The Effect Of Matrix Gla Protein On Bone Formation And Wnt/β-catenin Signaling Pathway In MG63 Cell Lines

Objectives: 1. To observe the effect on function of MG63 cell line(a kind of osteogenesis cell) by change the expression of matrix Gla protein(MGP) directly; 2. Observe the influence of MGP on Wnt/β-catenin signaling pathway in vitro, to clarify the potential mechanism of MGP in postmenopausal osteoporosis.Methods: 1. Transfection: MG63 cells were divided into overexpressing group and knockdown group. The overexpression group used recombinant plasmid pIRES2-EGFP-MGP(overexpression group, OE) and pIRES2-EGFP(the negative control of overexpression group, OE-NC), the knockdown group used p KLO-MGP-shRNA(knockdown group, KD) and p KLO-EGFP(the negative control of knockdown group, KD-NC); 2. The function of MG63 cells: Proliferation of MG63 was measured by CCK-8 assay, and ALP staining and assay was used to detected the differentiation, MG63 mineralization was detected by Alizarin Red S assay, and quantitative analyzed by cetylpyridinium chloride; 3.Detection of Wnt/β-catenin signaling pathway related factors: Western blot and QRT-PCR was used to detect protein and mRNA levels of members contained in Wnt/β-catenin signaling pathway, such as wnt3 a, β-catenin and Runx2.Results: 1. The results of CCK-8 assay showed the MG63 cells of OE grew to higher levels at 48, 72 and 96 hour compared with OE-NC(*P < 0.05), while down-regulate of MGP inhibited MG63 proliferation, difference was not statistically significance(P >0.05); 2. Both ALP staining and assay showed a significantly enhanced in the OE group compared with the OE-NC group. The ALP activity in OE group increased about 3-fold than OE-NC group; on the contrary, ALP activity was reduced after MGP knockdown, the KD group was about 2-folds lessened than KD-NC group, the difference was statistically significant(*P<0.05, **P<0.01); 3. The Alizarin Red S assay showed that overexpressed MGP lead to prominently up-regulated mineralization of MG63; conversely, knockdown MGP decreased mineralized level MG63, both were statistically significant diferences(*P<0.05); 4. Western blot and QRT-PCR showed that oversxpressing MGP can upregulate wnt3 a, β-catenin and Runx2(three members of Wnt/β-catenin signaling pathway), the expression of wnt3 a, β-catenin and Runx2 mRNA were 5.94-fold, 4.03-fold, 9.23-fold, the expression of protein were 2.73-fold, 2.32-fold, 2.28-fold, the difference was statistically significant(*P<0.05, **P<0.01), however, knockdown MGP, wnt3 a, β-catenin and Runx2 reduced consequently.Conclusions: 1. Promoting osteoblast proliferation, differentiation and mineralization may be a mechanism for MGP to prevent and treat osteoporosis; 2. MGP may promote the osteogenic effects via the Wnt/β-catenin signaling pathway.