METTL3介导的PDK1 mRNA m6A修饰通过Akt/mTOR信号通路促进肺上皮细胞增殖
METTL3-mediated m6A Modification of PDK1 mRNA Improves Lung Epithelial Cell Proliferation via Akt/mTOR Signaling
靳艾 1李梦宇 2孙青竹3
作者信息
- 1. 山西医科大学公共卫生学院营养与食品卫生学教研室,太原 030001;山西医科大学营养与食品科学研究所,太原 030001;山西医科大学煤炭环境致病与防治教育部重点实验室,太原 030001;山西医科大学黄河流域生态公共卫生安全研究中心
- 2. 山西医科大学公共卫生学院营养与食品卫生学教研室,太原 030001
- 3. 西北农林科技大学动物科技学院动物科学系,陕西杨凌 712100
- 折叠
摘要
腺苷N6-位点甲基化(m6A)在细胞增殖过程中发挥重要作用.RNA甲基转移酶3(METTL3)作为催化m6A关键酶,其介导m6A修饰在肺上皮细胞增殖中的作用机制尚不明确.本研究旨在探讨METTL3介导m6A修饰调控肺上皮细胞增殖的效应及机制.结果显示,在肺上皮细胞中敲低METTL3显著抑制细胞生长,而过表达METTL3则促进了细胞增殖(P＜0.05).进一步的蛋白质免疫印迹结果显示,细胞生长和增殖的关键蛋白质PCNA在METTL3敲降的肺上皮细胞中蛋白质水平的表达显著下调,并且Akt以及mTOR的磷酸化水平显著降低(P＜0.05).细胞免疫荧光结果发现,METTL3敲降的肺上皮细胞中m6A修饰水平显著降低(P＜0.05).实时荧光定量PCR及蛋白质免疫印迹结果表明,Akt-mTOR信号通路上游调控分子PDK1的mRNA和蛋白质表达水平在METTL3敲降的肺上皮细胞中显著下降(P＜0.05).机制上,m6A-IP-qPCR和RIP-qPCR结果进一步表明,METTL3催化PDK1 mRNA的3'UTR区域m6A修饰,进而被YTH N6-甲基腺苷RNA结合蛋白1(YTHDF1)识别,增强其mRNA的稳定性.总之,本研究揭示了 METTL3通过增强PDK1 m6A修饰,进而激活Akt-mTOR信号通路,促进细胞增殖.本研究为METTL3在上皮细胞增殖中的新角色提供了证据,同时为治疗肺上皮细胞损伤修复提供了新的治疗靶点.
Abstract
Adenosine N6-methylation(m6A)has been shown to be associated with the cell proliferation.The role of RNA methyltransferase 3(METTL3),a key enzyme catalyzing m6A,in mediating m6A modification in lung epi-thelial cell proliferation remains unclear.This study aims to explore the effects and mechanisms of METTL3-media-ted m6A modification in regulating lung epithelial cell proliferation.Results showed that knockdown of METTL3 sig-nificantly inhibited cell growth in lung epithelial cells,while overexpression of METTL3 promoted cell proliferation(P＜0.05).Further Western blotting results demonstrated that the expression levels of the key proteins PCNA in-volved in cell growth and proliferation were significantly downregulated in METTL3 knockdown lung epithelial cells,along with a significant decrease in phosphorylation levels of Akt and mTOR(P＜0.05).Immunofluorescence stai-ning revealed a significant decrease in m6A modification levels in METTL3 knockdown lung epithelial cells(P＜0.05).Real-time quantitative PCR and Western blotting results indicated that the mRNA and protein expression levels of PDK1,an upstream regulator of the Akt-mTOR signaling pathway,were significantly decreased in MET-TL3 knockdown lung epithelial cells(P＜0.05).Mechanistically,m6A-IP-qPCR and RIP-qPCR results further demonstrated that METTL3 catalyzed m6A modification in the 3'UTR region of PDK1 mRNA,which was then rec-ognized by YTH N6-methyladenosine RNA-binding protein 1(YTHDF1),enhancing the stability of its mRNA.In conclusion,this study reveals that METTL3 promotes cell proliferation by enhancing PDK1 m6 A modification,thereby activating the Akt-mTOR signaling pathway.It provides evidence for a novel role of METTL3 in epithelial cell proliferation and offers new therapeutic targets for lung epithelial cell injury repair.
关键词
甲基转移酶3/RNA/m6A修饰/细胞增殖/肺上皮细胞/磷酸肌醇依赖性蛋白激酶-1Key words
methyltransferase 3(METTL3)/RNA m6A modification/cell proliferation/lung epithelial cells/phosphate inositol dependent protein kinase-1(PDK1)引用本文复制引用
基金项目
国家自然科学基金(82304146)
山西省基础研究计划(202303021212125)
山西省高等学校科技创新计划项目(2023L061)
出版年
2024
NETL
NSTL
万方数据