miR-206-3p在缺氧复氧诱导的心肌细胞损伤中的作用及机制的研究

Effect of miR-206-3p in H9c2 cardiomyocyte injury induced by hypoxia/reoxygenation

目的探究miR-206-3p在缺氧/复氧(H/R)诱导的大鼠H9c2心肌细胞损伤中的作用及机制。方法体外培养H9c2细胞,建立H/R损伤心肌细胞模型,将细胞分为对照组(Control),H/R模型组,H/R+miRNA阴性对照组(H/R+miR-NC),H/R+miR-206-3p模拟物组(H/R+miR-206-3p mimics),H/R+miR-206-3p模拟物组+pcDNA阴性对照组(H/R+miR-206-3p mimics+pcDNA3.1)和H/R+miR-206-3p模拟物组+PDCD4过表达组(H/R+miR-206-3p mimics+pcDNA3.1-PDCD4),实时荧光定量PCR检测各组细胞中miR-206-3p和PDCD4的相对表达水平,Western blot检测各组细胞中PDCD4蛋白表达,Targetscan和双荧光素酶实验分析PDCD4与miR-206-3p的靶向关系,MTT法检测各组细胞活性,流式细胞术检测各组细胞凋亡。结果与对照组相比,H/R组miR-206-3p表达降低,PDCD4表达升高(P<0.05);与miR-NC组相比,miR-206-3p模拟物组中miR-206-3p表达升高,PDCD4表达降低(P<0.05)。Targetscan网站分析及双荧光素酶实验证实PDCD4为miR-206-3p的靶基因。相比miR-NC组,miR-206-3p模拟物组细胞活力升高,凋亡率下降;相比miR-206-3p mimics+pcDNA3.1组,PDCD4过表达组细胞活力下降,凋亡率升高。结论过表达miR-206-3p可以靶向负调控PDCD4在缺氧复氧诱导的心肌细胞损伤中发挥保护作用。

Objective To investigate the role and mechanism of miR-206-3p in hypoxia/reoxygenation(H/R)-induced H9c2 cell injury.Methods The H9c2 cells were cultured in vitro to establish H/R injury myocardial cell model,and were divided into control,H/R,H/R+miR-NC,H/R+miR-206-3p mimics,H/R+miR-206-3p mimics+pcDNA3.1 and H/R+miR-206-3p mimics+pcDNA3.1-PDCD4 group.The relative expression of miR-206-3p and PDCD4 was detected by qRT-PCR.The expression of PDCD4 was detected by Western blot.The targeting relationship between PDCD4 and miR-206-3p was analyzed by Targetscan and double luciferase experiment.The viability of H9c2 cells was detected by MTT.The apoptosis rate of H9c2 cells was detected by flow cytometry.Results Compared with the control group,the expression of miR-206-3p was decreased and the expression of PDCD4 was increased in H/R group(P<0.05).Compared with the miR-NC group,the expression of miR-206-3p was increased and the expression of PDCD4 was decreased in the miR-206-3p mimics group(P<0.05).Targetscan website analysis and double luciferase assay confirmed PDCD4 was the target gene of miR-206-3p.Compared with the miR-NC group,the cell viability of miR-206-3p mimics group was increased and the apoptosis rate was decreased.Compared with miR-206-3p mimics+pcDNA3.1 group,the cell viability of PDCD4overexpression group was decreased and the apoptosis rate was increased.Conclusion Over-expression of miR-206-3p can target to negatively regulate PDCD4 and play a protective role in hypoxia-reoxygenation-induced myocardial cell injury.