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MicroRNA miR-24-3p Reduces Apoptosis and Regulates Keap1-Nrf2 Pathway in Mouse Cardiomyocytes Responding to
Xu Xiao1, Zhigang Lu2, Victor Lin2
1Department of Emergency Medicine, Sichuan Provincial People's Hospital, Chengdu, Sichuan, China.
Abstract:
In recent years, microRNAs (miRNAs) have received increasing attention for their role in ischemia/reperfusion injury (I/RI), and many miRNAs have been demonstrated to play a very important role in cardiac I/RI. The miRNA miR-24-3p is a tumor suppressor that regulates multiple tumors; however, it remains unclear whether the expression level of miR-24-3p is altered in cardiac cells under I/RI. In this study, we used mouse primary cardiomyocytes and the H9C2 cardiomyocyte cell line to perform in vitro stimulated ischemia/reperfusion (SI/R) and then detected miR-24-3p expression level using quantitative real-time PCR (qRT-PCR). We discovered that the expression of miR-24-3p was significantly increased in cardiomyocytes following SI/R, and that the miR-24-3p level was inversely correlated to the ischemia marker HIF-1a. Furthermore, we transfected cardiomyocytes with miR-24-3p mimic or inhibitor to explore the role of miR-24-3p in cardiomyocyte ischemia/reperfusion injury in vitro. We performed flow cytometry to detect the apoptotic rate of H9C2 cardiomyocytes and found that the transfection of miR-24-3p mimic resulted in the decrease of the apoptosis rate of cardiomyocytes after SI/R, whereas the transfection of miR-24-3p inhibitor increased the number of apoptotic cardiomyocytes. These data suggest that the overexpression of miR-24-3p could reduce in vitro myocardial cell apoptosis induced by I/R injury. Finally, we applied the dual luciferase reporter gene system to verify whether miR-24-3p targets the Keap1 gene, and found that the luciferase signal intensity from a vector carrying the Keap1 wild-type reporter gene was significantly reduced after transfection with miR-24-3p mimic. The Keap1 protein level was also reduced following the transfection of miR-24-3p. The results from this study suggest a novel function of miR-24-3p in protecting cardiomyocytes from ischemia/reperfusion injury by the activation of the Nrf2-Keap1 pathway.
Insights
MicroRNA miR-24-3p protects heart cells from ischemia/reperfusion injury by reducing apoptosis. This microRNA (miRNA) targets Keap1, activating the Nrf2-Keap1 pathway for cellular protection.
Area of Science:
- Cardiovascular Biology
- Molecular Biology
- Cellular Physiology
Background:
- MicroRNAs (miRNAs) are key regulators in cellular processes, with growing interest in their role in cardiac ischemia/reperfusion injury (I/RI).
- The specific role of microRNA miR-24-3p in cardiac I/RI remains largely uncharacterized, despite its known functions in other cellular contexts.
Purpose of the Study:
- To investigate the expression levels of miR-24-3p in cardiomyocytes under simulated ischemia/reperfusion (SI/R) conditions.
- To elucidate the functional role of miR-24-3p in regulating cardiomyocyte apoptosis during I/RI.
- To identify potential molecular targets of miR-24-3p involved in cardiac I/RI.
Main Methods:
- Primary mouse cardiomyocytes and H9C2 cell line subjected to in vitro SI/R.
- Quantitative real-time PCR (qRT-PCR) to measure miR-24-3p expression.
- Transfection with miR-24-3p mimic or inhibitor, followed by flow cytometry for apoptosis analysis.
- Dual luciferase reporter assay and Western blot to validate miR-24-3p targets.
Main Results:
- miR-24-3p expression was significantly upregulated in cardiomyocytes following SI/R, inversely correlating with the ischemia marker HIF-1a.
- Overexpression of miR-24-3p reduced SI/R-induced cardiomyocyte apoptosis, while inhibition increased apoptosis.
- miR-24-3p directly targets the Keap1 gene, leading to reduced Keap1 protein levels.
Conclusions:
- miR-24-3p plays a protective role against in vitro ischemia/reperfusion injury in cardiomyocytes.
- The protective mechanism involves the downregulation of Keap1, suggesting activation of the Nrf2-Keap1 pathway.
- miR-24-3p represents a potential therapeutic target for mitigating cardiac I/R injury.
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