Regulation Of Adipogenic Differentiation By MiR-20a And The Mechanisms Involved In The Processes

Objective:MicroRNAs(miRNAs)are a class of short(about 22nt)non-coding RNA molecules that can regulate gene expression primarily through post-transcriptional repression.In recent years,researchers figured out that miRNAs play an important role in a broad range of biological processes,including cell fate determination in embryonic stem cells,cell proliferation,differentiation and apoptosis.Exploring the biological function of miRNAs in regulating adipocyte formation and osteoblast formation will provide a new insight on differerntiation mechanism of MSCs.In this study,we are doing to investigate the effect of miR-20a on adipogenic differentiation of mice bone marrow-derived MSCs,try to identify the target gene of miR-20a and then clarify the mechanisms of miR-20a promoting adipogenic differentiation.Methods:1.To identify the miRNA expression signature in the marrow stromal cells in response to the osteogenic or adipogenic culture medium,miRNA profiles of the cells were analyzed using TaqMan Rodent MicroRNA Array.Moreover,by using Real Time PCR,we tested the expression of miR-20a in the mesenchymal cell line C3H10T1/2 and the preadipocyte 3T3-L1 after treatment with adipogenic medium or in stromal ST2 cells after treatment with osteogenic medium.2.We studied the effects of miR-20a mimics and inhibitor on adipocyte formation by counting the numbers of differentiated adipocytes,analysing the mRNA and protein expression levels of adipocyte-specific transcription factors and marker genes.We also made the lentivirus that overexpresses miR-20a precursor and infected the 3T3-L1 cells with the virus.The expression levels of adipocyte-specific transcription factors and marker genes were detected by Real Time PCR.3.To clarify molecular mechanisms underlying the regulation of adipogenesis by miR-20a,bioinformatics prediction of miRNA targets was performed with TargetScan and PicTar.In order to test whether the potential target genes are really targeted by miR-20a,the 3'UTR fragments of the mRNAs containing the target sequences were PCR-amplified and cloned into the luciferase reporter vector.The effects of miR-20a on the luciferase activity of the 3'UTR constructs were assayed.The role of the target gene in controling adipogenesis was invesigated by carrying out loss-of function studies using 3T3-L1 cells.Results:1.The microRNA array revealed that miR-17 family were found to be increased during adipocyte differentiation and remained unchanged after treatment with the basic osteogenic treatment.It was also induced in mesenchymal cell line C3H10T1/2 and preadipocyte 3T3-L1 after adipogenic treatment.Coversely,it was reduced in mouse stromal line ST2 at early stage after osteogenic treatment.2.By transfecting the miR-20a mimics into 3T3-L1,we found that over-expression of miR-20a could promote lipid droplets accumulation and enhance the expression of the key adipogenic transcription factors PPARy,C/EBPa and C/EBP? and adipocyte marker gene aP2.Conversely,miR-20a inhibitor reduced lipid droplets accumulation and decreased the expression of PPARy,C/EBPa,C/EBP? and ap2.To further investigate the adipogenic effect of miR-20a,we made the lentivirus that over-expressed miR-20a precursor and infected the 3T3-L1 cells with the virus.The lentivirus duplicate the adipocyte promoting program of the the mimics.3.Tcf4,Bmp2,Smad5 and PPARy were predicted to be potentially associated with adipocyte differentiation.Luciferase assay revealed that the miR-20a miRNA significantly decreased the luciferase activity of Tcf4 3'UTR construct and removal of the putative miR-20a response sequence from the 3'UTR construct abolished the inhibitory effect of miR-20a on the construct.Furthermore,suppression of endogenous miR-20a significantly increased Tcf4 protein levels.To further demonstrate that Tcf4 controls adipogenesis,we carried out Tcf4 loss-of function studies using 3T3-L1 cells.Furthermore,upon exposure to the AIM treatment,the knockdown lentivirus resulted in the potentiation of adipocyte differentiation program,evidenced by the increased number of oil-red O positive adipocytes.The expression levels of PPAR?,C/EBP?,C/EBP? and aP2 were dramatically enhanced.Conclusion:1.miR-20a is induced during adipocyte formation and reduced during osteoblast differentiation.2.miR-20a favors adipocyte differentiation.3.Tcf4 is a direct target of miR-20a.Tcf4 restrains adipogenesis and miR-20a reciprocally regulates adipocyte differentiation by directly targeting Tcf4.