Tang Xiaolin, Lin Jiantao, Wang Guanhai, Lu Jianlin
Department of Medical Science, Shunde Polytechnic, Foshan, China.
Traditional Chinese Medicine and New Drug Research Institute, Guangdong Medical University, Dongguan, China.
PLoS One. 2017 Jun 19;12(6):e0179860. doi: 10.1371/journal.pone.0179860. eCollection 2017.
Dickkopf-1 (DKK1) is a powerful antagonist of canonical WNT signaling pathway, and is regarded as a biomarker for osteoporosis. Its expression is highly correlated with bone mass and osteoblasts maturation. In this study, mouse primary bone marrow cells and osteoblast cell lines were used. Luciferase reporter assay and western blotting methods were employed to validate if miRNA-433-3p epigenetically regulated DKK1 translation. Rat bone marrow derived osteoblasts were infected with lentivirus vector in which miR-433-3p was constructed. The authors constructed lentivirus mediated miRNA-433-3p stable expression and examined the alkaline phosphatase (ALP) activity and mineral deposition level in vitro. In situ hybridization method was used to observe miR-433-3p in primary osteoblasts. We built up an OVX rat model to mimic postmenopausal osteoporosis, and found aberrant circulating miR-433-3p and miR-106b, which were not reported previously. Results showed that miR-433-3p potentially regulated DKK1 mRNA, Furthermore, the correlation of serum DKK1 with circulating miR-433-3p level was significant (r = 0.7520, p = 0.046). In the luciferase reporter assay, we found that miR-433-3p siRNA decreased luminescence signal, indicating direct regulation of miR-433-3p on DKK1 mRNA. When the miR-433-3p binding site in DKK1 3'UTR was mutant, such reduction was prohibited. Western blotting result validated that miR-433-3p inhibited over 90% of DKK1 protein expression. Similarly, the change of protein expression was not observed in mutant group. The stable expression of lentivirus mediated miR-433-3p increased ALP activity and mineralization both in human and rat derived immortalized cells. We found that primary osteoblasts had higher miR-433-3p level compared with immortal cells through real-time PCR, as well as in situ hybridization experiment. Conclusively, our findings further emphasized the vital role of miR-433-3p in DKK1/WNT/β-catenin pathway through decreasing DKK1 expression and inducing osteoblasts differentiation.
Dickkopf-1（DKK1）是经典WNT信号通路的强效拮抗剂，被视为骨质疏松症的生物标志物。其表达与骨量和成骨细胞成熟高度相关。在本研究中，使用了小鼠原代骨髓细胞和成骨细胞系。采用荧光素酶报告基因检测和蛋白质印迹法来验证miRNA-433-3p是否通过表观遗传调控DKK1的翻译。用构建了miR-433-3p的慢病毒载体感染大鼠骨髓来源的成骨细胞。作者构建了慢病毒介导的miRNA-433-3p稳定表达，并在体外检测了碱性磷酸酶（ALP）活性和矿化沉积水平。采用原位杂交法观察原代成骨细胞中的miR-433-3p。我们建立了去势（OVX）大鼠模型来模拟绝经后骨质疏松症，发现了循环中miR-433-3p和miR-106b异常，这在之前未曾报道过。结果显示，miR-433-3p可能调控DKK1 mRNA，此外，血清DKK1与循环miR-433-3p水平的相关性显著（r = 0.7520，p = 0.046）。在荧光素酶报告基因检测中，我们发现miR-433-3p siRNA降低了发光信号，表明miR-433-3p对DKK1 mRNA有直接调控作用。当DKK1 3'UTR中的miR-433-3p结合位点发生突变时，这种降低被阻止。蛋白质印迹结果证实，miR-433-3p抑制了超过90%的DKK1蛋白表达。同样，在突变组中未观察到蛋白质表达的变化。慢病毒介导的miR-433-3p稳定表达增加了人和大鼠来源的永生化细胞中的ALP活性和矿化。通过实时PCR以及原位杂交实验，我们发现原代成骨细胞中的miR-433-3p水平高于永生化细胞。总之，我们的研究结果进一步强调了miR-433-3p通过降低DKK1表达并诱导成骨细胞分化在DKK1/WNT/β-连环蛋白通路中的重要作用。
