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