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作者	单位	E-mail
李　尹	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；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	liyin_ita@qq.com
许心怡	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；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
陈钰倩	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；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
任炼辞	南京医科大学第一临床医学院江苏南京 210029
李聚学	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；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

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中文摘要:

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mRNA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 mRNA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 mRNA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）CLK2 5'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 mRNA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CLK2 5'UTR区域的m6A修饰水平从而抑制CLK2 mRNA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。

英文摘要:

ABSTRACT 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-L1 cells were explored by adipogenic differentiation induction, subsequent oil red staining and oil red quantification; (2) The changes of FTO and CLK2 protein levels and mRNA 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 mRNA were determined by merip qPCR; (5) The degradation rate of CLK2 mRNA 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 mRNA; (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. Conclusion: FTO inhibits the degradation of CLK2 mRNA and promotes protein expression of CLK2 by reducing the m6A modification level in the CLK2 5'UTR region. CLK2, in turn, promotes adipogenic differentiation of 3T3-L1 cells by maintaining insulin AKT pathway activity.

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