Reciprocal Regulation Of Adipogenic And Osteogenic Differentiation By MiR-30e And The Mechanisms Involved In The Processes

Objective:Mouse mesenchymal stem cells(MSCs)are multipotent cells which can differentiate into different types of cells under the control of cell fate regulators,such as osteoblast,adipocyte,chondrocyte and so on.A mutually exclusive relationship exists between adipogenesis and osteogenesis due to the derivation from the common progenitor cell.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.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 quantitative RT-PCR,we tested the expression of miR-30e in the mesenchymal cell line C3H10T1/2 and the preadipocyte 3T3-L1 48 h after treatment with adipogenic medium or in stromal ST2 cells and preosteoblast MC3T3-E1 after treatment with osteogenic medium.2)The effect of the miR-30e on the proliferation of 3T3-L1 cells was determined by the MTT assay following transfection of synthetic mimics or inhibitor of miR-30e.We also studied the effects of miR-30e 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.The effects of miR-30e on osteoblast differntiation was studied as well by transfecting the inhibitor of miR-30e into the preosteoblast MC3T3-E1 followed by alkaline phosphate staining of the cells and RT-PCR quantification of the-specific transcription factors and marker genes.3)To clarify molecular mechanisms underlying the regulation of adipogenesis and osteogensis by miR-30e,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-30e,the 3'-UTR fragments of the mRNAs containing the target sequences were PCR-amplified and cloned into the luciferase reporter vector,respectively.The effects of miR-30e on the luciferase activity of the 3'-UTR constructs were assayed.The role of the validated 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 14 miRNAs were found to be increased during adipocyte differentiation and at the same time decreased during osteoblast differentiation,and miR-30e was among the most significantly changed.It was also induced in primarily cultured mouse bone marrow stromal cell,mesenchymal cell line C3H10T1/2 and preadipocyte 3T3-L1 after adipogenic treatment.Coversely,it was reduced in mouse stromal line ST2 and preosteoblast MC3T3-E1 after osteogenic treatment.2)Transfection of miR-30e mimics inhibited cell proliferation in 3T3-L1,and conversely,miR-30e inhibitor showed the opposite effect.By transfecting the miR-30e mimics into 3T3-L1,we found that over-expression of miR-30e 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-30e 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-30e,we made the lentivirus that over-expressed miR-30e precursor and infected the 3T3-L1 cells with the virus.The lentivirus duplicate the adipocyte promoting program of the the mimics.To verify if miR-30e could regulate osteoblast differentiation,we silenced the expression of miR-30e in preosteoblast MC3T3-E1 by transfecting the miR-30e inhibitor into the cells.Osteoblast differentiation was potentiated,as evidenced by the enhanced ALP staining.Consistently,real-time PCR analysis revealed that the mRNA levels of Runx2,osterix,osteocalcin,ALP and BSP were significantly increased upon exposure to osteoblast differentiation medium.3)Runx2,Wnt7b and Lrp6 were predicted to be potentially associated with adipocyte and osteoblast differentiation.Luciferase assay revealed that the miR-30e miRNA significantly decreased the luciferase activity of Lrp6 3'UTR construct and removal of the putative miR-30e response sequence from the 3'UTR construct abolished the inhibitory effect of miR-30e on the construct.Furthermore,enhanced expression of miR-30e in 3T3-L1 cells led to a decrease in Lrp6 and ?-catenin protein in 3T3-L1 cells.In contrast,suppression of endogenous miR-30e significantly increased Lrp6 and ?-catenin protein levels.4)To further demonstrate that LRP6 controls adipogenesis,we carried out LRP6 loss-of function studies using 3T3-L1 cells.Western blotting showed that knockdown of LRP6 led to a striking decrease in ?-catenin protein.Moreover,Topflash assay revealed a dramatic decrease of luciferase activity following the knockdown of LRP6 in 3T3-L1 cells.Furthermore,upon exposure to the AIM treatment,the knockdown adenovirus 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.Conclusions:1)miR-30e is induced during adipocyte formation and reduced during osteoblast differentiation.2)miR-30e suppresses cell proliferation in adipocyte progenitors and favors adipocyte differentiation.Conversely,miR-30e blocks osteoblast differentiation.3)Lrp6 is a direct target of miR-30e.4)Lrp6 restrains adipogenesis and miR-30e reciprocally regulates adipocyte and osteoblast differentiation by directly targeting Lrp6.