Li Huazhi, Zheng Fu, Tao Anqi, Wu Tong, Zhan Xinxin, Tang Hongyi, Cui Xinyu, Ma Zeyun, Li Cuiying, Jiang Jiuhui, Wang Yixiang
Department of Orthodontics, Peking University School and Hospital of Stomatology, No. 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, PR China; National Center of Stomatology & National Clinical Research Center for Oral Diseases, No. 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, PR China; National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, No. 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, PR China; Beijing Key Laboratory of Digital Stomatology, No. 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, PR China; Research Center of Engineering and Technology for Computerized Dentistry Ministry of Health, No. 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, PR China.
Department of Orthodontics, Peking University School and Hospital of Stomatology, No. 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, PR China.
Bone. 2025 Mar;192:117340. doi: 10.1016/j.bone.2024.117340. Epub 2024 Nov 29.
Osteoporosis is a prevalent metabolic bone disease. Osteoporotic fractures can lead to severe functional impairment and increased mortality. Long noncoding RNA H19 has emerged as a pivotal player in bone remodeling, serving both as a biomarker and a regulator. While previous research has elucidated H19's role in promoting osteogenic differentiation through diverse mechanisms, its involvement in osteoclast differentiation remains largely unknown.
In this study, we used lentiviral vectors to stably overexpress or knockdown H19 in RAW264.7 cell lines. Quantitative reverse polymerase chain reaction, Western blot, tartrate resistant acid phosphatase staining and bone resorption assay were performed to assess the level of osteoclast differentiation and bone resorption capacity. And fluorescence in situ hybridization, dual-luciferase reporter and RNA immunoprecipitation were used to explore the specific mechanism of H19 regulating osteoclast differentiation in vitro. Then, ovariectomized osteoporosis models in wild type mice and H19 knockout mice were conducted. And micro-CT analysis, HE staining, and immunohistochemistry were performed to verify the mechanism of H19 regulating osteoclast differentiation in vivo. Bone marrow derived monocytes and bone mesenchymal stem cells were extracted from mice and assayed for osteoclastic and osteogenic-related assays, respectively.
In vitro, H19 promoted osteoclast differentiation and bone resorption of RAW264.7 cells, while miR-29c-3p inhibited them. Both H19 and cathepsin K were the target genes of miR-29c-3p. In vivo, H19 knockout mice have increased femur bone mineral density, decreased osteoclast formation, and reduced cathepsin K expression. MiR-29c-3p agomir could increase bone mineral density in osteoporotic mice on the premise of H19 knockout.
H19 upregulates cathepsin K expression through sponging miR-29c-3p, which promoting osteoclast differentiation and enhancing bone resorption. This underscores the potential of H19 and miR-29c-3p as promising biomarkers for osteoporosis.
骨质疏松症是一种常见的代谢性骨病。骨质疏松性骨折可导致严重的功能障碍和死亡率增加。长链非编码RNA H19已成为骨重塑中的关键因子，既是生物标志物又是调节因子。虽然先前的研究已经阐明H19通过多种机制促进成骨细胞分化，但其在破骨细胞分化中的作用仍 largely未知。
在本研究中，我们使用慢病毒载体在RAW264.7细胞系中稳定过表达或敲低H19。进行定量逆转录聚合酶链反应、蛋白质印迹、抗酒石酸酸性磷酸酶染色和骨吸收测定，以评估破骨细胞分化水平和骨吸收能力。并使用荧光原位杂交、双荧光素酶报告基因和RNA免疫沉淀来探索H19在体外调节破骨细胞分化的具体机制。然后，构建野生型小鼠和H19基因敲除小鼠的去卵巢骨质疏松模型。并进行显微CT分析、苏木精-伊红染色和免疫组织化学，以验证H19在体内调节破骨细胞分化的机制。从小鼠中提取骨髓来源的单核细胞和骨间充质干细胞，分别进行破骨细胞和成骨细胞相关检测。
在体外，H19促进RAW264.7细胞的破骨细胞分化和骨吸收，而miR-29c-3p抑制它们。H19和组织蛋白酶K都是miR-29c-3p的靶基因。在体内，H19基因敲除小鼠的股骨骨密度增加，破骨细胞形成减少，组织蛋白酶K表达降低。在H19基因敲除的前提下，miR-29c-3p激动剂可增加骨质疏松小鼠的骨密度。
H19通过海绵化miR-29c-3p上调组织蛋白酶K表达，促进破骨细胞分化并增强骨吸收。这突出了H19和miR-29c-3p作为骨质疏松症有前景的生物标志物的潜力。
