MiR-20a-5p Modulates Adipogenic Differentiation In Mouse Bone Marrow Derived Stromal Cells By Targeting Klf3

Objective: MicroRNA(miRNA)are a class of endogenous single-stranded non-coding RNA of 19~ 22 nucleotides in length that can regulate gene expression by directing the silencing complex to degradate m RNA or repress its translation.miRNA are involved in the regulation of various developmental and homeostatic events,including embryonic development,energy balance,metabolism and tumorigenesis.A large number of reports have suggested that miRNA could regulate adipocyte differentiation by affecting the expression of various transcription factors associated with adipogenic differentiation.In our previous study,miR-20a-5p has been shown to promote adipogenic differentiation in ST2 stromal cells and C3H10T1/2 mesenchymal cells.However,its role and molecular mechanisms in the regulation of bone marrow derived stromal cells(BMSCs)lineage commitment to adipocyte remained unclear.This study focused on exploring the functions and mechanisms of miR-20a-5p in the regulation of adipogenic differentiation from primary cultured BMSCs.Methods: 1.Quantitative RT-PCR(RT-q PCR)analysis was used to detect the expressions of miR-20a-5p and adipogenic differentiation marker genes during adipocyte differentiation in primary cultured BMSCs;we performed gain-of-function study with overexpression lentivirus or synthetic mimics of miR-20a-5p,and loss-of-function study with sponge lentivirus or synthetic inhibitor of miR-20a-5p to investigate the effect of miR-20a-5p on adipogenic differentiation in mouse BMSCs using RT-q PCR,Western blotting and Oil-red O staining.2.For clarifying the molecular mechanism of miR-20a-5p regulating adipocyte differentiation,the candidate target genes of miR-20a-5p were predicted by prediction program Target Scan and were identified by dual luciferase reporter assay,GFP reporter assay and Western blotting.Moreover,the expressions of the other KLF family members(Klf5,Klf7)were measured after transfecting miR-20a-5p mimics and inhibitor into BMSCs respectively.3.Using loss-of-function and rescue experiments to verify the role of the candidate target gene Klf3 in miR-20a-5p-induced adipogenic differentiation.In addition,we determined whether Klf3,Kdm6 b and Tgfbr2(another two valid target genes of miR-20a-5p)attenuated miR-20a-5p-induced adipogenic differentiation synergistically through with RT-q PCR analyses and Oil-red O staining.Results: 1.The expressions of adipogenic differentiation marker genes peroxisome proliferator-activated receptor ?(PPAR?),CCATT enhancer binding protein alpha(C/EBP?),and adipose fatty acid binding protein 4(FABP4 or a P2)were significantly increased in BMSCs after adipogenic treatment.The expression of miR-20a-5p was up-regulated gradually for the first 3 days,and kept in a high level at following 2 days compared to day 0.2.We constructed the lentivectors that either overexpression miR-20a-5p precursor or downregulate endogenous miR-20a-5p successfully.Supplementing the activity of miR-20a-5p in mouse BMSCs by miR-20a-5p overexpression lentivirus or miR-20a-5p mimics promoted adipocyte differentiation,and increased adipogenic markers genes PPAR?,C/EBP? and a P2 protein expression levels significantly;conversely,downregulating the endogenous miR-20a-5p level by miR-20a-5p sponge lentivirus or miR-20a-5p inhibitor reduced lipid droplets accumulation and decreased the expression of PPAR?,C/EBP? and a P2.3.Klf3 was predicted to be a candidate target gene of miR-20a-5p.We successfully constructed recombinant luciferase/GFP reporter vectors containing the candidate target gene 3'UTR region and its mutant respectively.With dual luciferase report assay and GFP reporter gene assay,we found miR-20a-5p mimics significantly decreased luciferase activity in group transfected with Klf3-3'UTR luciferase reporter and down-regulated GFP expression in group transfected with Klf3-3'UTR GFP reporter respectively.In addition,miR-20a-5p mimics decreased the expression of Klf3 but miR-20a-5p inhibitor increased it.4.The manipulation of miR-20a-5p mimics or inhibitor didn't affect the protein levels of the other two KLF family members Klf5 and Klf7 in BMSCs when Klf3 expression was altered,indicating that there were not any compensatory changes of these two KLF family members upon Klf3 upregulation or downregulation.5.SiRNA-mediated Klf3 silence could promote the differentiation of mouse BMSCs into adipocytes,which consistented with the function of miR-20a-5p overexpression.In rescue-experiment,it showed that compared with overexpression of Klf3 alone,overexpression of mimics and Klf3 simultaneously enhanced adipogenic differentiation of BMSCs: Compared with overexpression of mimics alone,overexpression of mimics and Klf3 simultaneously inhibited adipogenic differentiation.These results suggested that miR-20a-5p may play a role in promoting adipogenic differentiation through Klf3.6.Compared with miR-20a-5p mimics and Klf3 co-transfection group,the number of adipocyte differentiation in miR-20a-5p mimics and Klf3,Kdm6 b and Tgfbr2 co-transfection group was significantly reduced;accordingly,the adipogenic marker genes PPAR?,C/EBP?,a P2 expression were down-regulated.Conclusion: 1.The expression of miR-20a-5p in the mouse primary cultured BMSCs was up-regulated after adipogenic treatment.2.MiR-20a-5p overexpression lentivirus and miR-20a-5p mimics promoted the adipogenic differentiation from BMSCs.In contrast,miR-20a-5p sponge lentivirus and miR-20a-5p inhibitor inhibited the adipogenic differentiation from BMSCs.3.Klf3 was the direct target gene of miR-20a-5p.4.There were not any compensatory changes of Klf5 and Klf7 when miR-20a-5p affected the expression of Klf3.5.Klf3 inhibited adipogenic differentiation from BMSCs;miR-20a-5p could promote adipocyte differentiation through targeting Klf3.6.Simultaneous overexpression of Klf3,Kdm6 b and Tgfbr2 could more effectively neutralized the role of miR-20a-5p in promoting adipogenesis.