miR-425-5p通过靶向HSPB8表达介导心肌缺血再灌注损伤

miR-425-5p mediating myocardial ischemia-reperfusion injury by targeting HSPB8 expression

目的:探讨miR-425-5p及其下游靶点HSPB8对心肌缺血再灌注损伤(MIRI)的影响及相关机制。方法:将AC16细胞随机分为control组,H/R组,H/R+miR-NC组,H/R+miR-425-5p inhibitor组,H/R+miR-425-5p mimic组,H/R+OE-NC组,H/R+OEHSPB8组,细胞计数试剂盒-8(CCK-8)法测定AC16的增殖能力;酶联免疫吸附测定(ELISA)法检测超氧化物歧化酶(SOD)、丙二醛(MDA)、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6水平,实时荧光定量PCR(RT-qPCR)和蛋白质免疫印迹法(western blotting)分别检测凋亡相关蛋白Caspase-3、Bax、Bcl-2的mRNA和蛋白表达情况,荧光素酶报告基因检测验证miR-425-5p及HSPB8间的靶向关系。结果:miR-425-5p在MIRI患者中表达显著高于正常人群;HSPB8在MIRI患者中表达低于正常人群;与control组相比,H/R组、H/R+miR-NC组、H/R+miR-425-5p mimic组、H/R+OE-NC组细胞活力降低(P<0.01),凋亡、炎症及氧化应激水平上升(P<0.01);与control组相比,H/R+miR-425-5p inhibitor组和H/R+OE-HSPB8组细胞活力显著增强(P<0.01),凋亡、炎症及氧化应激水平下降(P<0.01);与H/R+OE-NC组相比,H/R+OE-HSPB8组凋亡、炎症和氧化应激水平显著降低(P<0.01);靶向关系结果显示,miR-425-5p和HSPB8之间存在靶向作用关系。结论:miR-425-5p可以靶向HSPB8介导MIRI,抑制miR-425-5p可通过上调HSPB8来减轻MIRI。

Objective:To investigate the effect of miR-425-5p and its downstream target HSPB8 on myocardial ischemia-reperfusion injury(MIRI)and the related mechanisms.Methods:AC16 cells were randomly divided into control group,H/R group,H/R+miR-NC group,H/R+miR-425-5p inhibitor group,H/R+miR-425-5p mimic group,H/R+OE-NC group,and H/R+OE-HSPB8 group.The proliferation capacity of AC16 was determined by cell counting kit-8(CCK-8)method.The levels of superoxide dismutase(SOD),malonaldehyde(MDA),tumor necrosis factor-α(TNF-α)and interleukin(IL)-6 were detected by enzyme-linked immunosorbent assay(ELISA).The RNA and protein expression of Caspase-3,Bax and Bcl-2 was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and western blotting.The targeting relationship between miR-425-5p and HSPB8 was verified by luciferase reporter gene.Results:The expression of miR-425-5p in patients with MIRI was significantly higher than that in the normal population,and the expression of HSPB8 in patients with MIRI was significantly lower than that in the normal population.Compared with the control group,the cell viability of H/R group,H/R+miR-NC group,H/R+miR-425-5p mimic group,and H/R+OE-NC group decreased(P<0.01)and apoptosis,inflammation as well as oxidative stress levels increased(P<0.01).Compared with the control group,the cell viability of H/R+miR-425-5p inhibitor group and H/R+OE-HSPB8 inhibitor group significantly increased(P<0.01)and apoptosis,inflammation as well as oxidative stress levels decreased(P<0.01).Compared with the H/R+OE-NC group,apoptosis,inflammation as well as oxidative stress levels in the H/R+OEHSPB8 group significantly decreased(P<0.01).The results of targeting relationship showed that there was a targeting relationship between miR-425-5p and HSPB8.Conclusion:miR-425-5p can target HSPB8 to mediate MIRI,and inhibition of miR-425-5p can reduce MIRI by up-regulation of HSPB8.