Mettl3 promotes oxLDL‐mediated inflammation through activating STAT1 signaling

• Published in: Journal of clinical laboratory analysis 2022-01, Vol.36 (1), p.e24019-n/a
• Main Authors: Li, Zhenwei, Xu, Qingqing, Huangfu, Ning, Chen, Xiaomin, Zhu, Jianhua
• Format: Article
• Language: English
• Subjects: Antibodies; Apoptosis; Arteriosclerosis; Atherosclerosis; Cardiovascular disease; Cell culture; Cell cycle; Cerebrovascular diseases; Enzymes; Gene expression; Gene regulation; Immunoprecipitation; Inflammation; Inflammatory diseases; Low density lipoprotein; Macrophages; Methyltransferase; methyltransferase‐like protein 3; N6-methyladenosine; Proteins; RNA modification; Senescence; signal transducer and activator of transcription 1; Stat1 protein; Transcription; Tumor necrosis factor-TNF; Vascular diseases
• ISSN: 0887-8013; 1098-2825
• DOI: 10.1002/jcla.24019

Background Atherosclerosis (AS) is the main cause of cerebrovascular diseases, and macrophages act important roles during the AS pathological process through regulating inflammation. Modification of the novel N(6)‐methyladenine (m6A) RNA is reported to be associated with AS, but its role in AS is largely unknown. The aim of this study was to investigate the role and mechanism of m6A modification in inflammation triggered by oxidized low‐density lipoprotein (oxLDL) in macrophages during AS. Methods RAW264.7 macrophage cells were stimulated with 40 μg/ml ox‐LDL, Dot blot, Immunoprecipitation, western blot, Rip and chip experiments were used in our study. Results We found oxLDL stimulation significantly promoted m6A modification level of mRNA in macrophages and knockdown of Methyltransferase‐Like Protein 3 (Mettl3) inhibited oxLDL‐induced m6A modification and inflammatory response. Mettl3 promoted oxLDL‐induced inflammatory response in macrophages through regulating m6A modification of Signal transducer and activator of transcription 1 (STAT1) mRNA, thereby affecting STAT1 expression and activation. Moreover, oxLDL stimulation enhanced the interaction between Mettl3 and STAT1 protein, promoting STAT1 transcriptional regulation of inflammatory factor expression in macrophages eventually. Conclusions These results indicate that Mettl3 promotes oxLDL‐triggered inflammation through interacting with STAT1 protein and mRNA in RAW264.7 macrophages, suggesting that Mettl3 may be as a potential target for the clinical treatment of AS. oxLDL induces LOX‐1 expression and Mettl3 does not affect cell biological activity. (A) qRT‐PCR analysis of LOX‐1 mRNA expression in RAW264.7 cells treated with oxLDL with indicated concentrations for 24 h. (B) WB analysis of LOX‐1 in RAW264.7 cells treated with oxLDL or not for 24 h. (C) WB analysis of the indicated proteins in RAW264.7 cells treated with oxLDL or not for 24 h. (D) Cell viability analysis of RAW264.7 cells transfected with M3 or M3d vector for 24 h. (E) Apoptosis analysis of RAW264.7 cells transfected with M3 or M3d vector for 24 h. (F) Cell viability analysis of RAW264.7 cells transfected with si‐nc or si‐M3 for 24 h. (G) Apoptosis analysis of RAW264.7 cells transfected with si‐nc or si‐M3 for 24 h. (H) Cell cycle analysis of RAW264.7 cells transfected with si‐nc or si‐M3 for 24 h. (I) Cell senescence analysis of RAW264.7 cells transfected with si‐nc or si‐M3 for 24 h.