[1]王艳,赵然尊,刘德斌,等.miR-214 靶向抑制CaMKⅡ调控H9c2心肌细胞的氧化应激[J].第三军医大学学报,2019,41(13):1206-1215.
 WANG Yan,ZHAO Ranzun,LIU Debin,et al.MiR-214 regulates oxidative stress in H9c2 myocardial cells by targeted inhibition of CaMKⅡ[J].J Third Mil Med Univ,2019,41(13):1206-1215.

miR-214 靶向抑制CaMKⅡ调控H9c2心肌细胞的氧化应激(/HTML )




MiR-214 regulates oxidative stress in H9c2 myocardial cells by targeted inhibition of CaMKⅡ
贵州 遵义,遵义医科大学附属医院心内科
WANG Yan ZHAO Ranzun LIU Debin LIU Weiwei LI Chaofu WANG Zhenglong CHEN Wenming SHI Bei

Department of Cardiology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou Province, 563000, China

miR-214 H9c2 myocardial cells reactive oxygen species superoxide dismutase malondialdehyde CaMKⅡ
R322.11; R363.22; R394.3

目的探讨 miR-214靶向抑制CaMKⅡ调节缺氧复氧诱导H9c2心肌细胞氧化应激的作用。方法采用大鼠H9c2心肌细胞为研究对象,实验分4组,①正常组:正常培养的心肌细胞;②缺氧复氧组:心肌细胞经缺氧24 h复氧6 h处理;③miR-214模拟物组:心肌细胞转染miR-214模拟物后予缺氧复氧处理;④miR-214模拟物阴性对照组:心肌细胞转染miR-214模拟物阴性对照后予以缺氧复氧处理。予以CCK-8法检测各组细胞活性,qPCR检测各组细胞miR-214的变化,流式细胞术、TUNEL法及 SOD、MDA试剂盒检测各组细胞氧化应激水平及凋亡效应;双荧光素酶报告基因检测miR-214与CaMKⅡ相关性,qPCR和Western blot检测CaMKⅡ mRNA和蛋白表达变化。随后用腺病毒转染过表达细胞中CaMKⅡ进行回复实验,并采用上述方法检测细胞氧化应激水平及凋亡状态。结果CCK-8检测结果显示,与正常组比较,缺氧复氧组心肌细胞活性下降,miR-214模拟物组可明显改善该作用(P<0.05)。qPCR结果显示,与正常组比较,缺氧复氧组miR-214表达下降,而miR-214模拟物组miR-214高表达(P<0.05)。与正常组比较,缺氧复氧组细胞ROS、MDA表达水平及TUNEL阳性细胞数明显增高,SOD表达显著下降(P<0.05);与缺氧复氧组比较,miR-214模拟物组ROS、MDA水平及TUNEL阳性细胞数明显下降,SOD表达明显增高(P<0.05)。经TargetScan检测发现miR-214与CaMKⅡ存在结合位点,双荧光素酶报告基因也验证了在H9c2心肌细胞中miR-214可结合CaMKⅡ。此外,qPCR及Western blot结果显示,与正常组比较,缺氧复氧可致CaMKⅡ在mRNA及蛋白水平表达升高,miR-214可显著抑制缺氧复氧诱导的CaMKⅡ表达。采用腺病毒过表达细胞中CaMKⅡ后,miR-214抗氧化应激及凋亡效应被部分阻断。结论过表达 miR-214可通过抑制CaMKⅡ转录后翻译过程,改善H9c2心肌细胞氧化应激水平及凋亡状态。


ObjectiveTo explore the regulative effect of miR-214 on oxidative stress injury induced by hypoxia reoxygenation (H/R) in H9c2 myocardial cells though targeted inhibition of CaMKⅡ. MethodsRat myocardial H9c2 cells were randomly divided into 4 groups, ①control group: H9c2 cells without any treatment; ② H/R group: the myocardial cells were cultured in hypoxia for 24 h and then reoxygenation for 6 h; ③ mimics+H/R group: the myocardial cells were transfected with miR-214 and then cultured with H/R as above; ④mimics negative control (MNC)+H/R group: the myocardial cells were transfected with miR-214’s mimics negative control and then cultured with H/R as above. CCK-8 assay was used to detect the viability of cells. qPCR was adopted to detect the expression of miR-214. Flow cytometry, TUNEL assay, and SOD or MDA detect kits were used to determine the reactive oxygen species (ROS) levels and cell apoptosis. The association of miR-214 with CaMKⅡ was examined by double-luciferase reporter assay, and their changes at mRNA and protein levels were detected by qPCR and Western blotting. After H9c2 cells were transfected with recombinant adenovirus carrying CaMKⅡ gene or the control adenovirus, the regulative effect of miR-214 on oxidative stress of cells was examined by the above methods. ResultsCCK-8 assay showed that compared with normal group, the cell viability of H/R group was significantly decreased (P<0.05), while miR-214 overexpression could obviously increase the cell viability (P<0.05). qPCR results showed that the expression level of miR-214 was markedly downregulated in the H/R group than the control group, while notable increase of miR-214 was observed in the miR-214 mimics group (P<0.05). Compared with the normal group, the ROS production, MDA level and TUNEL positive cells were obviously increased, while the SOD level was significantly decreased in H/R group (P<0.05), but opposite trends were observed when  the HR group compared to and the miR-214 mimics group (P<0.05). TargetScan server indicated that there was binding site of miR-214 and CaMKⅡ. The dual luciferase reporter assay also confirmed that miR-214 could bind to CaMKⅡ in H9c2 cells. In addition, qPCR and Western blot results showed that H/R treatment increased the expression of CaMKⅡ at mRNA and protein levels, but miR-214 significantly inhibited the enhanced expression induced by H/R. Overexpression of CaMKⅡ in H9c2 cells resulted in partially blocked effects of miR-214 on oxidative stress and apoptosis induced by H/R treatment. ConclusionOverexpression of miR-214 can regulate oxidative stress and apoptosis of H9c2 cells by inhibiting CaMKⅡ at the transcription level.


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更新日期/Last Update: 2019-07-08