| Objective:Myocardial ischemia/reperfusion(I/R)is a common and lethal disease that threatens people's life worldwide.MiR-322/503,an X-chromosome miRNA cluster,is specifically expressed in the developing heart tube and functions to drive the formation of precocious cardiomyocyte formation.Relevant rearches has identified smad ubiquitin regulatory factor 2(Smurf2)which is an E3 ubiquitin ligase as a target of miR-322/503.In this rearch,H9c2 cells were used as the research object to simulate myocardial ischemia/reperfusion(I/R)injury treatment as an in vitro model,predicting that Smurf2 is a downstream target of miR-322/503,and exploring the role and mechanism of miR-322/503 in protecting myocardial ischemia/reperfusion(I/R)injury by regulating Smurf2 expression,in order to provide a theoretical basis for the application of miR-322/503 in cardiovascular diseases.Methods:1.H9c2 cells were subjected with the oxygen and glucose deprivation followed by reperfusion(OGD/R)as an in vitro model to study myocardial ischemia/reperfusion(I/R)injury.2.qRT-PCR analysis of miR-322,miR-503,EZH2 mRNA levels in H9c2 cells subjected to I/R.3.Cell proliferation was determined by MTT assay in H9c2 cells after miR-322/503 transfection.4.Cell apoptosis was determined by Flow analysis in H9c2 cells after miR-322/503 transfection.5.qRT-PCR analysis of biotinylated miR-322,levels of Smurf2 mRNAs pulled down by biotin-miR-3226.qRT-PCR analysis of biotinylated miR-503,levels of Smurf2 mRNAs.7.Dual luciferase assay was performed to measure the luciferase activity in H2c9 cells co-transfected with miR-322/503 mimic control/miR-322/503 mimic and Wt-Smurf2/Mut-Smurf2.8.qRT-PCR analysis of miR-322 and miR-503 48 h after transfection of pre-miR-322 or pre-miR-503.9.Western blot analysis of Smurf2 protein level after miR-322/503 transfection.10.qRT-PCR analysis of miR-322 and miR-503 48 h after transfection with oligomers targeting miR-322(anti-miR-322)or miR-503(anti-miR-503).11.Western blot analysis of Smurf2 protein in cells transfected with anti-miR-322 or anti-miR-503.Result:1.miR-322/503 is down-regulated in vitro I/R models?1)mRNA levels of miR-322 and miR-503 were significantly decreased in H9c2 cells subjected to I/R(60% and 40%,respectively,).Further,EZH2 mRNA level also went down upon I/R stress.2)overexpression of miR-322 and miR-503 significantly increased cell proliferation after I/R injury.3)overexpression of miR-322 and miR-503 significantly decreased cell apoptosis after I/R injury.2.Smurf2 is a downstream target of miR-322/503,miR-322/503 directly bind with Smurf2 mRNA and knockdown Smurf2.1)The levels of miR-322 and miR-503 levels significant increases compared to scramble which acted as control group(almost 4 folds and 8 folds,respectively).2)more Smurf2 mRNAs pulled down in conditions of miR-322 and miR-503 overexpression compared to control(almost 1.25 folds and 3.72 folds,respectively).3)miR-322/503 significantly decreased the luciferase of Wt-Smurf2 but not the Mut-Smurf2 in which the binding sites were mutated.3.miR-322/503 inhibits Smurf2 translation.1)miR-322 and miR503 levels were greatly higher in cells transfected with miR-322 and miR-503 respectively compared to control group(10.3 folds and 12.4folds,respectively).2)Smurf2 protein expressions were significantly reduced in cells transfected with miR-322/503.3)miR-322 and miR-503 levels were much decreased after two days' transfection with anti-miR-322 and anti-miR-503,respectively(65.4% and 72.4%,respectively).4)Smurf2 protein expressions were significantly up-regulated in cells transfected with anti-miR-322 and anti-miR-503(1.6 folds and 2.1 folds,respectively).Conclusion:1.The expression of miR-322/503 was reduced during I/R process.2.miR-322/503 plays an active role in myocardial ischemia-reperfusion(I/R)injury.,miR-322/503 directly binds to Smurf2 mRNA and inhibits Smurf2 translation to protect cells from ischemia-reperfusion(I/R)injury. |
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