缺血再灌注损伤中miR-126的作用及对心肌细胞凋亡的影响
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摘要
目的:通过体内外实验探讨微小RNA(miR)-126在心肌缺血再灌注损伤中的作用及机制。方法:体外部分:心肌细胞H9C2分6组,建立心肌细胞模拟缺血/再灌注(simulated ischemia/reperfusion,SI/R)损伤模型,并分别转染miR-126mimic和miR-126inhibitor。qRT-PCR检测心肌细胞miR-126表达,流式细胞术检测细胞凋亡,Western blot检测caspase-3及其裂解片段的蛋白表达。体内部分:雌性Wistar健康大鼠分5组,建立缺血/再灌注损伤(ischemia/reperfusion,I/R)模型,并分别转染含有GFP的空病毒、miR-126mimic以及miR-126inhibitor的慢病毒。测定各组大鼠心肌梗死范围,TUNEL测定心肌细胞凋亡。结果:与对照组相比,心肌细胞H9C2SI/R后miR-126表达(0.32±0.02)明显降低(P=0.000)、细胞凋亡率(3.8±0.6)明显上升(P=0.000);与SI/R相比,采用miR-126mimic上调miR-126表达后细胞凋亡率(5.3±0.8)明显增加(P=0.007),caspase-3表达(0.450±0.003)明显降低(P=0.000),而采用miR-126inhibitor下调miR-126表达后细胞凋亡率(2.9±0.5)明显降低(P=0.000),caspase-3表达(1.80±0.03)明显升高(P=0.002)。与假手术组相比,I/R组和I/R+GFP组心肌梗死范围[(59.4±2.5),(60.2±2.8)]明显增加(P=0.000,P=0.000),而与I/R组相比,I/R+miR-126mimic组心肌梗死范围(69.0±2.4)明显增加(P=0.013),而I/R+miR-126inhibitor组心肌梗死范围(39.5±2.5)明显降低(P=0.000);TUNEL检测结果显示,与假手术组相比,I/R组和I/R+GFP组心肌细胞凋亡率[(33.4±1.7),(33.7±1.8)]明显升高(P=0.000,P=0.000);而与I/R组相比,I/R+miR-126mimic组心肌细胞凋亡率(42.2±2.3)明显增加(P=0.003),而I/R+miR-126inhibitor组心肌细胞凋亡率(25.5±1.5)明显降低(P=0.000)。结论:心肌缺血再灌注损伤会引起miR-126表达降低,而上调miR-126表达会促进心肌细胞凋亡,相反抑制miR-126表达可能有助于预防心肌缺血再灌注引起的心肌细胞凋亡。
Objective:To explore the mechanism of miR-126 in myocardial ischemia reperfusion injury. Methods:In vitro,myocardial cells H9C2 were divided into 6 groups,and the myocardial cell simulated ischemia/reperfusion(SI/R)injury model was established,transfecting with the miR-126 mimic and miR-126 inhibitor. The expression of miR-126 was detected with real time-PCR in myocardial cells;apoptosis was detected with flow cytometry;and the expression of caspase-3 and its fragmentation were detected with Western blot. In vivo,healthy female Wistar rats were divided into 5 groups,and ischemia/reperfusion injury model was established.They were respectively disposed with the transfection of empty GFP,miR-126 mimic and miR-126 inhibitor virus. Myocardial infarction range was detected and the myocardial cell apoptosis was detected with TUNEL. Results:Compared with those of control group,the expression of miR-126 in myocardial cell H9C2 after SI/R was significantly reduced and the rate of apoptosis increased significantly. Up-regulation of miR-126 expression by miR-126 mimic could raise apoptosis rate and reduce caspase-3 expression significantly. On the contrary,down-regulation miR-126 expression by miR-126 inhibitor could reduce apoptosis rate and raise caspase-3 expression significantly. Compared with that of control group,myocardial infarction range increased significantly in the I/R group and the I/R +GFP group. Compared with that of I/R group,myocardial infarction range in the I/R+miR-126 mimic group reduced significantly. TUNEL test results showed that compared with that of control group,the myocardial cell apoptosis rate in I/R group and the I/R+GFP group increased significantly. Compared with that of I/R group,the myocardial cell apoptosis rate in I/R+miR-126 mimic group increased significantly,while myocardial cell apoptosis rate in the I/R + miR-126 inhibitor group decreased significantly,with statistically significant differences(P<0.05).Conclusion:miR-126 is down-regulated in myocardial ischemia-reperfusion injury and that the promotion of miR-126 may promote myocardial cell apoptosis,while the inhibition of miR-126 may protect against myocardial cell apoptosis caused by ischemia-reperfusion.
