MicroRNA-15b deteriorates hypoxia/reoxygenation-induced cardiomyocyte apoptosis by downregulating Bcl-2 and MAPK3
MicroRNA-15b deteriorates hypoxia/reoxygenation-induced cardiomyocyte apoptosis by downregulating Bcl-2 and MAPK3
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DOI：
10.1136/jim-2017-000485
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发表时间：
2018-01-01
影响因子：
2.6
通讯作者：
Wang, Xiaodong
中科院分区：
文献类型：
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作者：
Liu, Yaling;Yang, Liqun;Wang, Xiaodong
为了探讨miRNA-15b在急性心肌梗死(AMI)缺血再灌注损伤后心肌细胞凋亡中的作用，我们采用左前降支结扎法复制了大鼠急性心肌梗死模型，并在H9c2细胞上进行了缺氧/复氧实验。用定量逆转录聚合酶链式反应(qRT-PCR)检测miRNA-15b的表达。用末端脱氧核苷酸转移酶dUTP缺口末端标记法检测心肌细胞凋亡率。用脂质体将合成的miRNA-15b模拟物和抑制物导入H9c2细胞。采用定量逆转录聚合酶链式反应(qRT-PCR)检测B细胞淋巴瘤/白血病-2(Bcl2)和丝裂原活化蛋白激酶3(MAPK3)的RNA表达，并用免疫印迹法检测其蛋白表达。大鼠心肌缺血再灌注后，miRNA-15b表达增加，心肌细胞凋亡加重。在H9c2细胞中，缺氧/复氧诱导心肌细胞在缺氧24小时后miRNA-15b表达增加，心肌细胞凋亡率增加。与赋形剂组相比，miRNA-15b模拟物进一步上调了miRNA-15b的水平，增加了心肌细胞的凋亡，而miRNA-15b抑制剂抑制了miRNA-15b的表达，保护了心肌细胞的凋亡。虽然靶基因Bcl2和MAPK3的mRNA表达没有明显变化，但经miRNA-15b模拟处理后，这两个基因的蛋白表达显著降低，而在转染miRNA-15b抑制剂后，这两个基因的蛋白表达显著增加。结论：miRNA-15b通过转录后下调Bcl2和MAPK3的表达而加重心肌细胞的凋亡。
To investigate the role of miRNA-15b in cardiomyocyte apoptosis after ischemia reperfusion injury in acute myocardial infarction (AMI), we conducted the AMI rat model by using left anterior descending ligation and performed hypoxia/ reoxygenation experiments in H9c2 cells. MiRNA-15b was measured by quantitative reverse transcription PCR (qRT-PCR). Cardiomyocyte apoptosis was determined by terminal deoxynucleotide transferase dUTP nick end labeling staining. Synthesized miRNA-15b mimic and inhibitor were transfected into H9c2 cells by Lipofectamine regent. RNA expression of B cell lymphoma/leukemia-2 (Bcl-2) and mitogen-activated protein kinase 3 (MAPK3) was examined by qRT-PCR and their protein expression was determined by western blot. lschemia reperfusion increased miRNA-15b expression in the ischemic rat heart and resulted more severe cardiomyocytes apoptosis. In H9c2 cells, hypoxia/reoxygenation induced increased miRNA-15b expression and augmented cardiomyocyte apoptosis observed at 24 hours after 24-hour hypoxia. Compared with the vehicle group, miRNA-15b mimic further raised miRNA-15b level and increased cardiomyocyte apoptosis, whereas miRNA-15b inhibitor suppressed miRNA-15b expression and protected cardiomyocytes from apoptosis. Although the mRNA expression of the target genes Bcl-2 and MAPK3 was not changed significantly, the protein expression of these two genes were markedly reduced after miRNA-15b mimic treatment and significantly increased after transfected with miRNA-15b inhibitors. In conclusion, miRNA-15b deteriorates cardiomyocyte apoptosis by post-transcriptionally downregulating the expression of Bcl-2 and MAPK3.