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RBM15 promotes hypoxia/reoxygenation-induced ferroptosis in human cardiomyocytes by mediating m6A modification of
Yi Cheng1, Jiamin Wan1, Yingyue Xu1
1Department of Geriatric Medicine, Bishan Hospital of Chongqing, Bishan Hospital of Chongqing Medical University, No. 9, Double Star Avenue, Biquan Street, Bishan District, Chongqing, 402760, China.
Background:
Acute myocardial infarction (AMI) refers to the acute necrosis of part of the myocardium caused by persistent and severe myocardial ischemia. The aim of the study was to investigate the effect of RNA binding motif protein 15 (RBM15) and acyl-CoA synthetase long chain family member 4 (ACSL4) on ischemia/reperfusion (I/R)-induced ferroptosis of cardiomyocytes.
Methods And Results:
AC16 cells were treated with hypoxia/reoxygenation (H/R) to establish an in vitro myocardial infarction cell model. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assay were used to determine gene expression. Cell Counting Kit-8 (CCK-8) assay was conducted to investigate cell viability. Ferroptosis level was evaluated by commercial kits. N6-methyladenosine (m6A) level was examined by M6A quantification analysis. RNA immunoprecipitation (RIP) assay, methylated RNA Immunoprecipitation (meRIP) assay and dual-luciferase reporter assay were adopted to verify the combination between RBM15 and ACSL4. ACSL4 mRNA stability was analyzed by Actinomycin D treatment. RBM15 mRNA level was increased in AMI patients' serums and H/R-induced AC16 cells. Silencing of RBM15 promoted H/R-mediated AC16 cell viability and inhibited H/R-induced AC16 cell oxidative stress and ferroptosis. Moreover, it was demonstrated that RBM15 knockdown inhibited m6A modification of ACSL4 and suppressed the stability of ACSL4 mRNA. Furthermore, ACSL4 overexpression restored the effects of RNM15 silencing on H/R-induced AC16 cell oxidative injury and ferroptosis.
Conclusion:
RBM15 silencing repressed H/R-induced ferroptosis in human cardiomyocytes through regulating m6A modification of ACSL4.
Insights
RNA binding motif protein 15 (RBM15) silencing reduces ferroptosis in cardiomyocytes after myocardial infarction. This occurs by regulating N6-methyladenosine (m6A) modification of acyl-CoA synthetase long chain family member 4 (ACSL4) mRNA stability.
Area of Science:
- Cardiovascular Research
- Molecular Biology
- Cellular Pathology
Background:
- Acute myocardial infarction (AMI) involves cardiomyocyte necrosis due to severe ischemia.
- Ischemia/reperfusion (I/R) injury triggers ferroptosis in cardiomyocytes.
- The roles of RBM15 and ACSL4 in I/R-induced ferroptosis require investigation.
Purpose of the Study:
- To investigate the effect of RBM15 and ACSL4 on I/R-induced ferroptosis in cardiomyocytes.
- To elucidate the underlying molecular mechanisms, including m6A modification.
Main Methods:
- Established an in vitro myocardial infarction model using AC16 cells subjected to hypoxia/reoxygenation (H/R).
- Utilized qRT-PCR, Western blot, CCK-8 assay, and commercial kits to assess gene expression, cell viability, and ferroptosis.
- Employed m6A quantification, RIP, meRIP, and dual-luciferase reporter assays to analyze RBM15-ACSL4 interactions and m6A modification.
Main Results:
- RBM15 mRNA levels were elevated in AMI patient serums and H/R-treated cells.
- RBM15 silencing enhanced cell viability and reduced oxidative stress and ferroptosis.
- RBM15 knockdown decreased m6A modification of ACSL4, suppressed ACSL4 mRNA stability, and prevented ferroptosis.
Conclusions:
- RBM15 silencing protects human cardiomyocytes against H/R-induced ferroptosis.
- This protection is mediated by the regulation of ACSL4 mRNA stability through m6A modification.
- RBM15 serves as a potential therapeutic target for myocardial infarction.