Objective:To explore the mechanism of miR-126 in myocardial ischemia reperfusion injury. Methods:In vitro,myocardial cells H9C2 were divided into 6 groups,and the myocardial cell simulated ischemia/reperfusion(SI/R)injury model was established,transfecting with the miR-126 mimic and miR-126 inhibitor. The expression of miR-126 was detected with real time-PCR in myocardial cells;apoptosis was detected with flow cytometry;and the expression of caspase-3 and its fragmentation were detected with Western blot. In vivo,healthy female Wistar rats were divided into 5 groups,and ischemia/reperfusion injury model was established.They were respectively disposed with the transfection of empty GFP,miR-126 mimic and miR-126 inhibitor virus. Myocardial infarction range was detected and the myocardial cell apoptosis was detected with TUNEL. Results:Compared with those of control group,the expression of miR-126 in myocardial cell H9C2 after SI/R was significantly reduced and the rate of apoptosis increased significantly. Up-regulation of miR-126 expression by miR-126 mimic could raise apoptosis rate and reduce caspase-3 expression significantly. On the contrary,down-regulation miR-126 expression by miR-126 inhibitor could reduce apoptosis rate and raise caspase-3 expression significantly. Compared with that of control group,myocardial infarction range increased significantly in the I/R group and the I/R +GFP group. Compared with that of I/R group,myocardial infarction range in the I/R+miR-126 mimic group reduced significantly. TUNEL test results showed that compared with that of control group,the myocardial cell apoptosis rate in I/R group and the I/R+GFP group increased significantly. Compared with that of I/R group,the myocardial cell apoptosis rate in I/R+miR-126 mimic group increased significantly,while myocardial cell apoptosis rate in the I/R + miR-126 inhibitor group decreased significantly,with statistically significant differences(P<0.05).Conclusion:miR-126 is down-regulated in myocardial ischemia-reperfusion injury and that the promotion of miR-126 may promote myocardial cell apoptosis,while the inhibition of miR-126 may protect against myocardial cell apoptosis caused by ischemia-reperfusion.
引文
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[16]Jiang C,Ji N,Luo G,et al.The effects and mechanism of mi R-92a and mi R-126 on myocardial apoptosis in mouse ischemia-reperfusion model[J].Cell Biochem Biophys,2014,70(3):1901-1906.
[2]Chen L,Wang J,Wang B,et al.Mi R-126 inhibits vascular endothelial cell apoptosis through targeting PI3K/Akt signaling[J].Ann Hematol,2016,95(3):365-374.
[3]Wu K,Yang Y,Zhong Y,et al.The effects of microvesicles on endothelial progenitor cells are compromised in type 2 diabetic patients via downregulation of the mi R-126/VEGFR2 pathway[J].Am J Physiol Endocrinol Metab,2016,310(10):E828-837.
[4]Wang L,Lee AY,Wigg JP,et al.mi R-126 regulation of angiogenesis in age-related macular degeneration in CNV mouse model[J].Int J Mol Sci,2016,17(6):pii E895.
[5]刘付平,姚宏伟,李俊,等.大鼠心肌缺血再灌注损伤模型的改进[J].安徽医科大学学报,2003,38(3):234-236.
[6]Penna C,Brancaccio M,Tullio F,et al.Overexpression of the muscle-specific protein,melusin,protects from cardiac ischemia/reperfusion injury[J].Basic Res Cardiol,2014,109(4):418.
[7]Yang J,Fan Z,Yang J,et al.micro RNA-22 attenuates myocardial ischemia-reperfusion injury via an anti-inflammatory mechanism in rats[J].Exp Ther Med,2016,12(5):3249-3255.
[8]Wu Z,Qi Y,Guo Z,et al.mi R-613 suppresses ischemia-reperfusion-induced cardiomyocyte apoptosis by targeting the programmed cell death 10 gene[J].Biosci Trends,2016,10(4):251-257.
[9]Zuo Y,Wang Y,Hu H,et al.Atorvastatin protects Myocardium against ischemia-reperfusion injury through inhibiting mi R-199a-5p[J].Cell Physiol Biochem,2016,39(3):1021-1030.
[10]Gao CK,Liu H,Cui CJ,et al.Roles of Micro RNA-195 in cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion injury[J].J Genet,2016,95(1):99-108.
[11]Liu PY,Tian Y,Xu SY.Mediated protective effect of electroacupuncture pretreatment by mi R-214 on myocardial ischemia/reperfusion injury[J].J Geriatr Cardiol,2014,11(4):303-310.
[12]郑华峰,陶晶,张斌,等.Mi R-126在大鼠心肌缺血再灌注早期的表达及抗凋亡作用[J].中国心血管病研究,2015,13(11):1041-1046,1055-1056.
[13]Long G,Wang F,Li H,et al.Circulating mi R-30a,mi R-126 and let-7b as biomarker for ischemic stroke in humans[J].BMC Neurol,2013,16(13):178.
[14]郑志伟,朱志伟,郑滢,等.mi R-126对EA.hy926细胞血管内皮生长因子表达的调控作用[J].中国病理生理杂志,2014,30(1):30-34.
[15]Li Z,Li N,Wu M,et al.Expression of mi R-126 suppresses migration and invasion of colon cancer cells by targeting CXCR4[J].Mol Cell Biochem,2013,381(1-2):233-242.
[16]Jiang C,Ji N,Luo G,et al.The effects and mechanism of mi R-92a and mi R-126 on myocardial apoptosis in mouse ischemia-reperfusion model[J].Cell Biochem Biophys,2014,70(3):1901-1906.