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Effects of miR?103a?3p on the autophagy and apoptosis of cardiomyocytes by regulating Atg5.

ABSTRACT: Autophagy and apoptosis are associated with cardiovascular diseases. Emerging evidence shows that microRNAs (miRs) are critical in the development of pathological processes underlying cardiovascular diseases by regulating the induction of apoptosis and autophagy. The present study aimed to investigate the role of miR?103a?3p in cardiomyocyte injury through autophagy and apoptosis. H9c2 cells were cultured under hypoxia and reoxygenation (H/R) conditions and were used to mimic cells under ischemia. The transfection of cells with miR?103a?3p (mimics and inhibitors) was performed to examine its function in cardiomyocytes. The expression levels of miR?103a?3p were evaluated by reverse transcription?quantitative polymerase chain reaction analysis. Cell viability was determined using an MTT assay, and the lactate dehydrogenase assay (LDH) was used to investigate cell injury. The expression levels of B?cell lymphoma 2 (Bcl?2), Bcl?2?associated X protein, Beclin?1, autophagy?related 5 (Atg5), cleaved caspase?3 and cleaved caspase?9 were detected using western blotting. Immunofluorescence assays were performed to detect the expression of LC3 as a marker of autophagy. The target gene of miR?103a?3p was identified using dual?luciferase reporter assays. The results revealed that the expression levels of miR?103a?3p were significantly downregulated in cardiomyocytes under H/R conditions. Injury of the cardiomyocytes was evaluated under H/R conditions. Following transfection of the cells with miR?103a?3p inhibitors, cell injury was increased, as determined by LDH and MTT assays. The expression levels of apoptotic proteins were consistent with the results obtained in the LDH and cell viability assays. The induction of autophagy was increased in cells under H/R conditions and cells with miR?103a?3p inhibitor transfection, whereas the induction of autophagy was decreased in cells transfected with miR?103a?3p mimics. In addition, the data indicated that miR?103a?3p directly targeted Atg5, which regulated the induction of autophagy and apoptosis. Taken together, these findings indicate that, following the inhibition of miR?103a?3p, Atg5 promotes autophagy and apoptosis in cardiomyocytes by directly targeting Atg5. Therefore, miR?103a?3p can be considered a potential therapeutic target for myocardial ischemia.

SUBMITTER: Zhang C

PROVIDER: S-EPMC6443343 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Project description:Background:Microvascular obstruction (MVO) can result in coronary microcirculation embolism and myocardial microinfarction. Myocardial injury induced by MVO is characterized by continuous ischemia and hypoxia of cardiomyocytes. Autophagy and apoptosis are closely associated with various cardiovascular diseases. Based on our previous study, we observed a decrease in miR-30e-3p expression and an increase in Egr-1 expression in a rat coronary microembolization model. However, the specific function of miR-30e-3p in regulating autophagy and apoptosis in an ischemia/hypoxia (IH) environment remains to be deciphered. We exposed cardiomyocytes to an IH environment and then determined whether miR-30e-3p was involved in promoting cardiomyocyte autophagy and inhibiting apoptosis by regulating Egr-1. Methods:Cardiomyocytes were isolated from rats for our in vitro study. miR-30e-3p was either overexpressed or inhibited by transfection with lentiviral vectors into cardiomyocytes. 3-Methyladenine (3-MA) was used to inhibit autophagy. RT-qPCR and western blotting were used to determine the expression levels of miR-30e-3p, Egr-1, and proteins related to the autophagy and apoptosis process. Autophagic vacuoles and autophagic flux were evaluated using transmission electron microscopy (TEM) and confocal microscopy, respectively. Cardiomyocyte viability was evaluated using the MTS assay. Cell injury was assessed by lactate dehydrogenase (LDH) leakage, and apoptosis was determined by flow cytometry. Results:Both miR-30e-3p expression and autophagy were significantly inhibited, and apoptosis was increased in cardiomyocytes after 9 hours of IH exposure. Overexpression of miR-30e-3p increased autophagy and inhibited apoptosis, as well as suppressed Egr-1 expression and decreased cell injury. In addition, inhibition of miR-30e-3p reduced autophagy and increased apoptosis and cell injury. Conclusions:miR-30e-3p may be involved in promoting cardiomyocyte autophagy and inhibiting apoptosis by indirectly regulating Egr-1 expression in an IH environment.

Project description:BackgroundGlycolysis plays an essential role in the growth and metastasis of solid cancer and has received increasing attention in recent years. However, the complex regulatory mechanisms of tumour glycolysis remain elusive. This study aimed to explore the molecular effect and mechanism of the noncoding RNA miR-103a-3p on glycolysis in colorectal cancer (CRC).MethodsWe explored the effects of miR-103a-3p on glycolysis and the biological functions of CRC cells in vitro and in vivo. Furthermore, we investigated whether miR-103a-3p regulates HIF1A expression through the Hippo/YAP1 pathway, and evaluated the role of the miR-103a-3p-LATS2/SAV1-YAP1-HIF1A axis in promoting glycolysis and angiogenesis in CRC cells and contributed to invasion and metastasis of CRC cells.ResultsWe found that miR-103a-3p was highly expressed in CRC tissues and cell lines compared with matched controls and the high expression of miR-103a-3p was associated with poor patient prognosis. Under hypoxic conditions, a high level of miR-103a-3p promoted the proliferation, invasion, migration, angiogenesis and glycolysis of CRC cells. Moreover, miR-103a-3p knockdown inhibited the growth, proliferation, and glycolysis of CRC cells and promoted the Hippo-YAP1 signalling pathway in nude mice in a xenograft model. Here, we demonstrated that miR-103a-3p could directly target LATS2 and SAV1. Subsequently, we verified that TEAD1, a transcriptional coactivator of Yes-associated protein 1 (YAP1), directly bound to the HIF1A promoter region and the YAP1 and TEAD1 proteins co-regulated the expression of HIF1A, thus promoting tumour glycolysis.ConclusionsMiR-103a-3p, which is highly expressed in CRC cells, promotes HIF1A expression by targeting the core molecules LATS2 and SAV1 of the Hippo/YAP1 pathway, contributing to enhanced proliferation, invasion, migration, glycolysis and angiogenesis in CRC. Our study revealed the functional mechanisms of miR-103a-3p/YAP1/HIF1A axis in CRC glycolysis, which would provide potential intervention targets for molecular targeted therapy of CRC.

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Effects of miR?103a?3p on the autophagy and apoptosis of cardiomyocytes by regulating Atg5.

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