MiR-181a regulates the apoptosis of SH-SY5Y cells induced by OGD/R model by targeting Bcl-2

Abstract

Objective The cortex is one of the most sensitive tissues in response to cerebral hypoxia-ischemia. Based on the previous deep sequencing technology, we screened and obtained cortical target genes miR-181a and Bcl-2 that closely respond to cerebral hypoxia ischemia stress. The aim of this study is to verify the above targeted relationships and functions in SH-SY5Y cell line induced by the oxygen-glucose deprivation/reperfusion (OGD/R), and to clarify the role of miR-181a-Bcl-2 regulatory network in OGD/R induced nerve cell apoptosis. Methods The model of hypoxic-ischemic and reperfusion injury in rats was established by suture method, and the model was evaluated by TTC staining and behavioral score. Target gene expression was verified by qRT-PCR and Western Blot. Bioinformatics was used to analyze the target binding sites of miR-181a and Bcl-2 and compare the conservation of the binding sites. The specificity of targeted binding between miR-181a and Bcl-2 was confirmed through dual-luciferase reporter gene experiments. The OGD/R model was used to simulate cerebral ischemia-reperfusion injury in vitro. Cell apoptosis was detected by apoptosis-related protein expression and Hoechst fluorescence staining. Results The expression of miR-181a and Bcl-2 was reversed after middle cerebral artery occlusion in rats. RNA hybird software predicted that miR-181a could bind to the 3'-UTR region of Bcl-2 mRNA, and the nucleotides in the binding region were highly conserved. Dual luciferase reporter gene assay showed that compared with the Bcl-2 3'UTR-WT and mimic-NC co-transfection group, the fluorescence activity of Bcl-2 3'-UTR and miR-181a mimic co-transfection group was lower (P<0.001). There was no significant difference in the fluorescence activity of Bcl-2-Mut co-transfected with miR-181a mimic group (P>0.05). After the OGD/R model was constructed, SH-SY5Y cells were transfected with miR-181a mimics and inhibitors, respectively. It was found that miR-181a could inhibit the expression levels of Bcl-2 mRNA and protein (P<0.001). Overexpression of miR-181a significantly increased the apoptosis of SH-SY5Y cells (P<0.001), while inhibition of miR-181a expression significantly decreased the apoptosis of SH-SY5Y cells (P<0.001). Conclusion miR-181a can target Bcl-2 and down-regulation of miR-181a can inhibit the OGD/R injury induced apoptosis of SH-SY5Y nerve cells by up-regulating the expression of Bcl-2.

Keywords

miR-181a / Bcl-2 / SH-SY5Y / OGD/R / apoptosis