The Role Of MiR-196b-5p In Adipogenic Differentiation And Lipogenesis And The Mechanisms Involved

Objectives MicroRNAs are a class of evolutionarily conserved small non-coding RNAs that play a regulatory role at the post-transcriptional level.It is reported that microRNAs regulate adipogenic differentiation of mesenchymal stem cells,but the roles in lipogenesis,lipolysis and the molecular mechanisms remain to be explored.This study probed the function and mechanism of miR-196b-5p in adipogenic differentiation,lipogensis and lipolysis in vitro.Methods 1.miR-196b-5p,in ST2 and C3H10T1/2 transfected of miR-196b-5p mimics,was detected by qRT-PCR technology.The level of miR-196b-5p was increased or decreased in two cell lines,and the cell differentiation effect of miR-196b-5p was detected,by the ways of oil red O staining,triglyceride assay,qRT-PCR and Western blotting.2.miR-196b-5p' target was identified by Targetscan and dual luciferase test.The relationship between miR-196b-5p and target-related signaling pathway was explored by Western blotting.The role of the target was explored by functional acquired experiments and loss experiments.It's further determined the regulatory relationship between miR-196b-5p and the signaling pathway.Results 1.miR-196b-5p mimics increased the level of intracellular miR-196b-5p.Supplementing miR-196b-5p stimulated adipogenic differentiation,increased levels of TG.Moreover,several factors invoved were promoted,the key factors in adipocyte differentiation including PPAR?,C/EBP?,aP2 and Adipoq;the lipogenesis key factors comprised of Scd1,Fasn,Srebp1 c and Acc1?perilipin and the lipolysis-specific factors consisted of Acox1,Atgl and Lpl.Conversely,inhibition of endogenous miR-196b-5p inhibited adipogenic differentiation and the gene expression involved.2.Tsc1 and Rictor were predicted as the potential targets of miR-196b-5p via targetscan.The plasmids of Tsc1 and Rictor 3'UTR containing the miR-196b-5p binding site were constructed,the double luciferase assay proved that miR-196b-5p reduced the luciferase activity by binding to Tsc1 not to Rictor 3'UTR,and the luciferase activity was increased after the binding site mutated.Moreover,miR-196b-5p inhibited the protein expression of Tsc1,which proved that Tsc1 was the target of miR-196b-5p.miR-196b-5p boosted the phosphorylational protein level of mTOR,S6K1,RPS6 and 4EBP1,proving that miR-196b-5p activated mTORC1 signaling pathway.The Tsc1 overexpression plasmid was constructed for functional acquisitional experiments,and the siRNA was synthesized for functional loss experiments.The target gene function test showed that overexpression of Tsc1 could inhibit ST2 cells to adipogenic differentiation,following down-regulated lipogenesis and lipolysis genes.Reversely,inhibiting endogenous Tsc1 could significantly promote the adipogenic differentiation of ST2 cells,and the related factors were obviously up-regulated.After inhibition of the mTORC1 signaling pathway by rapamycin,the rising trend of adipogenic differentiation was rescued.Conclusion 1.miR-196b-5p mimics can obviously increase the level of miR-196b-5p in ST2 and C3H10T1/2 cells.miR-196b-5p mimics can promote cell adipogenic differentiation,lipogensis and lipolysis;whereas miR-196b-5p inhibitor can block adipogenic differentiation,lipogensis and lipolysis.2.Tsc1 is the target gene of miR-196b-5p.3.The overexpression of miR-196b-5p activates the mTORC1 signaling pathway.4.The overexpression of Tsc1 can inhibit ST2 cells differentiation into adipocyte,and downregulates lipogensis and lipolysis related factors,but the suppression of Tsc1 obviously up-regulate adipocyte differentiation,lipogenic and lipolysis related factors.5.miR-196b-5p regulates adipogenic differentiation via the mTORC1 signaling pathway,which was verified by Rapamycin.