微小RNA-124-3p通过靶向抑制RIPK1的表达对缺氧/复氧心肌细胞损伤中的保护作用及其机制研究

Proventive effect and mechanism of microRNA-124-3p on hypoxia/reoxygenation injury in cardiomyocytes through targeting inhibiting RIPK1 expression

目的探讨微小RNA-124-3p(miR-124-3p)调控受体相互作用蛋白激酶1(RIPK1)对心肌细胞缺氧/复氧(H/R)损伤的保护作用。方法大鼠心肌细胞随机分为Con组、H/R组、H/R+miR-124-3p组、H/R+miR-NC组、H/R+si-RIPK1组、H/R+siNC组、H/R+miR-124-3p+pcDNA组、H/R+miR-124-3p+pcDNA-RIPK1组。采用qRT-PCR法检测miR-124-3p与RIPK1 mRNA表达水平,Western blot法检测RIPK1蛋白表达情况,双荧光素酶报告基因检测验证miR-124-3p与RIPK1的靶向关系。流式细胞术检测细胞凋亡能力变化,Western blot法检测Bcl-2、Bax蛋白表达情况。检测乳酸脱氢酶(LDH)活性及丙二醛(MDA)、超氧化物歧化酶(SOD)含量。结果H/R组心肌细胞miR-124-3p表达水平显著低于Con组(P<0.05),而RIPK1 mRNA及蛋白水平均显著升高(P<0.05);H/R组心肌细胞LDH活性、MDA水平及Bax蛋白水平均显著高于Con组(P<0.05),细胞凋亡率显著升高(P<0.05),而SOD水平及Bcl-2蛋白水平均显著低于Con组(P<0.05);miR-124-3p过表达与抑制RIPK1表达可降低细胞凋亡率、LDH活性、MDA水平及Bax蛋白水平,而提高SOD水平及Bcl-2蛋白水平;双荧光素酶基因检测结果证实RIPK1是miR-124-3p的靶基因;RIPK1过表达逆转了miR-124-3p过表达对H/R心肌细胞损伤的作用。结论MiR-124-3p过表达可通过下调RIPK1表达进而减轻心肌细胞H/R损伤进而对心肌细胞发挥保护作用。

Objective To investigate the preventive effect of microRNA-124-3p(miR-124-3p)on hypoxia/reoxygenation(H/R)injury in cardiomyocytes through regulating receptor-interacting protein kinases1(RIPK1).Methods Rat cardiomyocytes were divided randomly into Con group,H/R group,H/R+miR-124-3p group,H/R+miR-NC group H/R+si-RIPK1,H/R+siNC group,H/R+miR-124-3p+pcDNA group and H/R+miR-124-3p+pcDNA-RIPK1 group.The mRNA expressions of miR-124-3p and RIPK1 were detected by using qRTPCR,and protein expression of RIPK1 was detected by using Western blotting assay.The targeting relationship between miR-124-3p and RIPK1 was detected by using dual-luciferase reporter gene assay.The apoptosis ability was detected by using flow cytometry(FCM),and protein expressions of B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)were detected by using Western blotting assay.The activity of lactic dehydrogenase(LDH)and content of malondialdehyde(MDA)and superoxide dismutase(SOD)were detected.Results The expression of miR-124-3p was significantly lower(P<0.05)and mRNA and protein expressions of RIPK1 were significantly higher(P<0.05)in H/R group than those in Con group.LDH activity and levels of MDA and Bax were significantly higher(P<0.05),apoptosis rate was significantly higher(P<0.05),and levels of SOD and Bcl-2 were significantly lower(P<0.05)in H/R than those in Con group.The overexpression of miR-124-3p and inhibition of RIPK1 expression decreased apoptosis,LDH activity and levels of MDA and Bax,and increased levels of SOD and Bcl-2.The results of dual-luciferase reporter gene assay showed that RIPK1 was the targeting gene of miR-124-3p,and overexpression of RIPK1 reversed the damage effect of overexpression of miR-124-3p on H/R cardiomyocytes.Conclusion The overexpression of MiR-124-3p can relieve H/R injury in cardiomyocytes through down-regulating RIPK1 expression,and play a protective role to cardiomyocytes.