Study On MiR-141 Regulating OP Mice BMSCs On Osteogenic Differentiation And The Intervention Effect Of JianGu Particles

ObjectiveTo clarify the mechanisms of ovariectomized mouse bone marrow mesenchymal stem cells(BMSCs) on osteogenic differentiation between miR-141 and Dlx5/Msx2/Runx2 in BMP signaling pathways, and reveal the mechanism of Jiangu particles preventing postmenopausal osteoporosis from the aspect of miRNA adjustment function.Methods1 The mechanism of miR-141 regulating OP mice BMSCs on osteogenic differentiation.1.1 Correlation study on miR-141 expression and postmenopausal osteoporosis (PMOP): Mice PMOP model was built by ovariectomy,divided into model and sham group. BMSCs were isolated and cultured by whole bone marrow culture method, phenotypic identification was used by flow cytometry. calculating the cell morphology and growth status by measuring the growth curve and clone formation were observed to.RT-PCR were applied to detect mRNA expression of miR-141 in the two cases.1.2 The study of expression on miR-141 and osteogenic gene Dlx5/Msx2/Runx2 in osteogenic differentiation process of BMSCs:model group,sham group BMSCs was induced by osteogenic inducing medium with 14d, RT-PCR detected miR-141 and osteogenic gene Dlx5,Msx2,Runx2 expression at3/8/13d, cell morphology was observed, alkaline phosphatase (ALP) was dyed and quantitated simultaneously,RT-PCR results was verified by Western blot. Staining calcified nodules was dyed by Alizarin Red at 13 d.1.3 To clarify the mechanism of miR-141 regulating Dlx5/Msx2/Runx2 signal axes on osteogenic differentiation process of BMSCs:promoting the miR-141 with miR-141 mimics and reducing with miR-141 inhibitors by transfection reagent(Lipo2000) transfected BMSCs transiently,induced by osteogenic agent 3d after,NC group was established.Observing cell morphology and ALP quantitated after 8d,the expression of miR-141,DLx5,Msx2, Runx2 calcified nodules was dected after 13 d.2 The mechanism of Jiangu particles regulating OP mice BMSCs on osteogenic differentiation.2.1 The mechanism of Jiangu particles containing serum regulating OP mice BMSCs on osteogenic differentiation:The BMSCs of model group osteogenic divided into two groups, Jiangu particles and saline serum intervention were added in 3d latter (intervention 72 hours once),8d latter,row cell morphology,ALP staining and quantitative;Observing cell morphology and ALP quantitated after 8d, the expression of miR-141,DLx5,Msx2,Runx2 calcified nodules was dected after 13 d.2.2 The mechanism of miR-141 regulating mice BMSCs with Jiangu particles osteogenic differentiation in Animal experiment:The model of PMOP was established by ovariectomized after 2 months,and divided into sham group with 10,two groups ovariectomized mice of each 10, surgery after a week, two ovariectomized mice groups were fed with Jiangu particles and normal saline respectively.2 months after model established successfully,taking the mice bone with microCT osteoporosis indicators and three-dimensional images of bone tissue and biomechanics detectinon; and detecting miR-141 and Dlx5, Msx2, Runx2 expression in bone tissue.Results1 Ovariectomized group compared with the sham group after model established:Sparse trabecular bone with mass decreased and microarchitecture broken; Tibia maximum load decreased; BMSCs proliferation reduced 6 days and cell colonies significantly 8 days; MiR-141 expression was significantly higher. Phenotype of two BMSCs:CD29, CD44(+); CD34, CD45(-).2 Compared within each BMSCs group,Dlx5,Runx2 expression are rising after osteogenic induction; MiR-141, Msx2 continued to decline; ALP decreased gradually improve in the first 7 days peak then decline, was "A" type change. Calcified nodules appeared in the mid-term induced and late extremes. Ovariectomized group compared with the sham group: cell proliferation and differentiation reduced, especially in the mid-term; MiR-141, Msx2 expression increased and Dlx5, Runx2,ALP reduced.3 Website forecast and verified by the dual luciferase reporter gene assay:miR-141 and the 3’-UTR sequence of Dlx5 exist a binding site; After cells transfected with osteogenic formation:ALP and calcified nodules compared with the control group,mimics group decrease and inhibitor increased,the differences are significant.13 days after transfection Dlx5,Runx2 gene and protein expression of miR-141 inhibitor group were increased,and Msx2 decreased, miR-141mimics group had conversely result.4 When drug serum concentration was 10%, the number of bone cells of the drug and saline serum group were the maximum. The proliferation speed of drug serum cell fastly, osteogenic differentiation highly. The expression of Drug serum miR-141,Msx2 were lower, and Dlx5 and Runx2 higher.5 Mice fed with Jiangu particles compared with saline group:bone mass increased, trabecular number, width, length, shape and distribution of partial recovery partly, density and coupling increased, but can not return to the sham group level, cortical bone area and bone marrow cavity recovered to sham group level;Three-piont compression maximum load significantly increased, but slightly lower than the sham group;Expression of miR-141,Msx2 in bone tissue decreased and Dlx5,Runx2 increased.Conclusions1 The bone metabolism status of ovariectomy mice were similar of human with PMOP. Ovariectomy can make BMSCs significantly reduced clearly in the mid-late term, and make expression of miR-141 increased, which indicate that the endogenous expression of miR-141 were associated with PMOP.2 In osteogenic process, miR-141,Msx2 were osteogenic negative regulator factor, and Dlx5、Runx2 were positive ones; ALP, as a bone metabolism synthase in which, appear peak change along with cell proliferation,differentiation and the aging process. Calcified nodules, as osteogenic differentiation markers, were highest concentrations in the late stage.3 Dlx5 is a target gene of miR-141, and its relationship to post-transcriptional inhibition; MiR-141 inhibition reducing the expression of Dlx5,and Dlx5 make runx2 expression decreased,while Msx2 as Hox gene of Dlx5,take a competitive inhibition part in the expression to Runx2. Therefore, inhibition of miR-141 can be cascaded to increase Dlx5/Msx2/Runx2 expression of osteoblast signaling axis, then promoting osteogenesis.4 10% was the best concentration to promote BMSCs proliferation and osteogenic differentiation; Jiangu particles serum can inhibit expression of osteogenic negative regulator of miR-141 in ovariectomized mice BMSCs,which contributing to the cell differentiation.5 Jiangu particles can inhibit expression of osteogenic negative regulator of miR-141 in ovariectomized mice bone tissue, and improve Dlx5/Msx2/Runx2 expression of osteoblast axis signal to promote bone tissue osteogenesis.Jiangu particles increase bone strength mainly from improving trabecular bone volume and shape, and play a part in prevention or treatment of PMOP.