METTL3介导的m6A修饰对ox-LDL诱导的巨噬细胞焦亡的影响

Effects of methyltransferase-like 3-mediated N6 methyladenosine modification on oxidized low-density lipoprotein-induced macrophage pyroptosis

目的探究METTL3(methyltransferase like 3,METTL3)介导的m6A(N6甲基腺苷)修饰对氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)诱导的小鼠单核巨噬细胞白血病细胞(RAW264.7)焦亡的影响。方法ox-LDL(50μg/mL)诱导RAW264.7细胞24 h后,对METTL3进行敲低或过表达,检测培养细胞RNA m6A甲基化水平,荧光定量PCR和Western blot法检测METTL3和细胞焦亡相关基因NLRP3、NF-κB p65、GSDMD-N、Caspase-1 mRNA和蛋白表达水平,乳酸脱氢酶(LDH)细胞毒性检测细胞死亡率,酶联免疫吸附法检测炎症细胞因子IL-1β、IL-18、IL-6、TNF-α和IL-10的含量。结果ox-LDL能够促进RAW264.7巨噬细胞METTL3的表达和RNA m6A甲基化水平,同时也能促进炎症因子IL-1β、IL-18、IL-6、TNF-α的分泌(P<0.05),降低IL-10(P<0.05)的表达。在ox-LDL诱导基础上,METTL3过表达后,RNA m6A甲基化水平升高,NLRP3、NF-κB p65、GSDMD-N、Caspase-1表达显著增加,促炎细胞因子IL-6、TNF-α、IL-1β、IL-18水平升高,抗炎细胞因子IL-10水平降低,LDH释放增加,METTL3敲低后结果相反。结论METTL3介导的m6A修饰能够显著促进ox-LDL诱导的巨噬细胞焦亡和炎症反应。

Objective To investigate the effect of methyltransferase-like 3(METTL3)-mediated N6 methyladenosine(m6A)modification on oxidized low-density lipoprotein(ox-LDL)-induced mouse monocyte-macrophage leukemic cell(RAW264.7)pyroptosis.Methods RAW264.7 cells were treated with 50μg/mL ox-LDL for 24 h,and then METTL3 overexpression and knockdown experiments were conducted,and RNA m6A methylation levels were analyzed.Fluorescence quantitative PCR and Western blot were used to measure mRNA and protein expression levels of METTL3,NLRP3,NF-κB p65,GSDMD-N and Caspase-1.Lactate dehydrogenase(LDH)cytotoxicity was used to detect LDH release.Enzyme-linked immunosorbent assays(ELISA)to measure the levels of inflammatory cytokines IL-1β,IL-6,TNF-α,IL-10 and IL-18.Results After ox-LDL stimulation,METTL3 expression and the RNA m6A methylation level were upregulated.Secreted IL-1β,IL-6,IL-18 and TNF-αwere significantly increased,while IL-10 expression was decreased after ox-LDL stimulation of RAW264.7 macrophages(P<0.05).On the basis of ox-LDL induction,after METTL3 overexpression,the RNA m6A methylation level was increased,mRNA and protein expression of NLRP3,NF-κB p65,GSDMD-N and Caspase-1 were significantly increased,the levels of proinflammatory cytokines IL-6,TNF-α,IL-1βand IL-18 were increased,the level of anti-inflammatory cytokine IL-10 was decreased,and LDH release was increased.METTL3 knockdown the showed the opposite trends.Conclusions METTL3-mediated m6A modification promotes ox-LDL-induced macrophage pyroptosis and inflammatory responses.