MiR-873对缺氧复氧诱导的心肌细胞凋亡的影响及其机制

目的：探讨微小RNA(micro RNA，miR)-873在心肌细胞缺氧复氧(hypoxia reoxygenation，H/R)损伤中的作 用及其相关机制。方法：采用体外培养大鼠H9c2心肌细胞构建H/R模型，并转染miR-873模拟物(mimic)，实验分为对 照组、H/R组、阴性对照组及miR-873 mimic组。Real-time PCR检测各组miR-873的表达水平；蛋白质印迹法检测egl-9 家庭缺氧诱导因子3(egl-9 family hypoxia inducible factor 3，Egln3)、B淋巴细胞瘤-2基因(B-cell lymphoma-2，Bcl-2)和Bcl-2 相关X蛋白(Bcl-2 associated X protein，Bax)的表达水平；ELISA试剂盒以及天冬氨酸特异性半胱氨酸蛋白酶-3(cysteinecontaining， aspartate-specific proteases-3，caspase-3)活性检测试剂盒分别用来检测细胞凋亡和caspase-3活性；采用双荧 光素酶报告基因系统检测miR-873对Egln3的作用靶点，并将实验分为阴性对照组、携带野生型(wild type，WT)报告 基因的Egln3 3'-非编码区(3'-untranslated regions，3'-UTR)组(WT组)和携带突变型(mutant type，MUT)报告基因的Egln3 3'-UTR组(MUT组)。为进一步检测Egln3对miR-873 mimic作用的影响，实验构建了Egln3过表达细胞，并将其分为H/R 组、H/R+miR-873 mimic组、H/R+pcDNA3-Egln3(pcEgln3)组和H/R+miR-873 mimic+pcEgln3组。结果：与对照组相比， H/R组中miR-873表达显著下调，差异有统计学意义(P<0.05)。与H/R组相比，miR-873 mimic组中H9c2心肌细胞凋亡 和caspase-3活性下降，Bax/Bcl-2比值下降，差异均有统计学意义(均P<0.05)；荧光素酶活性检测结果表明：与阴性对 照组相比，WT组荧光素酶活性显著下调，差异有统计学意义(P<0.05)，而MUT组荧光素酶活性无明显变化，差异无 统计学意义(P>0.05)。过表达实验结果表明：与H/R组相比，miR-873 mimic组细胞凋亡和Bax/Bcl-2比值均显著下降(均 P<0.05)；与miR-873 mimic组相比较，H/R+pcEgln3组和H/R+miR-873 mimic+pcEgln3组细胞凋亡和Bax/Bcl-2比值显著上 调，差异均有统计学意义(均P<0.05)。结论：MiR-873通过靶向作用于Egln3而抑制H/R心肌细胞凋亡。

Effect of miR-873 on cardiomyocyte apoptosis induced by hypoxia reoxygenation and its mechanism

Objective: To explore the role of miR-873 in cardiomyocyte injury induces by hypoxiareoxygenation (H/R) and its related mechanisms. Methods: H/R model was established by culturing mouse cardiac H9c2 cells in vitro, and miR-873 mimic was transfected. The experiments were divided into a control group, a H/R group, a negative control group and a miR-873 mimic group. The expression of miR-873 was measured using realtime PCR. The protein expression levels of egl-9 family hypoxia inducible factor 3 (Egln3), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) were evaluated by Western blotting. Cell apoptosis ELISA kit and cysteine-containing, aspartate-specific proteases-3 (caspase-3) activity kit was used to detect cell apoptosis and caspase-3 activity, respectively. The targeting effect of miR-873 on Egln3 were examined by the dual luciferase report gene assay, and the experiments were divided into a negative control group, a Egln3 3'-untranslated regions (3’-UTR) WT group (WT group) and a Egln3 3'-UTR MUT group (MUT group). In order to further detect the effects of Egln3 on miR- 873 mimics, the Egln3 overexpressed cells were constructed, and the experiments were divided into a H/R group, a H/R+miR-873 mimic group, a H/R+pcDNA3-Egln3 (pcEgln3) group and a H/R+ miR-873 mimic+pcEgln3 group. Results: Compared with the control group, the expression level of miR-873 was significantly decreased in the H/R group (P<0.05). Compared with the H/R group, H9c2 cell apoptosis, caspase-3 activity and the ratio of Bax/Bcl-2 were significantly reduced in the miR-873 mimic group (all P<0.05). Compared with the negative control group, the luciferase activity was significantly down-regulated in the WT group (P<0.05), while the luciferase activity was not significantly changed in the MUT group (P>0.05). In the over-expression experiment, compared with the H/R group, the cell apoptosis and the ratio of Bax/Bcl-2 were significantly reduced in the miR-873 mimic group (both P<0.05). Compared with miR-873 mimic group, the cell apoptosis and the ratio of Bax/Bcl-2 were significantly up-regulated in the H/R+pcEgln3 group and the H/R+miR-873 mimic+pcEgln3 group (all P<0.05). Conclusion: MiR-873 can inhibit H/R- induced apoptosis of cardiomyocyte via targeting Egln3.