METTL3介导的m6A甲基化调控脂多糖诱导的内皮细胞通透性变化  

METTL3-mediated m6A methylation regulates lipopolysaccharide-induced endothelial cell permeability changes

作　　者：王建丰 余慧林 余又新[1] 宋均辉 孟承颖 蒋薇 胡德林[1] Wang Jianfeng;Yu Huilin;Yu Youxin;Song Junhui;Meng Chengying;Jiang Wei;Hu Delin(Dept of Burns,The First Affiliated Hospital of Anhui Medical University,Hefei 230022;Dept of Critical Care Medicine,Anqing First People′s Hospital Affiliated to Anhui Medical University,Anqing 246003)

机构地区：[1]安徽医科大学第一附属医院烧伤科,合肥230022 [2]安徽医科大学附属安庆市第一人民医院重症医学科,安庆246003

出　　处：《安徽医科大学学报》2024年第6期1023-1028,共6页Acta Universitatis Medicinalis Anhui

基　　金：安徽省省级临床重点专科建设项目(编号:060102027);安徽省重点研究和开发计划项目(编号:1804h08020230)。

摘　　要：目的探讨甲基化转移酶3(METTL3)介导的N6-甲基腺苷(m6A)甲基化修饰参与调控脂多糖(LPS)诱导的内皮细胞通透性变化的分子生物学机制。方法选用人脐静脉内皮细胞(HUVECs)进行体外培养,使用50、125、250、500、1000、2000 ng/ml的LPS干预HUVECs 24 h,应用Real-time PCR检测METTL3 mRNA的表达;选用500 ng/ml的LPS干预HUVECs 24 h,检测m6A甲基化水平,应用细胞通透性实验检测细胞通透性,应用Real-time PCR和Western blot检测细胞间连接蛋白(Claudin-5、Occludin和VE-caherin)mRNA和蛋白表达;构建METTL3过表达稳转细胞株,检测METTL3过表达时内皮细胞m6A甲基化水平及通透性的变化。结果与对照组相比,LPS抑制HUVECs METTL3 mRNA表达,使得内皮细胞m6A甲基化下调,细胞通透性升高,细胞间连接蛋白(Claudin-5、Occludin和VE-caherin)mRNA和蛋白表达下降;当METTL3过表达时,内皮细胞m6A甲基化水平增强,LPS诱导的内皮细胞通透性增加得以改善。结论METTL3介导的m6A甲基化能够改善脓毒症诱导的内皮细胞通透性增加。Objective To explore the molecular mechanism of N6-methyladenosine(m6A)methylation mediated by methyltransferase 3(METTL3)in regulating lipopolysaccharide(LPS)-induced endothelial cell permeability changes.Methods Human umbilical vein endothelial cells(HUVECs)were cultured in vitro.HUVECs were treated with LPS 50,125,250,500,1000,2000 ng/ml for 24 h.METTL3 mRNA expression was detected by Real-time PCR.After HUVECs were intervened with 500 ng/ml for 24 h,the methylation level of m6A was detected,and cell permeability was measured by cell permeability test.Real-time PCR and Western blot were used to detect mRNA and protein expression of intercellular junction proteins(Claudin-5,Occludin and VE-caherin).METTL3 overexpressed stable cell lines were constructed to measure the changes of m6A methylation level and permeability of endothelial cells during METTL3 overexpression.Results Compared to the control group,LPS inhibited the expression of HUVECs METTL3 mRNA,decreased the methylation of m6A,increased the cell permeability,and decreased the mRNA and protein expression of intercellular junction proteins(Claudin-5,Occludin and VE-Caherin).When METTL3 was overexpressed,the m6A methylation levels of endothelial cells were enhanced,and the increase of endothelial cell permeability induced by LPS was reversed.Conclusion METTL3-mediated m6A methylation can improve the permeability of endothelial cells induced by sepsis.

关 键 词：脓毒症 m6A METTL3 内皮细胞通透性 脂多糖 

分 类 号：R644[医药卫生—外科学]