MicroRNA-182 regulates intracellular calcium homeostasis during myocardi-al infarction by targeting SNX17
Objective To explore the changes in the expression of microRNA-182(miR-182)during myocardial infarction(MI)and reveals its mechanism in regulating calcium homeostasis in cardiomyocytes by targeting sorting nexin 17(SNX17).Methods Twelve male Wistar rats were randomly divided into sham operation group and myocardial infarction group(n=6).The sham group underwent thoracotomy without coronary artery ligation,while the myocardial in-farction group was subjected to ligation of the left anterior descending artery to induce myocardi-al infarction.Primary cultures of neonatal rat cardiomyocytes were prepared,and an in vitro hy-poxia model was constructed.miR-182 mimics and AMO-182 were transfected to overexpress and inhibit miR-182,respectively;SNX17 plasmid was transfected to overexpress SNX17.Cell viability was measured using the cell counting kit-8(CCK-8);miR-182 and SNX17 mRNA lev-els were determined by real-time quantitative PCR;SNX17 protein levels were detected by Western blotting;a luciferase reporter assay was conducted to verify the direct binding site be-tween miR-182 and the 3′-UTR region of SNX17;Intracellular calcium concentrations were ob-served using Fluo-3-AM fluorescent dye and laser confocal microscopy.Results Compared with the sham group,miR-182 expression in myocardial tissue of the myocardial infarction group was significantly upregulated(P<0.05).Compared with the control group,hypoxia-treated neonatal rat cardiomyocytes showed significantly reduced viability,upregulated miR-182 expression,and downregulated SNX17 expression(P<0.05).The luciferase reporter assay showed that miR-182 directly binds to the 3′-UTR region of SNX17 and inhibits its translation(P<0.01).After transfection with miR-182 mimics,SNX17 protein expression was further downregulated(P<0.05),while transfection with AMO-182 significantly increased SNX17 protein levels(P<0.05).Compared with the control group,cardiomyocytes overexpressing miR-182 showed significantly increased resting calcium levels(P<0.01),reduced calcium transient amplitude(P<0.01),and prolonged calcium reuptake time(P<0.01);overex-pression of SNX17 significantly reversed the calcium homeostasis disorders caused by miR-182 overexpression(P<0.05).
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