摘要： 目的 探讨微RNA （miR）-133a调控生长相关转录因子2 （RUNX2）/骨形态蛋白2 （BMP2）信号通路参与激素性股骨头坏死（SONFH）的机制。方法 收集本院接受髋关节置换SONFH患者（SONFH组）骨髓及股骨颈骨折不愈合患者（对照组）骨髓，提取骨髓间充质干细胞（BMSCs），经表型鉴定、成骨、成脂分化鉴定后检测BMSCs miR-133a、RUNX2 mRNA和蛋白表达水平，双荧光素酶鉴定miR-133a与RUNX2的靶向关系。实验分组包括对照组、SONFH组、抑制剂对照组（inhibitor con组）、miR-133a抑制剂组（miR-133a inhibitor组）、miR-133a inhibitor+si-RUNX2组。实时定量PCR （RT-qPCR）检测细胞中miR-133a、RUNX2 mRNA水平； Western blotting检测BMSCs中RUNX2、BMP2、骨钙素（OCN）、Ⅰ型胶原蛋白（COL-Ⅰ）蛋白水平； CCK-8检测细胞增殖情况；茜素红染色鉴定细胞矿化能力。结果 BMSCs细胞表型鉴定结果显示，细胞表面CD71、CD44表达，CD34、人类白细胞抗原DR等位基因不表达；成骨分化鉴定显示BMSCs出现矿化结节，结节呈红色散落分布；成脂分化鉴定显示BMSCs中脂滴呈橙红色，脂滴沉着细胞，且数量较多，部分细胞内脂滴融合变大成泡状，细胞核位于中央或挤向外周，提示BMSCs提取成功。miR-133a与RUNX2靶向关系存在特异性结合位点。与对照组比较，SONFH组BMSCs中miR-133a水平升高（P < 0.05）； RUNX2 mRNA和蛋白水平，24、48、72 h时BMSCs的吸光度值，BMSCs矿化结节相对数量，BMP2、BGP、COL-Ⅰ蛋白水平均显著降低（均P < 0.05）。与SONFH组、inhibitor con组比较，miR-133a inhibitor组BMSCs中miR-133a水平降低（P < 0.05）； RUNX2 mRNA和蛋白水平，24、48、72 h时BMSCs的吸光度值，BMSCs矿化结节相对数量，BMP2、BGP、COL-Ⅰ蛋白水平均显著升高（均P < 0.05）。与miR-133a inhibitor组比较，miR-133a inhibitor+si-RUNX2组BMSCs中miR-133a水平升高（P < 0.05），RUNX2 mRNA和蛋白水平，24、48、72 h BMSCs的吸光度值，BMSCs矿化结节相对数量，BMP2、BGP、COL-Ⅰ蛋白水平均显著降低（均P < 0.05）。结论 miR-133a在SONFH患者BMSCs中高表达，抑制miR-133a表达可靶向促进RUNX2的表达，从而促进BMSCs成骨分化和细胞增殖。

miR-133a调控RUNX2/BMP2信号通路参与激素性股骨头坏死的机制

杨森林, 张国秋, 李超, 李克文

青海大学附属医院骨科, 西宁 810000

收稿日期:2023-03-02出版日期:2023-11-30发布日期:2023-11-07
通讯作者:杨森林E-mail:ropz233@163.com
作者简介:杨森林(1984-),男,主治医师,硕士.
基金资助:青海省基础研究计划项目（2018-ZJ-763）


关键词: 微RNA-133a, 生长相关转录因子2/骨形态蛋白2信号通路, 激素性股骨头坏死, 骨髓间充质干细胞
Abstract: Objective To explore the mechanism of microRNA (miR) -133a regulating runt-related transcription factor 2 (RUNX2)/bone morphogenetic protein 2 (BMP2) signaling pathway involved in steroid-induced osteonecrosis of the femoral head (SONFH). Methods The bone marrow of SONFH patients (SONFH group), who underwent hip replacements in our hospital, and the bone marrow of nonunion femoral neck-bone fractures (control group) were collected. After extraction of bone marrow mesenchymal stem cells (BMSCs), and identification of phenotype, osteogenic and adipogenic differentiation, BMSCs miR-133a, RUNX2 mRNA and protein expression levels were detected. Moreover, the targeting relationship between miR-133a and RUNX2 was identified with dual luciferase. The experi-mental groups comprised a control, SONFH, inhibitor con, miR-133a inhibitor, and miR-133a inhibitor+si-RUNX2 groups. The levels of miR-133a and RUNX2 mRNA in cells were detected with real-time quantitative PCR (RT-qPCR); the protein levels of RUNX2, BMP2, osteocalcin (OCN) and typeⅠcollagen (COL-Ⅰ) in BMSCs were detected with Western blotting; the cell proliferation was detected with CCK-8; the cell mineralization ability was identified with Alizarin red staining. Results CD71 and CD44 were expressed on the cell surface, but CD34 and human leukocyte antigen DR allele were not expressed. BMSCs showed mineralized nodules, and these were scattered and distributed in red. Lipid droplets were stained orange-red in BMSCs and deposited in cells in large number. In some cases, the lipid droplets fused and became vesicles, and the nucleus was located in the center or squeezed out to the periphery. All indicated that the BMSCs extraction was successful. The targeting relationship between miR-133a and RUNX2 verified the existence of specific binding sites. Compared with the control group, the level of miR-133a in BMSCs in the SONFH group increased (P < 0.05), and the levels of RUNX2 mRNA and protein, the optical density at 450 nm (OD₄₅₀) of BMSCs at (24, 48, 72) h, the relative number of BMSCs mineralized nodules, the protein levels of BMP2, OCN and COL-Ⅰ decreased (P < 0.05). Compared with the SONFH group and the inhibitor con group, the level of miR-133a in BMSCs in the miR-133a inhibitor decreased (P < 0.05), and the levels of RUNX2 mRNA and protein, the OD₄₅₀ of BMSCs at (24, 48, 72) h, the relative number of BMSCs mineralized nodules, the protein levels of BMP2, OCN and COL-Ⅰincreased (P < 0.05). Furthermore, compared with the miR-133a inhibitor group, the level of miR-133a in BMSCs in the miR-133a inhibitor+si-RUNX2 group increased (P < 0.05), and the levels of RUNX2 mRNA and protein, the OD₄₅₀ of BMSCs at (24, 48, 72) h, the relative number of BMSCs mineralized nodules, the protein levels of BMP2, OCN and COL-Ⅰ decreased (P < 0.05). Conclusion miR-133a is highly expressed in SONFH. Inhibition of miR-133a expression can target and promote the expression of RUNX2, thereby promoting osteogenic differentiation and the cell proliferation of BMSCs in SONFH.
Key words: microRNA-133a, runt-related transcription factor 2/bone morphogenetic protein 2 signaling pathway, steroid-induced osteonecrosis of the femoral head, bone marrow mesenchymal stem cell
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