Molecular Mechanisms Of MicroRNA-125b Regulates Osteogenic Differentiation Of Mesenchymal Stem Cells By Targeting Cbfβ

Research background and Objective：MicroRNAs (miRNAs) are endogenous, non-coding and21-24nucleotide small RNAs,which regulate the body metabolism exerting their biological effect. They are also smallmolecules regulating factors which play an important role in the stem cell function. miRNAscombine with the3’-UTR of target genes to give rise to the degradation of mRNA orsuppression of translation. Thus, the expression of target genes is negative controlled. Recentstudies suggested that miRNAs promoted the directional differentiation of stem cells throughdown-regulation of certain genes to maintain stem cell in an undifferentiated state and activatespecific genes of stem cell lineage, such as osteogenic differentiation, chondrogenicdifferentiation and adipogenic differentiation. MicroRNA-125b (miR-125b) is one of theearliest discovered miRNAs that plays a key role in the cellular functions of many cellsespecially in the control of proliferation and differentiation.Bone marrow mesenchymal stem cells (MSCs) exist in the bone marrow and manymature cells, with a great potential of self-renewal and differentiation to cell lineagesincluding osteoblasts, chondrocytes, adipocytes. Multiple levels of regulation and controlmechanisms are involved in osteoblastic differentiation from the MSCs and some studies haveproven that a variety of hormones and paracrine and/or autocrine cell factors participated inthis process.It has recent been researched that miR-125b may an important regulatory factor in theosteoblast differentiation of bone marrow MSCs process. Combined with previousexperimental, we confirmed that Cbfβ is one of target genes of miR-125b, but the concreteregulation mechanism remains to be further explored. Therefore, this study aims to find outthat miR-125b controls function in osteoblastic differentiation of MSCs, preliminarydiscussion that miR-125b regulates mechanism in osteogenic differentiation of MSCs by targeting Cbfβ.Methods：1. To verify Cbfβ regulated influence of osteoblastic differentiation in MSC, weconstructed artificial microRNA vector intervening the Cbfβ gene and transfected C3H10T1/2cells, while we also obtained a stable cell strain. Empty vector as control group, amir-Cbfβ asexperimental group, expression of ALP was detected in C3H10T1/2cells by BMP-2treatmentfor3d. Observe amir-Cbfβ inhibition of Cbfβ and regulation effect of osteogenesisdifferentiation.2. Analysis the expression profile of miRNAs of C3H10T1/2cell lines (from mouseMSCs) in osteogenic differentiation by using qRT-PCR. Accordingly, we further verifed earlydetection of gene chip detection result. Non-induced cells are control group, C3H10T1/2cellcultured with diffenetiate medium after1d,3d,5d. We analyzed differential expression ofmiR-125b in two groups of three time points. Endogenous miR-125b expression level wasmeasured by qRT-PCR.3. Take Cbfβ, Runx2, ALP, OCN, OPN and PPAR-γ as research objects. We transfectedrespectively into C3H10T1/2cells by BMP-2treatment with miR-125mimic, miR-125binhibitor and negative control for1d,7d and14d. qRT-PCR and Western Blot detectedexpression of Cbfβ, Runx2, ALP, OCN, OPN and PPAR-γ. And we observed miR-125bregulated function of Runx2, ALP, OCN, OPN and PPAR-γ by target Cbfβ.4. To verify expression of ALP, OCN, OPN, we conducted ALPactivity detection and calciumnodule dye in C3H10T1/2cells and C57BL/6mice bone marrow mesenchymal stem cells.Results：1. Construction of artificial microRNA vector intervening the mice Cbfβ gene andobtainment and identification of steady cell line. amir-Cbfβ vector intervening wassuccessfully constructed and transfected C3H10T1/2cells. We obtained a stable cell strain.qRT-PCR and Western blot were used to detect expression level of Cbfβ, Runx2and ALP inthe cells with BMP-2-treatment after3d. The expression of result that transfected vectorintervening is lower than negative control.2. The qRT-PCR revealed that C3H10T1/2cells were cultured with differentiate mediumafter1d,3d,5d, endogenous expression level of miR-125b was down-regulated whencompared with none differentiate cells （P<0.05）. 3. The results of qRT-PCR and Western blot showed that the expression of Cbfβ, Runx2,ALP, OC and OPN in C3H10T1/2cells with transfected miR-125b mimic obvious decreased(for0d,7d), while the PPAR-γ expression increased. Transfection miR-125b inhibitor in thesame way, the expression of Cbfβ, Runx2, ALP, OC and OPN increased, while the PPAR-γexpression decreased. However, we could not find definite proof that miR-125b would haveany effect on the terminal differentiation of osteoblastic by long-time effecting (for14d). Theexperiment repeated three times, the result was statistically significant (P <0.05).4. Alkaline phosphatase measurement and Alizarin red staining: We transfectedrespectively into C3H10T1/2cells and C57BL/6mice bone marrow mesenchymal stem cellsby BMP-2treatment with miR-125mimic, miR-125b inhibitor and negative control. And wedetected ALP activity at14d, we found that the activity of transfected miR-125b inhibitorhigher than transfected miR-125b mimic. The experiment repeated three times, the result wasstatistically significant (P <0.05). Calcium nodule number in calcium nodules staining frombig to small: miR-125b inhibitor、control、miR-125b mimic.Conclusion：Construction the vector intervening of amir-Cbfβ may inhibit expression of Cbfβ, anddecreased expression of Runx2and ALP. We also obtained a stable cell strain. miR-125b wasreduced in time of osteoblastic differentiation in C3H10T1/2cells. miR-125b negativelyregulated expression ALP, OC, OPN, and positively regulated PPAR-γ expression.