MicroRNA-1和microRNA-21在缺血预处理、后处理及远端预处理中的表达变化

目的 通过离体缺血-再灌注心脏模型,观察缺血预处理(IPC)、缺血后处理(IPO)和肢体远端预处理(RIPC)后心脏microRNA1(miRNA-1)和microRNA21 (miRNA-21)的表达变化,以及它们所调控靶蛋白热休克蛋白70 (HSP70)和程序性细胞死亡4(PDCD4)表达变化,期望从miRNA调控水平揭示心脏的内源性保护机制.方法 取Sprague-Dawley (SD)大鼠心脏,建立离体Langendorff心肌缺血-再灌注模型,随机分为4组(每组12只),对照组、IPC组、IPO组和RIPC组.检测各组血流动力学指标,蛋白印迹法(Western blotting)检测PpDCD4、HSP70、B细胞淋巴瘤/白血病-2(Bc1-2)和Bc1-2相关X蛋白(Bax)含量,taqman探针法检测miRNA-1和miRNA-21含量,末端脱氧核苷酸转移酶介导的原位缺口标记法(TUNEL)检测心肌细胞凋亡,2,3,5-氯化三苯基四氮唑(TTC)法检测心肌梗死面积. 结果 IPC组心肌的miRNA-1和miRNA-21表达明显高于对照组,但RIPC组和IPO组心肌的miRNA-1表达较对照组明显降低( P＜0.05).IPC组、RIPC组和IPO组心肌中HSP70、PDCD4和Bax蛋白含量较对照组明显减少(P＜ 0.05),Bc1-2蛋白含量各组间差异无统计学意义.IPC组、RIPC组和IPO组左室心肌梗死面积/左室总面积以及心肌细胞凋亡率明显低于对照组(P＜ 0.05). 结论 miRNA-1和miRNA-21在缺血预处理、缺血后处理和远端预处理后,表达变化是不同的,同时各处理组中miRNA与其靶蛋白并不都是负性调节关系.

Expression of MicroRNA-1, 21 in Ischemic Preconditioning, Ischemic Postconditioning and Remote Ischemic Preconditioning in an Isolated Rat Heart Model

Objective To observe the expression changes of microRNA 1(miRNA-1)and microRNA 21(miRNA-21)after ischemic preconditioning(IPC),ischemic postconditioning(IPO)and remote ischemic preconditioning(RIPC)in an ischemia-reperfusion rat heart model in vitro,as well as the expression of their target protein heat shock protein 70(HSP70)and programmed cell death 4(PDCD4),and evaluate whether miRNA are involved in endogenous cardioprotective mechanism.Methods The Langendorff-perfused Sprague-Dawley rat hearts were randomly assigned into one of the four groups,control group(CON group,n=12),ischemia preconditioning group(IPC group,n=12),ischemia postconditioning group(IPO group,n=12)and remote ischemia preconditioning group(RIPC group,n=12).Cardiac function was digitalized and analyzed.The expression of HSP70,PDCD4,B-cell lymphoma/leukemia-2(Bcl-2)and Bax was detected by Western blotting.The expression of miRNA-1 and miRNA-21 was detected by real-time reverse transcriotionpolymerase chain reaction(RT-PCR).Assessment of cardiac infarct size and myocardial apoptosis was determined using triphenyltetrazolium chloride(TTC)assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL)assay respectively.Results The expressions of miRNA-1 and miRNA-21 were up-regulated in IPC group,but the expression of miRNA-1 was down-regulated in RIPC group and IPO group(P < 0.05).The expressionsof PDCD4,HSP70 and Bax were down-regulated in ‘conditioning’ groups compared with CON group(P < 0.05).The expression of Bcl-2 was not statistically different among the four groups.The infarct size and the myocardial apoptosis in‘conditioning’hearts were significantly decreased compared with CON group(P < 0.05).Conclusion The expressions of the miRNA-1 and miRNA-21 are different in IPC,RIPC and IPO groups,and their target proteins are not inversely correlated with the miRNAs in all the ‘conditioning’ groups.