulation of mi R-327 obviously inhibited the expression of proapoptotic molecules including cleaved Caspase-9,Caspase-8,Cyt-c,Bax,Fas and Fas L,and increased the expression of Bcl-2,whereas upregulation of mi R-327 displayed an opposite effect(All P<0.05).Conclusion Inhibition of mi R?327 could suppress both extrinsic and intrinsic apoptotic cascades by targeting ARC,which reduce cardiomyocytes apoptosis and protect myocardium from MI/RI. |
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