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 hy-poxia 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 in-duced 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 experi-ments.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 mid-dle 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 ex-pression significantly decreased the apoptosis of SH-SY5Y cells(P<0.001).Conclusion miR-181a can target Bcl-2 and down-reg-ulation of miR-181a can inhibit the OGD/R injury induced apoptosis of SH-SY5Y nerve cells by up-regulating the expression of Bcl-2.
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