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miR-30e-5p靶向BIM调控缺氧诱导细胞凋亡的应用研究
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Abstract:
目的：研究miR-30e-5p与H2O2损伤后心肌细胞凋亡的关系，探讨miR-30e-5p靶向BIM基因调控H2O2损伤后心肌细胞凋亡的作用，为心肌梗死的诊断以及治疗提供新的靶点。方法：将40天CA16分为正常组(N)、缺氧组(H)、过表达对照组(NC+H)、miR-30e-5p过表达组(miR+H)。N组置于正常培养箱下培养；H组置于缺氧培养箱培养24 h；NC+H组与miR+H组分别为稳定感染装载有miR-NC和miR-30e-5p的重组慢病毒后缺氧处理24 h。应用RT-qPCR检测CA16中miR-30e-5p和BIM的mRNA表达水平，应用Caspase-3活性实验和流式细胞技术检测hiPSC-CMs的凋亡水平。利用生物信息学miRNA数据库预测miR-30e-5p的靶基因，并应用双荧光素酶报告基因实验进行验证。结果：1) 与N组比较，H组miR-30e-5p表达水平明显下调；与NC+H组比较，miR+H组miR-30e-5p表达水平则明显上调。2) 与N组比较，H组Caspase-3活性水平明显升高；与NC+H组比较，miR+H组Caspase-3活性水平下降。3) 与N组比较，H组CA16细胞凋亡比例明显上升；与NC+H组比较，miR+H组CA16凋亡比例则明显下降。4) 与N组比较，H组蛋白BIM表达水平上调；与NC+H组比较，miR+H组蛋白BIM表达水平下调。5) 通过miRNA数据库分析发现BIM是miR-30e-5p的潜在靶基因之一。结论：1) miR-30e-5p调控缺氧诱导CA16的凋亡。2) BIM是miR-30e-5p的靶基因之一。3) miR-30e-5p靶向BIM调控缺氧诱导CA16的凋亡。4) miR-30e-5p在急性心肌梗死的发生、发展过程中可能发挥抑癌基因的作用。
Objective: To investigate the relationship between miR-30e-5p and myocardial cell apoptosis after H2O2 injury, and to explore the role of miR-30e-5p targeting BIM genes in regulating myocardial cell apoptosis after H2O2 injury, providing new targets for the diagnosis and treatment of myocardial infarction. Method: The 40 day CA16 was divided into normal group (N), hypoxia group (H), overexpression control group (NC+H), and miR-30e-5p overexpression group (miR+H). Group N was cultured in a normal incubator; Group H was incubated in a hypoxia incubator for 24 hours; The NC+H group and miR+H group were treated with hypoxia for 24 hours after stable infection with recombinant lentivirus loaded with miR-NC and miR-30e-5p, respectively. RT qPCR was used to detect the mRNA expression levels of miR-30e-5p and BIM in CA16, and Caspase-3 activity assay and flow cytometry were used to detect the apoptosis level of hiPSC-CMs. Using bioinformatics miRNA database to predict target genes of miR-30e-5p, and validating with dual luciferase reporter gene experiments. Result: 1) Compared with group N, the expression level of miR-30e-5p in group H was significantly down-regulated; Compared with the NC+H group, the expression level of miR-30E-5p in the miR+H group was significantly up-regulated. 2) Compared with group N, the activity level of Caspase-3 in group H was significantly increased; Compared with NC+H group, the activity level of Caspase-3 in miR+H group was decreased. 3) Compared with group N, the apoptosis ratio of CA16 cells in group H was significantly increased; Compared with NC+H group, the apoptosis ratio of CA16 in miR+H group was significantly decreased. 4) Compared with group N, BIM expression level of histone
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