Zeng Huan, Li Ying, Liu Xinzong, Li Xinxin, Zhou Tian, Cao Shanshan, Wang Mingjuan, Ju Mingfei
Department of Cardiac Function, The First College of Clinical Medical Science, China Three Gorges University & Yichang Central People's Hospital, Yichang, China.
Institute of Orthopedics and Traumatology, The People's Hospital of Three Gorges University, The First People's Hospital of Yichang, Yichang, China.
J Biochem Mol Toxicol. 2022 Dec;36(12):e23205. doi: 10.1002/jbt.23205. Epub 2022 Oct 12.
MicroRNAs are widely reported as biomarkers and therapeutic targets in cardiovascular diseases. This study is aimed to expound on the regulatory responsibility of miR-383-3p in H/R-induced injury of H9c2 cells. In this study, H9c2 cells were administrated with H/R. MiR-383-3p expression was measured using qRT-PCR. ELISA was used to determine lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) levels. Reactive oxygen species (ROS) were detected with 2,7-Dichlorodihydrofluorescein diacetate probe. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide, flow cytometry, and TUNEL experiments were conducted to measure cell viability and apoptosis. Cleaved caspase-3, caspase-3, Bax, Bcl-2, PTEN, PI3K, p-PI3K, Akt, p-AKT expression levels were examined by Western blot. Cleaved caspase-3 expression was also measured by immunofluorescence staining. Dual-luciferase reporter gene assay was applied to validate the binding sites in miR-383-3p and the 3'UTR of PTEN. We reported that, miR-383-3p expression in H9c2 cells treated with H/R was remarkably decreased. MiR-383-3p overexpression ameliorated oxidative stress and apoptosis and promoted cell viability in H9c2 cells treated with H/R, while miR-383-3p inhibitor showed the reverse effects. PTEN was identified as a target gene of miR-383-3p. Additionally, enhancement of PTEN expression abolished the influences of miR-383-3p on H9c2 cells. MiR-383-3p mimics could significantly decrease PTEN expression in H9c2 cells while increasing p-PI3K expression and p-AKT expression, while the miR-383-3p inhibitors showed the opposed effects. In conclusion, miR-383-3p protected H9c2 cells from H/R-induced injury via regulating PTEN/PI3K/AKT signal pathway.
微小RNA在心血管疾病中作为生物标志物和治疗靶点被广泛报道。本研究旨在阐述miR-383-3p在缺氧/复氧诱导的H9c2细胞损伤中的调控作用。在本研究中，对H9c2细胞进行缺氧/复氧处理。采用qRT-PCR检测miR-383-3p的表达。采用ELISA法测定乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）和丙二醛（MDA）水平。用2,7-二氯二氢荧光素二乙酸酯探针检测活性氧（ROS）。进行3-(4,5)-二甲基噻唑-2,5-二苯基四氮唑溴盐、流式细胞术和TUNEL实验以检测细胞活力和凋亡情况。通过蛋白质免疫印迹法检测裂解的半胱天冬酶-3、半胱天冬酶-3、Bax、Bcl-2、PTEN、PI3K、磷酸化PI3K、Akt、磷酸化Akt的表达水平。还通过免疫荧光染色检测裂解的半胱天冬酶-3的表达。应用双荧光素酶报告基因检测法验证miR-383-3p与PTEN的3'非翻译区中的结合位点。我们报道，缺氧/复氧处理的H9c2细胞中miR-383-3p的表达显著降低。miR-383-3p过表达改善了缺氧/复氧处理的H9c2细胞中的氧化应激和凋亡，并促进了细胞活力，而miR-383-3p抑制剂则表现出相反的作用。PTEN被鉴定为miR-383-3p的靶基因。此外，PTEN表达的增强消除了miR-383-3p对H9c2细胞的影响。miR-383-3p模拟物可显著降低H9c2细胞中PTEN的表达，同时增加磷酸化PI3K的表达和磷酸化Akt的表达，而miR-383-3p抑制剂则表现出相反的作用。总之，miR-383-3p通过调节PTEN/PI3K/Akt信号通路保护H9c2细胞免受缺氧/复氧诱导的损伤。
