MiR-224-5p overexpression inhibits oxidative stress by regulating the PI3K/Akt/FoxO1 axis to attenuate hypoxia/reoxygenation-induced cardiomyocyte injury
Objectives To investigate the regulatory role of miRNA-224-5p in hypoxia/reoxygenation(H/R)-induced H9c2 cardiomyocyte injury.Methods Plasma samples were collected from 160 patients with acute myocardial infarction and 80 healthy controls(HC)to measure miRNA-224-5p levels and other biochemical parameters.In cultured H9c2 cells with H/R injury,the effects of transfection with miR-224-5p mimics or a negative control sequence on cell viability,malondialdehyde(MDA)content,and superoxide dismutase 2(SOD2)and lactate dehydrogenase(LDH)activities were tested.Dual luciferase reporter gene assay was performed to verify the targeting relationship between miR-224-5p and PTEN.Bioinformatics methods were used to analyze the potential mechanisms of the target genes.The expression of miRNA-224-5p in the treated cells was detected with qRT-PCR,the protein expressions of PTEN,Bcl-2,Bax,cleaved caspase-3,SOD2,p-PI3K/PI3K,p-Akt/Ak and p-FoxO1/FoxO1 were determined using Western blotting,and cell apoptosis was analysed with flow cytometry.Results The levels of blood glucose,C-reactive protein,CK,CK-MB and cTnI were significantly higher in the AMI group compared with the HC group(P<0.05).The expression level of miR-224-5p was significantly lowered in patients with STEMI and NSTEMI and in H9c2 cells with H/R injury.The viability of H9c2 cells decreased time-dependently following H/R injury.PTEN was a target gene of miR-224-5p,and the PI3K/Akt pathway was the most significantly enriched pathway.H9c2 cells with H/R injury showed significantly decreased SOD2 activity,increased LDH activity and MDA content,increased cell apoptosis,decreased protein expression levels of p-PI3K,p-Akt,p-FoxO1,SOD2,and Bcl-2,and increased expressions of PTEN,Bax,and cleaved caspase-3.These changes were obviously attenuated by trasnfection of the cells with miR-224-5p mimics prior to H/R exposure.Conclusion MiR-224-5p overexpression upregulates the expression of the antioxidant gene SOD2 through the PI3K/Akt/FoxO1 axis to relieve H/R-induced oxidative stress and reduce apoptosis of H9c2 cells.
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