Department of Orthopedics, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, P.R. China.
J Cell Biochem. 2018 Nov;119(10):8346-8358. doi: 10.1002/jcb.26910. Epub 2018 Jun 12.
This study aims to explore the effects of miR-539 on osteoblast proliferation and differentiation and osteoclast apoptosis in a rat model of osteoporosis, and its mechanism involving the regulation of the AXIN1-mediated wingless-Int (Wnt) signaling pathway. A rat model of osteoporosis was successfully established by ovariectomy. With osteoblasts and osteoclasts of rats not receiving ovariectomy in the sham group as control, those of osteoporotic rats were treated with miR-539 inhibitor, miR-539 mimic, and AXIN1 shRNA. The expression of miR-53, AXIN1, the Wnt pathway related-genes, apoptosis related-genes, and osteogenic markers were measured by RT-qPCR and Western blot analysis, respectively. Alkaline phosphatase (ALP) activity in osteoblast and tartrate-resistant acid phosphatase (TRAP) activity in osteoclasts were determined after cell transfection. Osteoblast and osteoclast viability was assayed by CCK-8 assay. Cell cycle and apoptosis of osteoblasts and osteoclasts were detected by flow cytometry. Lastly, alizarin red S staining was used to detect mineralized nodules of osteoblasts. Firstly, we determined that miR-539 was down-regulated in osteoblast and osteoclast of osteoporotic rats and AXIN1 was negatively regulated by miR-539. Additionally, overexpression of miR-539 increased the expressions of β-catenin, LEF1, c-myc, cyclin D1, RUNX2, BGP, BMP-2 in osteoblast as well as β-catenin, RhoA, caspase-3, and Bcl-2 in osteoclasts. Finally, overexpression of miR-539 elevated ALP activity, proliferation, and mineralized nodules in osteoblast and osteoclast apoptosis, with reduced TRAP activity in osteoclasts. Our results demonstrate that miR-539 promotes osteoblast proliferation and differentiation as well as osteoclast apoptosis through the AXIN1-dependent Wnt signaling pathway in osteoporotic rats.
本研究旨在探讨 miR-539 对骨质疏松症大鼠模型中成骨细胞增殖和分化以及破骨细胞凋亡的影响，及其通过调节 AXIN1 介导的 Wnt 信号通路的作用机制。通过卵巢切除术成功建立了骨质疏松大鼠模型。以假手术组中未接受卵巢切除术的大鼠成骨细胞和破骨细胞作为对照，用 miR-539 抑制剂、miR-539 模拟物和 AXIN1 shRNA 处理骨质疏松大鼠的成骨细胞和破骨细胞。通过 RT-qPCR 和 Western blot 分析分别测量 miR-53、AXIN1、Wnt 通路相关基因、凋亡相关基因和成骨标志物的表达。转染后测定成骨细胞碱性磷酸酶（ALP）活性和破骨细胞抗酒石酸酸性磷酸酶（TRAP）活性。通过 CCK-8 测定法检测成骨细胞和破骨细胞的活力。通过流式细胞术检测成骨细胞和破骨细胞的细胞周期和凋亡。最后，用茜素红 S 染色检测成骨细胞的矿化结节。首先，我们确定 miR-539 在骨质疏松症大鼠的成骨细胞和破骨细胞中下调，AXIN1 受 miR-539 负调控。此外，miR-539 的过表达增加了成骨细胞中 β-catenin、LEF1、c-myc、cyclin D1、RUNX2、BGP、BMP-2 的表达以及破骨细胞中β-catenin、RhoA、caspase-3 和 Bcl-2 的表达。最后，miR-539 的过表达增加了成骨细胞和破骨细胞的 ALP 活性、增殖和矿化结节，并降低了破骨细胞的 TRAP 活性。我们的研究结果表明，miR-539 通过骨质疏松症大鼠中的 AXIN1 依赖性 Wnt 信号通路促进成骨细胞增殖和分化以及破骨细胞凋亡。
