Zhang Guo-Ping, Zhang Jing, Zhu Chao-Hua, Lin Lei, Wang Jian, Zhang Hai-Jing, Li Jun, Yu Xiao-Guang, Zhao Zhen-Shuan, Dong Wei, Liu Guo-Bin
Department of Orthopedics, The First Hospital of Hebei Medical University, Shijiazhuang, China.
Medical Physics Department of Basic Medical College of Hebei Medical University, Shijiazhuang, China.
J Cell Mol Med. 2017 Feb;21(2):254-264. doi: 10.1111/jcmm.12961. Epub 2016 Nov 18.
To study the effects of microRNA-98 (miR-98) on human bone mesenchymal stromal cells (hBMSCs). The patients undergoing hip arthroplasty were selected by inclusion/exclusion criteria for this study. The extracted hBMSCs were detected of osteogenic differentiation by alizarin red S staining, and of cell phenotype by flow cytometry. Bioinformatics, dual luciferase report, western blotting, RT-PCR and immunoblotting were used in our study. The hBMSCs were divided into miR-98 mimics, miR-98 negative control (NC), miR-98 inhibitors, Mock and miR-98 inhibitors + siBMP2 groups. Human bone mesenchymal stromal cells were extracted and purified in vitro and had specific cytological morphology, surface markers and abilities of self-renewal and differentiation. Compared with the NC group and Mock group, the miR-98 mimics group showed increased miR-98 level while the miR-98 inhibitors group decreased miR-98 level (both P < 0.01). Dual luciferase reporter showed BMP2 was the target gene of miR-98. The levels of mRNA and protein expression of BMP2, protein expression of RUNX2, alkaline phosphatase activity and osteocalcin content significantly decreased in the miR-98 mimics group while increased in the miR-98 inhibitors group and showed no changes in the NC group and Mock group (all P < 0.05). The miR-98 mimics group showed obviously declined stained red particles and the miR-98 inhibitors group showed opposite result. After lowering the expression of miR-98, osteogenic differentiation ability of hBMSCs rose, which was weakened by the transfection with siBMP2. miR-98 may regulate osteogenic differentiation of hBMSCs by targeting BMP2.
研究微小RNA-98(miR-98)对人骨髓间充质干细胞(hBMSCs)的影响。根据纳入/排除标准选取行髋关节置换术的患者进行本研究。采用茜素红S染色检测提取的hBMSCs的成骨分化情况,采用流式细胞术检测细胞表型。本研究运用了生物信息学、双荧光素酶报告基因检测、蛋白质印迹法、逆转录-聚合酶链反应(RT-PCR)和免疫印迹法。将hBMSCs分为miR-98模拟物组、miR-98阴性对照组(NC)、miR-98抑制剂组、Mock组和miR-98抑制剂+siBMP2组。人骨髓间充质干细胞在体外提取并纯化,具有特定的细胞形态、表面标志物以及自我更新和分化能力。与NC组和Mock组相比,miR-98模拟物组miR-98水平升高,而miR-98抑制剂组miR-98水平降低(均P<0.01)。双荧光素酶报告基因检测显示骨形态发生蛋白2(BMP2)是miR-98的靶基因。miR-98模拟物组中BMP2的mRNA和蛋白表达水平、RUNX2蛋白表达、碱性磷酸酶活性及骨钙素含量显著降低,而miR-98抑制剂组则升高,NC组和Mock组无变化(均P<0.05)。miR-98模拟物组茜素红染色红色颗粒明显减少,miR-98抑制剂组结果相反。降低miR-98表达后,hBMSCs的成骨分化能力增强,而转染siBMP2则削弱了这种能力。miR-98可能通过靶向BMP2调控hBMSCs的成骨分化。