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Title: [MiR-224-5p overexpression inhibits oxidative stress by regulating the PI3K/Akt/FoxO1 axis to attenuate hypoxia/reoxygenation-induced cardiomyocyte injury].
Author: Liang G, Tang H, Guo C, Zhang M.
Journal: Nan Fang Yi Ke Da Xue Xue Bao; 2024 Jun 20; 44(6):1173-1181. PubMed ID: 38977348.
Abstract:
OBJECTIVES: To investigate the regulatory role of miRNA-224-5p in hypoxia/reoxygenation (H/R) -induced H9c2 cardiomyocyte injury. METHODS: Plasma samples were collected from 160 patients with acute myocardial infarction and 80 healthy controls(HC) to measure miRNA-224-5p levels and other biochemical parameters. In cultured H9c2 cells with H/R injury, the effects of transfection with miR-224-5p mimics or a negative control sequence on cell viability, malondialdehyde (MDA) content, and superoxide dismutase 2 (SOD2) and lactate dehydrogenase (LDH) activities were tested. Dual luciferase reporter gene assay was performed to verify the targeting relationship between miR-224-5p and PTEN. Bioinformatics methods were used to analyze the potential mechanisms of the target genes. The expression of miRNA-224-5p in the treated cells was detected with qRT-PCR, the protein expressions of PTEN, Bcl-2, Bax, cleaved caspase-3, SOD2, p-PI3K/PI3K, p-Akt/Ak and p-FoxO1/FoxO1 were determined using Western blotting, and cell apoptosis was analysed with flow cytometry. RESULTS: The levels of blood glucose, C-reactive protein, CK, CK-MB and cTnI were significantly higher in the AMI group compared with the HC group (P < 0.05). The expression level of miR-224-5p was significantly lowered in patients with STEMI and NSTEMI and in H9c2 cells with H/R injury. The viability of H9c2 cells decreased time-dependently following H/R injury. PTEN was a target gene of miR-224-5p, and the PI3K/Akt pathway was the most significantly enriched pathway. H9c2 cells with H/R injury showed significantly decreased SOD2 activity, increased LDH activity and MDA content, increased cell apoptosis, decreased protein expression levels of p-PI3K, p-Akt, p-FoxO1, SOD2, and Bcl-2, and increased expressions of PTEN, Bax, and cleaved caspase-3. These changes were obviously attenuated by trasnfection of the cells with miR-224-5p mimics prior to H/R exposure. CONCLUSION: MiR-224-5p overexpression upregulates the expression of the antioxidant gene SOD2 through the PI3K/Akt/FoxO1 axis to relieve H/R-induced oxidative stress and reduce apoptosis of H9c2 cells. 目的: 探究miRNA-224-5p在缺氧/复氧（H/R）诱导心肌细胞损伤中的作用机制。方法: 收集160 例急性心肌梗死（AMI）患者和80 例健康体检者（HC）的血浆检测miRNA-224-5p及生化指标。H9c2细胞分为对照组（Control）、H/R组、H/R+miR-224-5pNC组、H/R+miR-224-5p mimics组。噻唑蓝（MTT）检测细胞活力；试剂盒检测丙二醛（MDA）、超氧化物歧化酶2（SOD2）和乳酸脱氢酶（LDH）；双荧光素酶报告基因实验验证miR-224-5p与PTEN的靶向关系；生物信息学方法对靶基因的潜在机制进行分析；qRT-PCR检测miRNA-224-5p mRNA表达；Western blotting检测PTEN、Bcl-2、Bax、Cleaved caspase-3、SOD2、p-PI3K/PI3K、p-Akt/Ak及p-FoxO1/FoxO1蛋白水平；流式细胞术检测细胞凋亡率。结果: 与 HC 组相比，AMI组的血糖、 C 反应蛋白、CK、CK-MB 和cTnI水平均显著高于对照组（P<0.05）。AMI组和H/R组miR-224-5p的表达低于对照组（P<0.05）；心肌细胞活力呈缺氧/复氧时间依赖性减少；PTEN是miR-224-5p的靶基因；PI3K/Akt通路是最显著富集的通路；与Control组相比，H/R组SOD2的活性降低，LDH的活性和MDA的含量均上升，细胞凋亡率上升（P<0.05），细胞中p-PI3K、p-Akt、p-FoxO1、SOD2和Bcl-2蛋白表达水平均降低，PTEN、Bax和Cleaved caspase-3蛋白表达均升高（P<0.05）；与H/R组比较，H/R+miR-224-5p mimics组SOD2的活性显著上升，LDH、MDA的水平和细胞凋亡率均显著降低（P<0.05），细胞中p-PI3K、p-Akt、p-FoxO1、SOD2和Bcl-2的表达均上调，PTEN、Bax和Cleaved caspase3蛋白表达水平均降低（P<0.05）。结论: miR-224-5p过表达通过PI3K/Akt/FoxO1轴上调抗氧化基因SOD2的表达，缓解H/R诱导的H9c2细胞的氧化应激，减少细胞凋亡。

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