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Title: [MicroRNA-26a-5p targets Wnt5a to regulate osteogenic differentiation of human periodontal ligament stem cell from inflammatory microenvironment].
Author: Zhang KK, Geng YD, Wang SB, Huo L.
Journal: Zhonghua Kou Qiang Yi Xue Za Zhi; 2019 Oct 09; 54(10):662-669. PubMed ID: 31607001.
Abstract:
Objective: 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. Group Ⅰ is 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 (1×10(7)/ml) in Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ groups, 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 Ⅲ were significantly decreased, while those in the GroupⅤ were significantly increased (P<0.05). Conclusions: MicroRNA-26a-5p could promote osteogenic differentiation of PPDLSC in vivo and in vitro, and its mechanism might be inhibiting the activation of Wnt/Ca(2+) signaling pathway by targeting Wnt5a. 目的： 探讨微RNA-26a-5p对炎症来源人牙周膜干细胞（human periodontal ligament stem cell，hPDLSC）成骨分化的影响及相关机制。 方法： 体外分离培养成年人健康牙周组织中的hPDLSC和牙周炎患者牙周组织中炎症微环境下的hPDLSC（hPDLSC from periodontitis patients，PPDLSC）。将PPDLSC细胞分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ组，Ⅰ组为空白对照组，Ⅱ、Ⅲ、Ⅳ、Ⅴ组PPDLSC细胞分别转染含miR-NC、miR-26a-5p、抗miR-NC和抗miR-26a-5p慢病毒载体。应用茜素红染色、碱性磷酸酶（alkaline phosphatase，ALP）活性测定实验及实时荧光定量PCR检测5组PPDLSC细胞体外成骨分化能力。构建裸鼠动物模型，将裸鼠分成5组，每组8只，分析miR-26a-5p对PPDLSC体内成骨分化能力的影响。将PPDLSC细胞分为A、B、C、D共4组，分别转染miR-26a-5p和Wnt5a-Wt、miR-NC和Wnt5a-Wt、miR-26a-5p和Wnt5a-Mut、miR-NC和Wnt5a-Mut，应用荧光素酶活性测定法分别检测其相对荧光素酶活性，分析miR-26a-5p与Wnt5a的靶向关系。应用蛋白质印迹法分析成骨分化相关蛋白的表达。 结果： ① hPDLSC及PPDLSC符合间充质干细胞特性，具有体外成骨分化能力；PPDLSC成骨能力[（31.46±6.56）%]显著低于hPDLSC[（89.87±8.12）%，P<0.05]。②与Ⅰ组PPDLSC细胞的ALP活性（0.88±0.34）、钙化结节（29.69±2.65）、成骨分化的标志基因[Runt相关转录因子2（Runt-related transcription factor 2，Runx2）、ALP、骨钙蛋白]的mRNA表达水平[（1.30±0.30）、（0.09±0.25）、（1.71±0.50）]相比，Ⅲ组[（1.88±0.59）、（79.88±5.92）、（2.40±0.70）、（2.10±0.60）、（3.00±0.90）]均显著升高，而Ⅴ组[（0.44±0.07）、（14.83±3.05）、（0.50±0.11）、（0.30±0.08）、（0.80±0.17）]均显著降低（P<0.05）。裸鼠体内研究发现，与Ⅰ组中成骨区域占总面积的比例[（23.28±3.03）%]相比，Ⅲ组显著升高[（34.96±5.65）%]，而Ⅴ组显著降低[（8.02±2.27）%]（P<0.05）。③ A组PPDLSC细胞的荧光素酶活性（0.46±0.06）显著低于B组（3.46±0.45，P<0.05）。与Ⅰ组中Wnt5a蛋白、钙调蛋白激酶Ⅱ、蛋白激酶C的蛋白表达水平相比，Ⅲ组显著降低，Ⅴ组显著增加（P<0.05）。 结论： miR-26a-5p可在体内及体外促进PPDLSC的成骨分化，其作用机制可能是通过靶向Wnt5a抑制Wnt/Ca(2+)信号通路的激活。.

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