Abstract

BACKGROUND: Oxidative stress in myocardial ischemia results in cardiomyocyte apoptosis. The expression of microRNA-141-3p (miR-141-3p) and the 105 kD toll-like receptor protein (TLR), RP105, have been identified in cardiomyocytes in vitro. This study aimed to investigate the effects of hypoxia in H9c2 rat cardiomyoblasts with and without the inhibition of miR-141-3p and to investigate the expression of RP105 and the PI3K/AKT signaling pathway.

MATERIAL AND METHODS: H9c2 rat cardiomyoblasts were cultured in conditions of hypoxia and treated with a specific miR-141-3p-inhibitor. RP105 short-interfering RNA (siRNA) was constructed, and LY294002 was used to inhibit the PI3KA/AKT pathway. The fluorescent probe, dihydroethidium (DHE), was used to detect reactive oxygen species (ROS). Flow cytometry evaluated ROS and apoptosis. Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot studied the expression of the PI3K/AKT pathway genes and proteins. Bioinformatics and dual-luciferase reporter assays were used to identify the targets for miR-141-3p.

RESULTS: A predictive TargetScan algorithm showed that the RP105 gene was a potential target of miR-141-3p. Expression of miR-141-3p was significantly increased in hypoxic H9c2 cells, and inhibition of miR-141-3p increased cell viability and reduced apoptosis. Also, miR-141-3p was shown to target 3’-UTR of RP105. Down-regulation of RP105 associated with hypoxia and its downstream PI3K/AKT pathway were significantly increased following miR-141-3p inhibition. The protective effect of miR-141-3p inhibition in hypoxic H9c2 cells was abolished by the absence of RP105 and inhibition of PI3K/AKT.

CONCLUSIONS: Inhibition of miR-141-3p reduced hypoxia-induced apoptosis in H9c2 cardiomyocytes in vitro by activating the RP105-dependent PI3K/AKT signaling pathway.

Keywords: Apoptosis, MicroRNAs, Myocardial Infarction, Reactive Oxygen Species, Antigens, CD, Base Sequence, Cell Hypoxia, Cell Line, Myocytes, Cardiac, Phosphatidylinositol 3-Kinases, Proto-Oncogene Proteins c-akt, Up-Regulation