MiR-421 inhibition protects H9c2 cells against hypoxia/reoxygenation-induced oxidative stress and apoptosis by targeting Sirt3

Abstract

Background: MicroRNAs (miRNAs) are involved in myocardial ischemia-reperfusion injury. miRNA-421 (miR-421) plays a significant role in the initiation of apoptosis and myocardial infarction. However, the molecular regulation of miR-421 in myocardial ischemia-reperfusion injury requires further elucidation. Methods: An in vitro hypoxia/reoxygenation model was established, and the expression levels of miR-421 and Sirtuin-3 (Sirt3) in H9c2 cells were quantified using quantitative real-time polymerase chain reaction. Flow cytometry was employed to measure the effects of miR-421 on myocardial apoptosis induced by hypoxia/reoxygenation. The activity of lactate dehydrogenase and superoxide dismutase and levels of malondialdehyde were measured. The binding sites of miR-421 on Sirt3 were predicted using TargetScan software. A luciferase reporter assay was used to validate the direct targeting of Sirt3 with miR-421. Protein expression levels of Sirt3 and its downstream proteins were evaluated using Western blot analysis. Results: Exposure of H9c2 cells to hypoxia/reoxygenation led to increased apoptosis, levels of malondialdehyde and lactate dehydrogenase, and decreased levels of superoxide dismutase. miR-421 knockdown resulted in decreased apoptosis, levels of lactate dehydrogenase and malondialdehyde, and increased superoxide dismutase levels in H9c2 cells. Hypoxia/reoxygenation significantly decreased the relative expression levels of Sirt3. Down-regulation of Sirt3 resulted from overexpression of miR-421, which directly targeted Sirt3. Knockdown of miR-421 up-regulated Sirt3 expression, inhibited activation of the Jun N-terminal kinase/activator protein 1 pathway and caspase 9/3-dependent cell death. Conclusion: The miR-421-Sirt3-Jun N-terminal kinase/activator protein 1 axis is a novel molecular mechanism that accommodates hypoxia/reoxygenation-induced oxidative stress and apoptosis and provides a new direction for the study and treatment of hypoxia/reoxygenation.

Effect of miR‑205 on proliferation and migration of thyroid cancer cells by targeting CCNB2 and the mechanism

“…Therefore, it is of great significance to explore the molecules that can affect the occurrence of TC. miRNA is a kind of non-coding RNA, non-coding protein, which is often used as upstream regulatory molecule to affect the biological process of many tumors and plays an important role in the occurrence and development of tumors (12)(13)(14). In TC, many studies have also reported that miRNA is involved in the regulation of its biological process.…”

MicroRNA and ROS Crosstalk in Cardiac and Pulmonary Diseases

“…Nevertheless, myocardial reperfusion can initiate oxidative stress through massive ROS generation, which further promotes myocardial injury inducing apoptosis and autophagy: this process is known as ischemia/reperfusion (I/R) injury. I/R injury has been adopted as a model to study MI and its mechanisms include a ROS burst during reperfusion, as well as calcium overload [61,62], resulting in increased apoptosis [63]. Conversely, SOD expression favors the post-ischemic cardiac function recovery, indicating the critical role of ROS and oxidative balance in MI and I/R.…”

“…In the cardiac I/R settings, Liu et al demonstrated that silencing of miR-421 in vitro decreased apoptosis, and both MDA and LDH levels, and increased SOD levels, ultimately reverting I/R effects. Sirt3, the miR-421 target, was found downregulated after I/R and its expression inversely correlated to increased expression of miR-421 [62]. In lung cancer, the regulation of ROS by miR-421 has been associated with resistance to the chemotherapy drug paclitaxel [183].…”

Extracellular vesicles enclosed‐miR‐421 suppresses air pollution (PM2.5)‐induced cardiac dysfunction via ACE2 signalling

“…To determine which sEV-linked mediators may cause cardiomyocytes apoptosis post-PM 2.5 exposure, we explored literature and public sequencing data systematically based on biological function, circulating and EVs-packed abundance of exogenous microRNAs. We identified a well conserved, PM 2.5 -associated EVs-miRNA (miR-421, 1.26 ± 0.32, p = 0.0003 of PM 2.5 compared to control), based on previous reports (Rodosthenous et al, 2016;Shu et al, 2015) that played important roles in regulating smoke-associated lung injury (Izzotti et al, 2011) and hypoxia-associated cardiac infarction (Liu et al, 2020;Wang et al, 2015). As shown in Figure S3, subacute respiratory exposure to PM 2.5 increased the content of miR-421 (mmu-miR421-3p) in heart tissue and serum-derived sEV (Figure S3A-S3B).…”