MicroRNA-221 promotes myocardial apoptosis caused by myocardial ischemia-reperfusion by down-regulating PTEN.

Abstract

OBJECTIVE\nThe aim of this study was to investigate whether microRNA-221 could promote cardiomyocyte apoptosis by down-regulating the expression of PTEN (gene of phosphate and tension homology deleted on chromosome ten), thereby participating in the development of myocardial ischemia-reperfusion.\n\n\nMATERIALS AND METHODS\nQuantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to analyze the expression levels of microRNA-221 and PTEN in human cardiomyocytes (HCM) cells treated with hypoxia/reoxygenation (H/R). The expressions of myocardial injury markers, including lactic dehydrogenase enzyme (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were determined by qRT-PCR as well. The binding relationship between microRNA-221 and PTEN was verified by the Dual-Luciferase reporter gene assay. Subsequently, microRNA-221 inhibitor and si-PTEN were transfected into cells. The proliferation and apoptosis of cells were analyzed using Cell Counting Kit-8 (CCK-8) and flow cytometry, respectively. In addition, the expression levels of apoptosis-related proteins were determined by Western blot.\n\n\nRESULTS\nThe qRT-PCR results confirmed that the expression level of microRNA-221 in H/R treated cells was significantly up-regulated when compared with the normoxic treated group, whereas PTEN expression was markedly down-regulated. After silencing microRNA-221, the expression levels of myocardial injury markers, including LDH, MDA, GSH-PX in H/R cells were significantly decreased. However, SOD levels were remarkably increased. At the same time, down-regulation of microRNA-221 markedly increased cell proliferation, whereas decreased apoptosis. However, microRNA-221 enhanced the expression of apoptosis-related genes, including Bax and cytochrome C. Meanwhile, the expression level of anti-apoptotic gene Bcl-2 was significantly inhibited. The Dual-Luciferase reporter gene assay showed that microRNA-221 could target bind to PTEN and inhibit its expression. Similarly, down-regulation of PTEN markedly decreased cell proliferation and increased cell apoptosis. Furthermore, PTEN down-regulation remarkably promoted protein expression of pro-apoptosis-related genes, whereas inhibited the protein expression of anti-apoptotic genes.\n\n\nCONCLUSIONS\nMicroRNA-221 promoted myocardial apoptosis induced by myocardial ischemia-reperfusion by down-regulating PTEN. Therefore, microRNA-221 might be a potential therapeutic target for myocardial ischemia-reperfusion injury.