MicroRNA?26a?5p targets Wnt5a to regulate osteogenic differentiation of human periodontal ligament stem cell from inflammatory microenvironment
10.3760/cma.j.issn.1002?0098.2019.10.003
- VernacularTitle:微RNA?26a?5p靶向Wnt5a调控人炎症来源牙周膜干细胞的成骨分化
- Author:
Keke ZHANG
1
;
Yudong GENG
;
Shubin WANG
;
Lei HUO
Author Information
1. 郑州大学第一附属医院口腔正畸科450052
- Keywords:
MicroRNAs;
Osteogenic differentiation;
Periodontal ligament stem cells;
Infla-mmatory microenvironment
- From:
Chinese Journal of Stomatology
2019;54(10):662-669
- CountryChina
- Language:Chinese
-
Abstract:
To investigate the effect of microRNA?26a?5p on osteogenic differentiation of human periodontal ligament stem cells (hPDLSC) and its related mechanisms. Methods hPDLSC in periodontal tissues from healthy adults and hPDLSC from periodontitis patients (PPDLSC) were isolated and cultured in vitro , respectively. The PPDLSC were divided into Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ groups. GroupⅠis control group, and the other four groups were transiently transfected with miR?NC, miR?26a?5p, antimiR?NC and antimiR?26a?5p lentiviral vectors, respectively. The osteogenic differentiation abilities of the cells in vitro were determined by alizarin red staining, alkaline phosphatase (ALP) activity assay and real?time quantitative PCR (qPCR). Totally 40 male mice (6?weeks) were equally divided into five groups with 8 mice in each group. The PPDLSCs cells (1×107/ml) inⅠ,Ⅱ,Ⅲ,ⅣandⅤgroups, which adhered to hydroxyapatine?tricalcium phosphate (HA?TCP), were implanted into the nude mice subcutaneously and the animal models were constructed to analyze the effect of miR?26a?5p on the osteogenic differentiation of PPDLSCs in vivo. PPDLSCs were divided into A, B, C, D groups, and transfected with miR?26a?5p+Wnt5a?Wt, miR?NC+Wnt5a?Wt, miR?26a?5p+Wnt5a?Mut and miR?NC+Wnt5a?Mut in each of the above mentioned 5 groups, respectively. The luciferase activity assay was used to detect the relative luciferase in A, B, C and D groups to analyze the targeting relationship between miR?26a?5p and Wnt5a. Osteogenic differentiation related proteins expression were analyzed by western blotting. Results hPDLSC and PPDLSC were observed consistent with the characteristics of mesenchymal stem cells and had osteogenic differentiation ability in vitro. Compared with hPDLSC [(89.87±8.12) %], the osteogenic capacity of PPDLSC [(31.46±6.56) %] was significantly lower (P<0.05). The ALP activity (1.88±0.59), calcified nodules (79.88± 5.92), the expression of the osteogenic differentiation markers Runt?related transcription factor 2 (Runx2) (2.40 ± 0.70), ALP (2.10 ± 0.60) and osteocalcin (3.00 ± 0.90) mRNA in the PPDLSC from Group Ⅲ were significantly higher in comparison with the control group [(0.88±0.34), (29.69±2.65), (1.30±0.30), (0.09± 0.25), (1.71 ± 0.50)], while those from Group Ⅴ[(0.44 ± 0.07), (14.83 ± 3.05), (0.50 ± 0.11), (0.30 ± 0.08) and (0.80±0.17)] were significantly lower (P<0.05). In vivo studies in nude mice showed that the proportion of the osteogenic region [(34.96±5.65) %] in the miR?26a?5p group was significantly increased in comparison with the control group [(23.28±3.03) %], while in the antimiR?26a?5p group [(8.02±2.27) %] was significantly lower (P<0.05). The luciferase activity of the Group A (0.46 ± 0.06) was significantly lower than Group B (3.46±0.45) (P<0.05). Compared with the control group, the expression levels of Wnt5a protein, calmodulin kinase Ⅱ and protein kinase C proteins in the Group Ⅲwere significantly decreased, while those in the GroupⅤwere significantly increased (P<0.05). Conclusions MicroRNA?26a?5p could promote osteogenic differentiation of PPDLSC in vivo and in vitr o, and its mechanism might be inhibiting the activation of Wnt/Ca2+signaling pathway by targeting Wnt5a.
