Duan Lijun, Zhao He, Xiong Yang, Tang Xiangsheng, Yang Yongdong, Hu Zhenguo, Li Chuanhong, Chen Sixue, Yu Xing
Department of Orthopedics, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China.
Department of Orthopedics, Bayannaoer City Hospital, Bayannaoer City, China.
Cell Physiol Biochem. 2018;51(3):1087-1102. doi: 10.1159/000495489. Epub 2018 Nov 26.
BACKGROUND/AIMS: Osteoporosis is a bone metabolic disease characterized by a systemic impairment of bone mass, which results in increased propensity of fragility fractures. A reduction in the differentiation of MSCs into osteoblasts contributes to the impaired bone formation observed in osteoporosis. Mesenchymal stem cells (MSCs) are induced to differentiate into preosteoblasts, which are regulated by the signaling cascades initiated by the various signals, including miRNAs. miR-16-2* is a newly discovered miRNA that participates in diagnosis and prognosis of hepatocellular carcinoma, cervical cancer and chronic lymphocytic leukemia. However, the effect of miR-16-2* on the regulation of osteoblast differentiation and the mechanism responsible are still unclear. Here we discuss the contribution of miR-16-2* to osteoporosis, osteoblast differentiation and mineralization.
The expression pattern of miR-16-2* during osteogenesis or in osteoporosis bone samples was validated by quantitative real-time PCR (qRT-PCR). The human bone marrow mesenchymal stem cells (hBMSCs) were induced to differentiate into osteoblasts by osteogenic induced medium containing dexamethasone, ascorbate-2-phosphat, beta-glycerophosphate and vitamin-D3. The target genes of miR-16-2* were predicted by TargetScan and PicTar. The mRNA and protein levels of osteogenic key markers were detected using qRT-PCR or western blot respectively. The WNT signal activity was analyzed by TOP/FOP reporter assay.
The expression of miR-16-2* in patient bone tissue with osteoporosis was negatively correlated with bone formation related genes. During osteoblast differentiation process, the expression of miR-16-2* was significantly decreased. Upregulation of miR-16-2* in hBMSCs impaired the osteogenic differentiation while the downregulation of miR-16-2* increased this process. Upregulation the expression of miR-16-2* could also block the WNT signal pathway by directly target WNT5A. Furthermore, knockdown of miR-16-2* could promote the activation of RUNX2, possibly by lifting the inhibitory effect of miR-16-2* on WNT pathway.
Taken together, we report a novel biological role of miR-16-2* in osteogenesis through regulating WNT5A response for the first time. Our data support the potential utilization of miRNA-based therapies in regenerative medicine.
背景/目的：骨质疏松症是一种骨代谢疾病，其特征为骨量的系统性损害，这会导致脆性骨折倾向增加。间充质干细胞（MSCs）向成骨细胞分化的减少导致了骨质疏松症中观察到的骨形成受损。间充质干细胞被诱导分化为前成骨细胞，这受包括微小RNA（miRNAs）在内的各种信号启动的信号级联调节。miR-16-2是一种新发现的微小RNA，参与肝细胞癌、宫颈癌和慢性淋巴细胞白血病的诊断和预后。然而，miR-16-2对成骨细胞分化的调节作用及其机制仍不清楚。在此，我们探讨miR-16-2*在骨质疏松症、成骨细胞分化和矿化中的作用。
通过定量实时聚合酶链反应（qRT-PCR）验证miR-16-2在成骨过程或骨质疏松症骨样本中的表达模式。用人骨髓间充质干细胞（hBMSCs）通过含有地塞米松、抗坏血酸-2-磷酸酯、β-甘油磷酸和维生素D3的成骨诱导培养基诱导分化为成骨细胞。通过TargetScan和PicTar预测miR-16-2的靶基因。分别使用qRT-PCR或蛋白质免疫印迹法检测成骨关键标志物的mRNA和蛋白质水平。通过TOP/FOP报告基因检测分析WNT信号活性。
骨质疏松症患者骨组织中miR-16-2的表达与骨形成相关基因呈负相关。在成骨细胞分化过程中，miR-16-2的表达显著降低。hBMSCs中miR-16-2的上调损害了成骨分化，而miR-16-2的下调则增强了这一过程。miR-16-2表达的上调还可通过直接靶向WNT5A阻断WNT信号通路。此外，敲低miR-16-2可能通过解除miR-16-2*对WNT通路的抑制作用来促进RUNX2的激活。
综上所述，我们首次报道了miR-16-2*通过调节WNT5A反应在成骨过程中的新生物学作用。我们的数据支持基于微小RNA的疗法在再生医学中的潜在应用。
