Inhibition of miR-337-3p involved in the protection of CoCl2 -induced injury in PC12 cells via activating JAK2/STAT3 signaling pathway

Abstract

Objective: To investigate the possibility of microRNA (miR)-337-3p in the protection of hypoxia-induced injury in PC12 cells via modulating the JAK2/STAT3 signaling pathway.

Methods: Dual-luciferase reporter assay analyzed the relationship between the miR-337-3p and JAK2. PC12 cells were divided into normal, CoCl₂ , CoCl2 + NC, CoCl2 + inhibitors, CoCl2 + JAK2, and CoCl2 + mimics + JAK2 groups. Then, PC12 cell viability and apoptosis were measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and Annexin-V-fluorescein isothiocyanate/propidium iodide methods. Quantitative real-time polymerase chain reaction and Western blot analysis were used to determine expressions. Besides, the intracellular reactive oxygen species (ROS) was examined by dichloro-dihydro-fluorescein diacetate (DCFH-DA) while the mitochondrial membrane potential (MMP) by using JC-1.

Results: The negative targeting relationship between miR-337-3p and JAK2 was confirmed. When compared with the normal group, miR-337-3p was increased while JAK2 and STAT3 were decreased in CoCl₂ -induced PC12 cells, with decreased cell viability. Moreover, either miR-337-3p inhibitor or JAK2 overexpression could partially reverse CoCl₂ -induced decrease in PC12 cell viability. Besides, CoCl₂ could also trigger PC12 cell apoptosis by increasing cleaved caspase 3 and Bax but decreasing Bcl-2 and Bcl-XL, which, however, were abolished with the transfection of miR-337-3p inhibitors or lentivirus transfection to activate JAK2. Compared with the CoCl₂ group, the average of fluorescent signals of ROS in the CoCl2 + inhibitors group and the CoCl2 + JAK2 group was lower, while the activities of superoxide dismutase, catalase, glutathione peroxidase, and total anti-oxidative capacity were higher, together with an increase in MMP.

Conclusion: Inhibiting miR-337-3p could activate the JAK2/STAT3 signaling pathway to suppress CoCl ₂ -induced cytotoxicity and apoptosis and ameliorate oxidative stress and MMP in PC12 cells.

Keywords: JAK2/STAT3; PC12 cell; miR-337-3p.