Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow, India.
J Endocrinol. 2019 Jun 1;241(3):249-263. doi: 10.1530/JOE-19-0015.
miRNAs have appeared as critical controllers of gene expression at post-transcriptional level either by degrading RNA transcripts or repressing translation. It is evident from the ever-growing scientific literature that miRNAs play a significant role in osteoblast commitment and differentiation. Here, we report that overexpression of miR-487b-3p leads to inhibition of osteoblastic differentiation. Using in silico approaches, Nrarp was found to be the direct target of miR-487b-3p, which was further validated by luciferase 3' UTR reporter assay. Nrarp inhibits Notch-1 signaling and promotes Wnt signaling by stabilization of LEF-1. Role of miR-487b-3p in regulating canonical Wnt and Notch signaling was determined by western blotting. Protein levels of Nrarp, RUNX-2, Lef1 and β catenin were reduced in osteoblasts cells transfected with miR-487b-3p, whereas protein levels of Notch1, Hes1 and P-β catenin were upregulated when osteoblast cells were transfected with miR-487b-3p. These outcomes were reversed after treating cells with anti-miR-487b-3p. Further silencing of miR-487b-3p in neonatal Balb/c mice attenuated all the inhibitory actions of miR-487b-3p on osteoblast differentiation. Importantly, in vivo action of anti-miR-487b-3p to ovariectomized osteopenic BALB/c mice steered to significant enhancement in trabecular bone microarchitecture. Furthermore, the bio-mechanical properties of isolated femurs were enhanced in anti-miR-487b-3p-treated mice. Overall, miR-487b-3p negatively regulates osteogenesis by suppressing Nrarp expression, which in turn, suppresses Runx-2 and Wnt signaling, both of which play a pivotal action in osteoblast differentiation.
miRNAs 作为转录后水平基因表达的关键调控因子，通过降解 RNA 转录本或抑制翻译来发挥作用。越来越多的科学文献表明，miRNAs 在成骨细胞的分化中起着重要作用。在这里，我们报告 miR-487b-3p 的过表达导致成骨细胞分化的抑制。通过计算方法，发现 Nrarp 是 miR-487b-3p 的直接靶标，这进一步通过荧光素酶 3'UTR 报告基因实验得到验证。Nrarp 通过稳定 LEF-1 抑制 Notch-1 信号并促进 Wnt 信号。通过 Western blot 确定 miR-487b-3p 在调节经典 Wnt 和 Notch 信号通路中的作用。转染 miR-487b-3p 的成骨细胞中 Nrarp、RUNX-2、Lef1 和βcatenin 的蛋白水平降低，而转染 miR-487b-3p 的成骨细胞中 Notch1、Hes1 和 P-βcatenin 的蛋白水平升高。用抗 miR-487b-3p 处理细胞后，这些结果得到逆转。在新生 Balb/c 小鼠中进一步沉默 miR-487b-3p 减弱了 miR-487b-3p 对成骨细胞分化的所有抑制作用。重要的是，体内抗 miR-487b-3p 对去卵巢骨质疏松 BALB/c 小鼠的作用导致小梁骨微结构显著增强。此外，抗 miR-487b-3p 处理的小鼠的离体股骨生物力学性能得到增强。总的来说，miR-487b-3p 通过抑制 Nrarp 表达负调控成骨作用，从而抑制 Runx-2 和 Wnt 信号，这两者在成骨细胞分化中起着关键作用。
