Dataset Information

MiR-30e-3p Promotes Cardiomyocyte Autophagy and Inhibits Apoptosis via Regulating Egr-1 during Ischemia/Hypoxia.

ABSTRACT: 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.

SUBMITTER: Su B

PROVIDER: S-EPMC7448244 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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miR-30e-3p Promotes Cardiomyocyte Autophagy and Inhibits Apoptosis via Regulating Egr-1 during Ischemia/Hypoxia.

Su Bo B Wang Xiantao X Sun Yuhan Y Long Manyun M Zheng Jing J Wu Wenhao W Li Lang L

BioMed research international 20200817

<h4>Background</h4>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 f ...[more]

PMID: 32879888

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Project description:BackgroundChlorogenic acid (CGA) is a polyphenolic compound with antioxidant and anti-inflammatory properties. CGA has been shown to improve neuroinflammation. This study is aimed at elucidating the exact mechanism by which CGA reduces neuroinflammation.MethodsOxygen and glucose deprivation (OGD) was utilized to treat BV2 microglia and HT-22 hippocampal neurons to engineer an in vitro model of hypoxic ischemia reperfusion. The levels of inflammatory factors (IL-1β, IL-6, TNF-α, IL-4, and IL-10) and oxidative stress factors (MDA, SOD, and GSH-PX) in microglia were determined by ELISA kits. The neuron proliferation was assessed by CCK-8 assay, and LDH kit was used to determine LDH release in neurons. The fluorescent dye DCF-DA was employed to measure ROS levels in neurons. Correlation of MIR497HG, miR-29b-3p, and SIRT1/NF-κB in neurons and microglia was determined by qRT-PCR. Expressions of inflammatory proteins (COX2, iNOS), oxidative stress pathways (Nrf2, HO-1), and apoptosis-related proteins (Bcl-2, Bax, caspase3, caspase8, and caspase9) in microglia or neurons were determined by western blot. The interactions between MIR497HG and miR-29b-3p, as well as between miR-29b-3p and SIRT1, were determined by dual luciferase assay and RIP assay.ResultsCGA attenuated OGD-mediated inflammation and oxidative stress in microglia and inhibited microglia-mediated neuronal apoptosis. CGA increased the levels of MIR497HG and SIRT1 and suppressed the levels of miR-29b-3p in BV2 and HT-22 cells. MIR497HG knockdown, miR-29b-3p upregulation, and SIRT1 inhibition inhibited CGA-mediated anti-inflammatory and neuronal protective functions. There is a targeting correlation between MIR497HG, miR-29b-3p, and Sirt1. MIR497HG sponges miR-29b-3p to regulate SIRT1 expression in an indirect manner.ConclusionCGA upregulates MIR497HG to curb miR-29b-3p expression, hence initiating the SIRT1/NF-κB signaling pathway and repressing OGD-elicited inflammation, oxidative stress, and neuron apoptosis.

Project description:ObjectivesTo explore the effects of miR-16-5p and PTPN4 on the apoptosis and autophagy of AC16 cardiomyocytes after hypoxia/reoxygenation treatment.MethodsAC16 cells were divided into the control group (NC), hypoxia/reoxygenation group (H/R), knockdown miR-16-5p negative control group (NC inhibitor), knockdown miR-16-5p group (miR-16-5p inhibitor), overexpression miR-16-5p negative control group (NC mimics), overexpression miR-16-5p group (miR-16-5p mimics), silent PTPN4 negative control group (sh-NC), silent PTPN4 group (sh-PTPN4), and silent PTPN4 + knockdown miR-16-5p group (sh-PTPN4 + miR-16-5p inhibitor). Real-time fluorescent quantitative PCR (RT-qPCR) and western blotting (WB) were used to measure the expression level of miR-16-3p, miR-16-5p, protein tyrosine phosphatase nonreceptor type 4 (PTPN4), and autophagy-related proteins (beclin-1, LC3 II/I, and P26) in AC16 cells. The apoptosis level of AC16 cells in each group was measured by flow cytometry and TUNEL. The dual-luciferase reporter gene experiment was also used to verify the targeting relationship between miR-16-5p and PTPN4.ResultsAfter H/R treatment, the levels of myocardial injury markers including LDH and CK-MB in AC16 cells were increased significantly (P < 0.05), and the levels of cell apoptosis and autophagy also increased significantly (P < 0.05). The level of miR-16-3p in AC16 cells did not change significantly after H/R treatment, whereas the level of miR-16-5p was increased significantly (P < 0.05). After miR-16-5p was knocked down, the levels of LDH and CK-MB in AC16 cells treated with H/R were significantly reduced (P < 0.05), and the rates of cell apoptosis and autophagy were also significantly reduced (P < 0.05). miR-16-5p negatively regulated the expression level of PTPN4 protein in AC16 cells (P < 0.05), and the dual-luciferase reporter gene experiment confirmed that PTPN4 was the downstream target of miR-16-5p. Silencing of PTPN4 significantly increased the damage of AC16 cells induced by H/R treatment (P < 0.05), but simultaneously inhibiting the expression of PTPN4 and miR-16-5p reversed the protective effect of miR-16-5p knockdown on AC16 cells (P < 0.05).ConclusionsThe expression of miR-16-5p is upregulated in AC16 cells after H/R treatment and the knockdown which can protect AC16 cells from H/R-induced cell damage that may be due to its regulation on the expression of PTPN4.

Project description: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.

Dataset Information

MiR-30e-3p Promotes Cardiomyocyte Autophagy and Inhibits Apoptosis via Regulating Egr-1 during Ischemia/Hypoxia.

Publications

miR-30e-3p Promotes Cardiomyocyte Autophagy and Inhibits Apoptosis via Regulating Egr-1 during Ischemia/Hypoxia.

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