miR-499与miR-1对心肌细胞增殖与凋亡调控作用及机制研究

Study on the regulation of miR-499 and miR-1 on cardiomyocyte proliferation and apoptosis and the mechanism

目的:探究心肌细胞增殖与凋亡过程中,miR-499与miR-1所发挥的调控作用及其机制。方法:采用Lipofectamine^TM2000将miR-499 mimics和miR-1 inhibitor转染至H9C2心肌细胞,并采用RT-PCR检测转染情况。实验分组:空白对照组、H2O2组(未转染的H9C2心肌细胞)、干预组A(miR-499 mimics转染的H9C2心肌细胞)、干预组B(miR-1 inhibitor转染的H9C2心肌细胞)、干预组C(miR-499 mimics+miR-1 inhibitor转染的H9C2心肌细胞)。通过H2O2建立心肌细胞氧化应激模型。采用CCK8法检测细胞增殖情况,流式细胞仪检测细胞凋亡情况,Western Blot检测Bcl-xl、Bax蛋白表达水平。结果:H2O2组细胞增殖较空白对照组减少(P<0.05),细胞凋亡率较空白对照组增加(P<0.05)。干预组A、干预组B、干预组C细胞增殖较H_2O_2组增加(P<0.05),而细胞凋亡率较H2O2组减少(P<0.05)。H2O2组的Bcl-xl蛋白表达水平较空白对照组降低(P<0.05),而Bax蛋白表达水平较空白对照组升高(P<0.05)。干预组A、干预组B、干预组C Bcl-xl蛋白表达水平较H2O2组升高(P<0.05),Bax蛋白表达水平较H2O2组降低(P<0.05)。结论:miR-499与miR-1对心肌细胞增殖与凋亡具有重要调控作用,miR-499可能通过上调Bcl-xl、下调Bax蛋白表达抑制心肌细胞凋亡,而miR-1则通过下调Bcl-xl、上调Bax蛋白表达促进心肌细胞凋亡。

Objective:To investigate the regulation of miR-499 and miR-1 on cardiomyocyte proliferation and apoptosis and the mechanism.Methods:MiR-499 mimics and miR-1 inhibitor were transfected into H9C2 cardiomyocytes by LipofectamineTM 2000.The blank control group,the H2O2 group(H9C2 cardiomyocytes not transfected),the intervention group A(H9C2 cardiomyocytes transfected with miR-499 mimics),the intervention group B(H9C2 cardiomyocytes transfected with miR-1 inhibitor)and the intervention group C(H9C2 cardiomyocytes transfected with miR-499 mimics+miR-1 inhibitor)were set up.The oxidative stress models of H9C2 cardiomyocytes were established by H2O2.The cell proliferation of each group was detected by CCK-8.The apoptosis of each group was detected by flow cytometry.The expression levels of Bcl-xl and Bax were detected by Western Blot.Results:Compared with the blank control group,the cell proliferation significantly decreased and the apoptosis ratesignificantly increased in the H2O2 group.The difference was statistically significant(P<0.05).Compared with the H2O2 group,the cell proliferation significantly increased and the apoptosis rate significantly decreased in the intervention group A and the intervention group B.The change was more significant in the intervention group C,and the difference was statistically significant(P<0.05).Compared with the blank control group,the expression of Bcl-xl significantly decreased while the expression of Bax significantly increasedin the H2O2 group.The difference was statistically significant(P<0.05).Compared with the H2O2 group,the expression of Bcl-xl significantly increased while the expression of Bax significantly decreased in the intervention group A and the intervention group B.The change was more significant in the intervention group C,and the difference was statistically significant(P<0.05).Conclusion:MiR-499 and miR-1 play an important role in regulating cardiomyocyte proliferation and apoptosis.MiR-499 inhibits cardiomyocyte apoptosis by up-regulating Bcl-xl and down-regulating Bax while miR-1 promotes cardiomyocyte apoptosis by down-regulating Bcl-xl and up-regulating Bax.