RBM15 promotes hypoxia/reoxygenation-induced ferroptosis in human cardiomyocytes by mediating m6A modification of ACSL4

Abstract

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.

Keywords: AC16; ACSL4; AMI; RBM15; m6A.

Fig. 1

High level of RBM15 in the serums of AMI patients. (A) The mRNA level of RBM15 in the serums of AMI patients and healthy controls was determined by qRT-PCR. (B and C) The linear correlation between RBM15 level and cTnI/CK-MB level in AMI patients was analyzed by Spearman’s correlation coefficient analysis. (D) Diagnostic value of RBM15 for AMI was estimated. ***P < 0.001

Fig. 2

Deficiency of RBM15 ameliorated H/R-stimulated oxidative damage in AC16 cells. AC16 cells were treated with H/R, H/R + si-NC or H/R + si-RBM15 and untreated cells were control. (A and B) The mRNA and protein levels in AC16 cells were measured by qRT-PCR assay and western blot assay, respectively. (C) AC16 cell viability was explored by CCK-8 assay. (D-G) The levels of LDH, ROS, MDA and SOD in AC16 cells were examined by related commercial kits. **P < 0.01, ***P < 0.001

Fig. 3

RBM15 knockdown repressed H/R-stimulated ferroptosis in AC16 cells. AC16 cells were divided into 4 groups: control, H/R, H/R + si-NC and H/R + si-RBM15. (A) Fe²⁺ level in AC16 cells was examined by indicated commercial kit. (B-F) The protein levels of GPX4, ACSL4, FTH1 and NCOA4 in AC16 cells were measured by western blot. (G-I) The levels of GSH, GSSG and GSH/GSSG in AC16 cells were examined with indicated commercial kits. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

RBM15 regulated ACSL4 expression through m6A modification. (A) SRAMP database showed that RBM15 contained the m6A modification sites of ACSL4. (B) The combination between RBM15 and ACSL4 was verified by RIP assay. (C) m6A levels in AC16 cells in control, H/R, H/R + si-NC and H/R + si-RBM15 controls were determined by m6A quantification assay kit. (D-F) The interaction between RBM15 and ACSL4 was verified by MeRIP assay and dual-luciferase reporter assay. (G and H) After Actinomycin D treatment for 0 h, 2 h. 4 h and 6 h, ACSL4 mRNA level in AC16 cells was determined by qRT-PCR. **P < 0.01, ***P < 0.001

Fig. 5

RBM15 knockdown repressed H/R-induced AC16 cell damage by altering ACSL4 expression. (A) The protein level of ACSL4 in AC16 cells transfected with pcDNA-NC or pcDNA-ACSL4 was measured by western blot. (B-K) AC16 cells were divided into 5 groups: control, H/R + si-NC, H/R + si-RBM15, H/R + si-RBM15 + pcDNA-NC and H/R + si-RBM15 + pcDNA-ACSL4. (B) AC16 cell viability was assessed by CCK-8 assay. (C-G) The levels of LDH, ROS, MDA, SOD and Fe²⁺ in AC16 cells were examined by commercial kits. (H) The protein levels of GPX4, ACSL4, FTH1 and NCOA4 in AC16 cells were measured by western blot. (I-K) The levels of GSH, GSSG and GSH/GSSG in AC16 cells were examined by the indicated kits. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

The abridged general view of RBM15/ACSL4 axis in regulating H/R-induced AC16 cell injury