miR-214-5p靶向PAK4对缺血再灌注大鼠的心肌损伤和免疫反应的调节作用

Regulatory effect of miR-214-5p on myocardial injury and immune response in ischemia-reperfusion rats by targeting PAK4

目的:探讨微小RNA-214-5p(miR-214-5p)靶向p21活化蛋白激酶4(PAK4)对缺血再灌注(I/R)大鼠的心肌损伤和免疫反应的作用。方法:将健康雄性SD大鼠随机分为假手术(sham)组、I/R组、Ad-Scramble组和Ad-miR-214组(n=9)。在大鼠左心室前壁6个不同的位点注入腺病毒;4 d后,缝合左前部下行冠状动脉构建I/R模型。RT-qPCR检测miR-214的表达水平,HE染色观察心肌损伤,ELISA检测心肌损伤标志物(CK-MB、Mb和cTnI)和炎症因子(IL-6、IL-1β和TNF-α)的水平,流式细胞术检测心肌细胞凋亡率,试剂盒检测MDA含量和SOD活性,基因软件预测靶基因并通过双萤光素酶报告验证,Western blot检测caspase-3、caspase-9、Bcl-2、Bax、PAK4、p-Akt和p-mTOR表达水平。结果:miR-214在I/R大鼠心肌细胞中低表达(P<0.01);miR-214-5p过表达减轻I/R大鼠心肌损伤程度,下调CK-MB、Mb和cTnI表达,降低心肌细胞凋亡率,上调Bcl-2表达,下调Bax、caspase-3和caspase-9表达,提高SOD活性,降低MDA、IL-6、IL-1β和TNF-α的含量(P<0.01);miR-214-5p与PAK4在3’-UTR存在结合位点,miR-214-5p过表达上调PAK4、p-Akt和p-mTOR表达(P<0.01)。结论:miR-214-5p过表达靶向PAK4减轻I/R大鼠的心肌损伤,并抑制心肌细胞凋亡、氧化应激反应和炎症反应,同时激活PI3K/Akt/mTOR通路。

AIM:To investigate the effect of microRNA-214-5 p(miR-214-5 p)on myocardial injury and immune response in rats with ischemia-reperfusion(I/R)by targeting p21-activated protein kinase 4(PAK4).METHODS:The rats were divided into sham group,I/R group,Ad-Scramble group,and Ad-miR-214 group(n=9).Adenovirus was injected into 6 different sites on the anterior wall of the left ventricle of the rats.Four days later,the I/R model was constructed by suturing the left anterior descending coronary artery.The expression level of miR-214 was detected by RT-qPCR.Myocardial injury was observed by HE staining.The levels of heart damage markers(CK-MB,Mb,and cTnI)and inflammatory factors(IL-6,IL-1βand TNF-α)were measured by ELISA.The rate of cardiomyocyte apoptosis was analyzed by flow cytometry.The content of MDA and the activity of SOD were detected by commercially available kits.Target genes were predicted by genetic software and verified by dual-luciferase reporter assay.The protein levels of caspase-3,caspase-9,Bcl-2,Bax,PAK4,p-Akt and p-mTOR were determined by Western blot.RESULTS:miR-214 was down-regulated in the cardiomyocytes of I/R rats(P<0.01).Over-expression of miR-214-5 p attenuated myocardial injury in the I/R rats,down-regulated the expression of CK-MB,Mb and cTnI,decreased the apoptotic rate of cardiomyocytes,up-regulated the expression of Bcl-2,down-regulated Bax,caspase-3 and caspase-9 expression,increased SOD activity,and decreased the content of MDA,IL-6,IL-1βand TNF-α(P<0.01).The binding sites of miR-214-5 p and PAK4 were pre-sent in the 3’-UTR,and over-expression of miR-214-5 p up-regulated the protein levels of PAK4,p-Akt and p-mTOR(P<0.01).CONCLUSION:miR-214-5 p over-expression attenuates myocardial injury in I/R rats by targeting PAK4,inhibits cardiomyocyte apoptosis,oxidative stress and inflammation,and activates the PI3 K/Akt/mTOR pathway.