摘要
目的 探讨Circ-SMG6对缺氧/复氧诱导的心肌细胞损伤的影响及对miR-132-3p/BTG2轴的调节作用.方法 心肌H9c2细胞分为:NC组(不做任何处理)、缺氧/复氧组、缺氧/复氧+si-NC组、缺氧/复氧+si-Circ-SMG6组、缺氧/复氧+si-Circ-SMG6+anti-NC组、缺氧/复氧+si-Circ-SMG6+anti-miR-132-3p组.RT-qPCR检测miR-132-3p、Circ-SMG6表达;Western blot检测BTG2、Bcl-2、Bax、cleaved-Caspase-3蛋白水平;ELISA法检测SOD、MDA以及IL-1β、IL-6、TNF-α水平CCK8法测定心肌细胞增殖;流式细胞术检测心肌细胞凋亡;双荧光素酶验证Circ-SMG6与miR-132-3p,miR-132-3p与BTG2靶向关系.结果 与NC组相比,缺氧/复氧组、缺氧/复氧+si-NC组BTG2 mRNA及蛋白、Circ-SMG6水平、MDA、IL-1β、IL-6、TNF-α含量、凋亡率及Bax、cleaved-Caspase-3蛋白水平显著增加(P<0.01),miR-132-3p水平、SOD含量、72 h的细胞活力、Bcl-2蛋白水平显著降低(P<0.01).与缺氧/复氧组相比,缺氧/复氧+si-Circ-SMG6组BTG2 蛋白、Circ-SMG6水平、MDA、IL-1β、IL-6、TNF-α含量、凋亡率及Bax、cleaved-Caspase-3蛋白水平显著降低(P<0.01),miR-132-3p水平、SOD含量、72 h的细胞活力、Bcl-2蛋白水平显著增加(P<0.01).与缺氧/复氧+si-Circ-SMG6组相比,缺氧/复氧+si-Circ-SMG6+anti-miR-132-3p组BTG2蛋白、MDA、IL-1β、IL-6、TNF-α含量、凋亡率及Bax、cleaved-Caspase-3蛋白水平显著增加(P<0.01),miR-132-3p水平、SOD含量、72 h的细胞活力、Bcl-2蛋白水平显著下降(P<0.01).结论 敲低Circ-SMG6可能通过调节miR-132-3p/BTG2轴减轻缺氧/复氧诱导的心肌细胞损伤.
Abstract
AIM To investigate the influence of Circ-SMG6 on myocardial cell injury induced by hypoxia/reoxygenation and its regulatory effect on miR-132-3p/BTG2 axis.METHODS Myocardial H9c2 cells were grouped into:NC group(without any treatment),hypoxia/reoxygenation group,hypoxia/reoxygenation+si-NC group,hypoxia/reoxygenation+si-Circ-SMG6 group,hypoxia/reoxygenation+si-Circ-SMG6+anti-NC group,and hypoxia/reoxygenation+si-Circ-SMG6+anti-miR-132-3p group.The expressions of miR-132-3p and Circ-SMG6 were detected using RT-qPCR and the protein levels of BTG2,Bcl-2,Bax and cleaved-Caspase-3 were detected using Western blot.The levels of SOD,MDA,IL-1β,IL-6 and TNF-α were detected using ELISA method and cardiomyocyte proliferation was measured using CCK8 method.Flow cytometry was applied to detect cardiomyocyte apoptosis and dual luciferase was applied to verify the targeting relationship between Circ-SMG6 and miR-132-3p,and between miR-132-3p and BTG2.RESULTS Compared with those in NC group,the levels of BTG2 mRNA and protein,level of Circ-SMG6,contents of MDA,IL-1β,IL-6,TNF-α,apoptosis rate,the protein levels of Bax and cleaved-Caspase-3 in hypoxia/reoxygenation group and hypoxia/reoxygenation+si-NC group increased obviously(P<0.01),and the level of miR-132-3p,content of SOD,72 h cell viability and the protein level of Bcl-2 decreased obviously(P<0.01).Compared with those in hypoxia/reoxygenation group,the levels of BTG2,level of Circ-SMG6,contents of MDA,IL-1β,IL-6,TNF-α,apoptosis rate,the protein levels of Bax and cleaved-Caspase-3 in hypoxia/reoxygenation+si-Circ-SMG6 group decreased obviously(P<0.01),and the level of miR-132-3p,content of SOD,72 h cell viability and the protein level of Bcl-2 increased obviously(P<0.01).Compared with those in hypoxia/reoxygenation+si-Circ-SMG6 group,the levels of BTG2 protein,contents of MDA,IL-1β,IL-6,TNF-α,apoptosis rate,the protein levels of Bax and cleaved-Caspase-3 in hypoxia/reoxygenation+si-Circ-SMG6+anti-miR-132-3p group increased obviously(P<0.01),and the level of miR-132-3p,content of SOD,72 h cell viability and the protein level of Bcl-2 decreased obviously(P<0.01).CONCLUSION Knockdown of Circ-SMG6 may alleviate hypoxia/reoxygenation-induced cardiomyocyte injury by regulating the miR-132-3p/BTG2 axis.
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