FTO通过m6A调控3T3-L1细胞分化的作用及其机制研究  

Effect and Mechanism of FTO in Regulating 3T3-L1 Cell Differentiation through m6A

作　　者：李尹 许心怡 陈钰倩 任炼辞 李聚学 LI Yin;XU Xin-yi;CHEN Yu-qian;REN Lian-ci;LI Ju-xue(Department of Biochemistry and Molecular Biology,School of Basic Medicine,Nanjing Medical University,Nanjing,Jiangsu,211166,China;Key Laboratory of Human Functional Genomics of Jiangsu Province,Nanjing Medical University,Nanjing,Jiangsu,211166,China;The Firtst Clinical Medical College of Nanjing Medical University,Nanjing,Jiangsu,210029,China)

机构地区：[1]南京医科大学基础医学院生物化学与分子生物学系,江苏南京211166 [2]南京医科大学江苏省人类功能基因组学重点实验室,江苏南京211166 [3]南京医科大学第一临床医学院,江苏南京210029

出　　处：《现代生物医学进展》2024年第20期3801-3808,3822,共9页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(82070872)。

摘　　要：目的:探究m6A去甲基化酶FTO及其下游基因CLK2对3T3-L1细胞成脂分化的影响,以及FTO影响CLK2表达水平的分子机制。方法:(1)通过成脂分化诱导和后续的油红染色以及油红定量探究FTO和CLK2对3T3-L1细胞成脂分化的影响;(2)通过蛋白免疫印迹和实时荧光定量PCR测定FTO和CLK2蛋白水平和m RNA水平的改变;(3)通过生物信息学分析筛选不同分化时期的3T3-L1细胞差异化m6A修饰位点;(4)通过MeRIP-qPCR测定CLK2 m RNA上的m6A修饰水平;(5)通过放线菌素D抑制新生转录本合成探究CLK2 m RNA的降解速率;(6)通过胰岛素刺激探究3T3-L1细胞Insulin-AKT通路激活情况。结果:(1)3T3-L1细胞的成脂分化依赖FTO的去甲基化酶活性和CLK2的激酶活性;(2)CLK25'UTR区域存在可被FTO去甲基化的m6A修饰,且该位点m6A修饰提高CLK2 m RNA的降解速率;(3)CLK2表达水平与FTO表达水平存在正相关,且CLK2和FTO抑制剂均抑制3T3-L1细胞Insulin-AKT通路的激活。结论:FTO通过降低CLK25'UTR区域的m6A修饰水平从而抑制CLK2 m RNA的降解,促进CLK2的蛋白表达,CLK2进而通过维持Insulin-AKT通路活性促进3T3-L1细胞成脂分化。Objective:To investigate the effects and the molecular mechanism of m6A demethylase FTO and its downstream gene CLK2 on adipogenic differentiation of 3T3-L1 cells.Methods:(1)The effects of FTO and CLK2 on adipogenic differentiation of 3T3-L1cells were explored by adipogenic differentiation induction,subsequent oil red staining and oil red quantification;(2)The changes of FTO and CLK2 protein levels and m RNA levels were determined by Western blot and real-time PCR;(3)The differential m6A modification sites of 3T3-L1 cells at different differentiation stages were screened by bioinformatics analysis;(4)M6A modification levels on CLK2 m RNA were determined by merip qPCR;(5)The degradation rate of CLK2 m RNA was explored by inhibiting the synthesis of nascent transcripts by actinomycin D;(6)Objective to explore the activation of insulin Akt pathway in 3T3-L1 cells by insulin stimulation.Results:(1)Adipogenic differentiation of 3T3-L1 cells was depened on the demethylase activity of FTO and the kinase activity of CLK2;(2)The m6A modification in the 5'UTR region of CLK2 could be demethylated by FTO,and the m6A modification at this site increased the degradation rate of CLK2 m RNA;(3)There was a positive correlation between CLK2 expression level and FTO expression level,and both CLK2 and FTO inhibitors inhibited the activation of insulin Akt pathway in 3T3-L1 cells.Conclusions:FTO inhibits the degradation of CLK2 m RNA and promotes protein expression of CLK2 by reducing the m6A modification level in the CLK25'UTR region.CLK2,in turn,promotes adipogenic differentiation of 3T3-L1 cells by maintaining insulin AKT pathway activity.

关 键 词：m6A修饰 成脂分化 FTO CLK2 

分 类 号：Q291[生物学—细胞生物学]