下调miR-203a-3p通过靶向Hspb8抑制心肌细胞缺氧/复氧损伤

贾元春

下调miR-203a-3p通过靶向Hspb8抑制心肌细胞缺氧/复氧损伤

贾元春

Investigation of the down-regulation of miR-203a-3p inhibiting myocardial hypoxia/reoxygenation injury by targeting Hspb8

JIA Yuanchun

摘要

摘要:
目的： 探讨miR-203a-3p在心肌细胞缺氧/复氧损伤中的作用及其机制。
方法： 构建缺氧/复氧的细胞（H/R）及缺血再灌注动物模型（I/R），实时荧光定量链式聚合酶反应（RT-PCR）检测各组miR-203a-3p的表达水平；随后将细胞随机分为4组：空白对照组、H/R组（H/R处理后的H9c2细胞系）、H/R + miR-NC组（H/R处理后的H9c2细胞系转染miR-NC）、H/R + miR-203a-3p-inhibitor组（H/R处理后的H9c2细胞系转染miR-203a-3p-inhibitor），利用流式细胞术及TUNEL法对各组细胞的凋亡率进行检测，对各组细胞外液中的CK及LDH水平进行生化检测；Western blotting检测缺氧/复氧处理后的H9c2及HCM细胞中Hspb8的表达水平；通过TargetScan在线工具预测到miR-203a-3p的潜在靶点Hspb8，并利用双荧光素酶报告实验对此进行验证；miR-203a-3p-mimic转染至H9c2细胞，Western blotting检测Hspb8的表达水平；为对Hspb8功能进行进一步验证，进行细胞转染实验，分为4组：空白对照组（Control + miR-NC + shRNA-Con）、H/R组（H/R + miR-NC + shRDNA-Con）、H/R + miR-203a-3p-inhibitor组（H/R + miR-203a-3p-inhibitor + shRNA-Con）、H/R + miR-203a-3p-inhibitor + shRNA-Hspb8组，利用流式细胞术对各组细胞的凋亡率进行检测，对各组细胞外液中的CK及LDH水平进行生化检测。
结果： miR-203a-3p在H/R处理的H9c2细胞系和I/R处理的大鼠心肌组织中的表达水平明显升高（P ＜ 0.01）；下调miR-203a-3p表达可抑制H/R诱导的H9c2细胞凋亡（P ＜ 0.01），降低细胞培养液中CK和LDH活性（P ＜ 0.01）；在WT组（野生型）H9c2和HCM细胞中，转染了miR-203a-3p-mimic的Hspb8相对荧光素酶活性明显降低（P ＜ 0.01）；miR-203a-3p过表达可增加细胞中Hspb8的表达（P ＜ 0.01）；与H/R + miR-203a-3p-inhibitor + shRNA-Con组相比，H/R + miR-203a-3p-inhibitor + shRNA-Hspb8组H9c2细胞的凋亡率明显增加（P ＜ 0.05），且细胞培养液中CK和LDH活性明显增加（P ＜ 0.01）。
结论： miR-203a-3p可通过靶向Hspb8抑制心肌细胞缺氧/复氧损伤。

Abstract:
Objective To investigate the role and mechanism of miR-203a-3p in myocardial hypoxia/reoxygenation injury.
Methods The hypoxia/reoxygenation cells (H/R) and ischemia/reperfusion animal models (I/R) were constructed, and the expression level of miR-203a-3p in each group was detected by quantitative real-time PCR (RT-PCR). The cells were divided into the control group, H/R groupH9c2 cell line treated with (H/R), H/R + miR-NC group (H/R processing H9c2 cell line transfected with miR-NC) and H/R + miR-203a-3p inhibitor group (H/R processing H9c2 cell line transfected with miR-203a-3p- inhibitor). The flow cytometry and TUNEL method were used to detect the apoptosis rate of cells in each group, and the levels of CK and LDH in extracellular fluid in four groups were biochemically detected. Western blotting was used to detect the expression levels of Hspb8 in H9c2 and HCM cells after hypoxia/reoxygenation treatment. The potential target of miR-203a-3p, Hspb8, was predicted by TargetScan online tool, and verified by double luciferase reporting experiment. The miR-203a-3p-mimic was transfected into H9c2 cells, and the expression level of Hspb8 was detected by Western blotting. To further verify the function of Hspb8, the cells were divided into the Control + miR-NC + shRNA-Con group, H/R + miR-NC + shRDNA-Con group, H/R + miR-203a-3p-inhibitor + shRNA-Con group and H/R + miR-203a-3p-inhibitor + shRNA-Hspb8 group, and the apoptosis rate of cells in each group was detected by flow cytometry, and the levels of CK and LDH in extracellular fluid of each group were biochemically detected.
Results The expression levels of miR-203a-3p significantly increased in the H/R-treated H9c2 cell lines and I/R-treated rat myocardial tissues (P ＜ 0.01). Down-regulating the expression of miR-203a-3p could inhibit the H/R-induced apoptosis in H9c2 cells (P ＜ 0.01) and decrease the CK and LDH activities in cell cultures (P ＜ 0.01). The relative luciferase activity of Hspb8 transfected with miR-203a-3p-mimic was significantly reduced in WT group (wild-type) H9c2 and HCM cells (P ＜ 0.01). The overexpression of miR-203a-3p could improve the Hspb8 expression in cells (P ＜ 0.01). Compared with the H/R + miR-203a-3p-inhibitor + shRNA-Con group, the apoptosis rate of H9c2 cells in the H/R + miR-203a-3p-inhibitor + shRNA-Hspb8 group significantly increased (P ＜ 0.05), and the activities of CK and LDH in the cell culture medium significantly increased (P ＜ 0.01).
Conclusions MiR-203a-3p inhibits hypoxia/reoxygenation injury of cardiomyocytes by targeting Hspb8.

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