Expression of MiR-125b in Osteoblastic Differentiation of C3H10T1/2Cell and its Target Gene Analysis
|School||Third Military Medical University|
|Keywords||miRNA miR-125b Dual luciferase reporter system mesenchymal stemcell Core-banding factor β|
Research background and Objective：MicroRNA(miRNA)is a group of approximately21-25nucleotide long single-strandednon-coding RNAs,which located in non-coding area of chromosome and can not betranslated in to proteins. Maturing miRNAs combine with the3’-UTR (3’-untranslationalregion) of target genes to cause the degradation of mRNA or inhibition of translation.Inrecent years, miRNAs have generally showed to be involved in regulating individualdevelopment, differentiation, proliferation and other biological process. Especially itsinfluence in self-renewal and differentiation of stem cells including MSCs has attractedmore attention.Osteoblasts were derived from MSCs.So far,many factors and mechanisms haveshown to be involved in this process. Studies showed that miR-125b can be the key factorwhich regulating osteoblastic differentiation of MSCs.However, the precise mechanismsinvolved still remain to be defined.Therefore,this study was aimed to detect miR-125bexpression in osteoblastic differentiation of MSCs,verify its target gene and discussed thepossible regulatory mechanisms of miR-125b in osteoblastic differentiation.Methods：1. Analysis the spectrum of miRNAs of C3H10T1/2cell lines(mouse MSCs) inosteoblastic differentiation by using gene Microarray. C3H10T1/2cell cultured withdiffenetiate medium after7d (group1)and14d(group2) as the experimental group,C3H10T1/2cell cultured with DMEM medium as the control group(group NC). In addition,C3H10T1/2cell cultured with diffenetiate medium after1d,3d,5d, endogenous expressionlevel of miR-125b was measured by qRT-PCR.2. To further define the miR-125b specific role of the target gene,we passedPictar,Target scan,miRanda bioinformatic analysis.At last,we select Cbfβ as miR-125b target gene and identified.3. To identify Cbfβ is the exact target gene of miR-125b,we will plug the predictedmiR-125b binding sites upstream and downstream part of Cbfβ into PmiR-report luciferaseexpression vector. pRL-TK vector as a control vector, analyse the results by dual luciferaseplasmid reporter system.Acording to the3’UTR of Cbfβ,there were three fragments wassynthesised:①Experimental fragment:wild type sequence (microRNA recognitionelement,MRE).②Positive control:completely complementary with mature miR-125b(Perfect Target,PT).③Negative control: wild type lack of seed sequence(MRE-mutation，mut).In order to acquire positive result,we created recombinant plasmid contained twocopies of each fragment(2×PT，2×MRE，2×mut). Previous studies have proven that suchplasmid constructed with multiple fragment is an available method.Then recombinantplasmid and pRL-TK vector were co-transfected with miR-125b mimic or anti-miR-125binto293T cell. The293T cell were harvest48h after transfection for dual luciferaseanalysis.4. Overexpressed the exogenous miR-125b and inhibit the endogenous miR-125bexpression in C3H10T1/2cells,Which were harvest48h later for Cbfβ expression analysisby Western-blot and RT-PCR.Results：1. By microRNA Microarray data analysis,we found that:①Comparing with groupNC,there were12miRNAs up-regulated and30miRNAs down-regulated in group1,84miRNAs up-regulated and98miRNAs down-regulated in group2. besides,comparing withgroup1,there were92miRNAs up-regulated and94miRNAs down-regulated in group2.(>2fold)②The relative expression level of miR-125b,group NC: group1: group2=1:0.8710:0.6378.The results of qRT-PCR showed that C3H10T1/2cells cultured withdiffenetiate medium after1d,3d,5d,endogenous expression level of miR-125b wasdown-regulated when compared with none differentiate cells.2. The results of bioinformatics prediction showed that the total number of target geneof mmu-miR-125b was342.Core banding factor β,a bone development regulator,was one ofthe putative target gene.By softwares forecasting, Cbf β3’-UTR contains one putativemmu-miR-125b seed site which is bound with imperfect complementation.3. recombinant plasmid pMIR-2×PT，pMIR-2×MRE，pMIR-2×mut were deal with a single restriction endonuclease HindⅢ,the products of digestion were observed byelectrophoresis. the results consistent with the theoretical value, and sequenced by Takaracompany, data confirm that the recombinant vector was successfully constructed.4. The dual luciferase results as follows:After we co-transfected PmiR-2×PT into293Tcells with miR-125b mimic or anti-miR-125b,we found that luciferase expressionsignificantly decreased or increased in the293T cells. Co-transfection miR-125b mimicwith wild-type PmiR-2×MRE resulted in a suppression of luciferase gene expression,butCo-transfection miR-125b mimic with PmiR-2×mut did not.In addition we found thatanti-miR-125b could overcome the suppression effect when it was co-transfected withPmiR-2×MRE and miR-125b mimic.But the effect of anti-miR-125b was abolished whenPmiR-2×mut was used instead of PmiR-2×MRE.5. As expected for the mechanisms of miR-125b regulation,measured by western blotand RT-PCR assay,Cbfβprotein and mRNA level were notably decreased in the cells ofmiR-125b over-expression and increased in the cells of miR-125b inhibition.Conclusion：miR-125b was down-regulated during osteoblastic differentiation. miR-125b couldregulate Cbfβprotein and mRNA expression by binding3’-UTR of CbfβmRNA.