Sun Tianhao, Li Chen-Tian, Xiong Lifeng, Ning Ziyu, Leung Frankie, Peng Songlin, Lu William W
Department of Orthopaedics and Traumatology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
Department of Spine Surgery, Shenzhen People's Hospital, Jinan University Second College of Medicine, Shenzhen, China.
PLoS One. 2017 Feb 3;12(2):e0171281. doi: 10.1371/journal.pone.0171281. eCollection 2017.
Wnt signaling pathways are essential for bone formation. Previous studies showed that Wnt signaling pathways were regulated by miR-375. Thus, we aim to explore whether miR-375 could affect osteogenesis. In the present study, we investigated the roles of miR-375 and its downstream targets. Firstly, we revealed that miR-375-3p negatively modulated osteogenesis by suppressing positive regulators of osteogenesis and promoting negative regulators of osteogenesis. In addition, the results of TUNEL cell apoptosis assay showed that miR-375-3p induced MC3T3-E1 cell apoptosis. Secondly, miR-375-3p targeted low-density lipoprotein receptor-related protein 5 (LRP5), a co-receptor of the Wnt signaling pathways, and β-catenin as determined by luciferase activity assay, and it decreased the expression levels of LRP5 and β-catenin. Thirdly, the decline of protein levels of β-catenin was determined by immunocytochemistry and immunofluorescence. Finally, silence of LRP5 in osteoblast precursor cells resulted in diminished cell viability and cell proliferation as detected by WST-1-based colorimetric assay. Additionally, all the parameters including the relative bone volume from μCT measurement suggested that LRP5 knockout in mice resulted in a looser and worse-connected trabeculae. The mRNA levels of important negative modulators relating to osteogenesis increased after the functions of LRP5 were blocked in mice. Last but not least, the expression levels of LRP5 increased during the osteogenesis of MC3T3-E1, while the levels of β-catenin decreased in bone tissues from osteoporotic patients with vertebral compression fractures. In conclusion, we revealed miR-375-3p negatively regulated osteogenesis by targeting LRP5 and β-catenin. In addition, loss of functions of LRP5 damaged bone formation in vivo. Clinically, miR-375-3p and its targets might be used as diagnostic biomarkers for osteoporosis and might be also as novel therapeutic agents in osteoporosis treatment. The relevant products of miR-375-3p might be developed into molecular drugs in the future. These molecules could be used in translational medicine.
Wnt信号通路对骨形成至关重要。先前的研究表明，Wnt信号通路受miR - 375调控。因此，我们旨在探究miR - 375是否会影响骨生成。在本研究中，我们调查了miR - 375及其下游靶点的作用。首先，我们发现miR - 375 - 3p通过抑制骨生成的正调节因子和促进骨生成的负调节因子来负向调节骨生成。此外，TUNEL细胞凋亡检测结果表明，miR - 375 - 3p诱导MC3T3 - E1细胞凋亡。其次，通过荧光素酶活性检测确定miR - 375 - 3p靶向低密度脂蛋白受体相关蛋白5（LRP5），Wnt信号通路的一个共受体，以及β - 连环蛋白，并且它降低了LRP5和β - 连环蛋白的表达水平。第三，通过免疫细胞化学和免疫荧光确定β - 连环蛋白蛋白水平的下降。最后，如基于WST - 1的比色法检测所示，成骨细胞前体细胞中LRP5的沉默导致细胞活力和细胞增殖降低。此外，包括μCT测量的相对骨体积在内的所有参数表明，小鼠中LRP5基因敲除导致小梁更疏松且连接更差。在小鼠中阻断LRP5的功能后，与骨生成相关的重要负调节因子的mRNA水平升高。最后但同样重要的是，在MC3T3 - E1的骨生成过程中LRP5的表达水平升高，而在患有椎体压缩骨折的骨质疏松症患者的骨组织中β - 连环蛋白水平降低。总之，我们发现miR - 375 - 3p通过靶向LRP5和β - 连环蛋白负向调节骨生成。此外，LRP5功能丧失在体内损害骨形成。临床上，miR - 375 - 3p及其靶点可能用作骨质疏松症的诊断生物标志物，也可能用作骨质疏松症治疗的新型治疗剂。miR - 375 - 3p的相关产品未来可能开发成分子药物。这些分子可用于转化医学。
