Department of Orthopaedics, Xiangya Hospital of Central South University, 410008, Changsha, Hunan Province, P.R. China.
Exp Mol Med. 2020 Aug;52(8):1310-1325. doi: 10.1038/s12276-020-0475-0. Epub 2020 Aug 11.
Our study aimed to determine how lncRNA DANCR, miR-320a, and CTNNB1 interact with each other and regulate osteogenic differentiation in osteoporosis. qRT-PCR and western blotting were performed to determine the expression of DANCR, miR-320a, CTNNB1, and the osteoporosis- or Wnt/β-catenin pathway-related markers T-cell factor 1 (TCF-1), runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Interactions between CTNNB1, DANCR, and miR-320a were predicted by bioinformatics approaches and validated using a luciferase assay. Osteoblastic phenotypes were evaluated by ALP staining, ALP activity assay and Alizarin Red staining. The bilateral ovariectomy method was used to establish an in vivo osteoporosis model. Bone morphological changes were examined using hematoxylin and eosin (H&E) and Alcian Blue staining. The expression levels of DANCR and miR-320a in BMSCs derived from osteoporosis patients were upregulated, whereas CTNNB1 expression was downregulated compared with that in healthy controls. Importantly, we demonstrated that miR-320a and DANCR acted independently from each other and both inhibited CTNNB1 expression, whereas the inhibitory effect was additive when miR-320a and DANCR were cooverexpressed. Moreover, we found that DANCR overexpression largely abrogated the effect of the miR-320a inhibitor on CTNNB1 expression and the Wnt/β-catenin signaling pathway in BMSCs during osteogenic differentiation. We further confirmed the results above in BMSCs derived from an osteoporosis animal model. Taken together, our findings revealed that DANCR and miR-320a regulated the Wnt/β-catenin signaling pathway during osteogenic differentiation in osteoporosis through CTNNB1 inhibition. Our results highlight the potential value of DANCR and miR-320a as promising therapeutic targets for osteoporosis treatment.
我们的研究旨在确定长链非编码 RNA（lncRNA）DANCR、miR-320a 和 CTNNB1 如何相互作用并调节骨质疏松症中的成骨分化。通过 qRT-PCR 和 Western blot 测定 DANCR、miR-320a、CTNNB1 以及骨质疏松症或 Wnt/β-catenin 通路相关标志物 T 细胞因子 1（TCF-1）、 runt 相关转录因子 2（RUNX2）、碱性磷酸酶（ALP）、骨钙素（OCN）和骨桥蛋白（OPN）的表达。通过生物信息学方法预测 CTNNB1、DANCR 和 miR-320a 之间的相互作用，并通过荧光素酶测定进行验证。通过 ALP 染色、ALP 活性测定和茜素红染色评估成骨细胞表型。采用双侧卵巢切除术方法建立体内骨质疏松模型。通过苏木精和伊红（H&E）和阿利新蓝染色检查骨形态变化。与健康对照组相比，骨质疏松症患者骨髓间充质干细胞（BMSCs）中 DANCR 和 miR-320a 的表达上调，而 CTNNB1 的表达下调。重要的是，我们证明 miR-320a 和 DANCR 彼此独立发挥作用，均抑制 CTNNB1 的表达，而 miR-320a 和 DANCR 共表达时抑制作用具有加性。此外，我们发现 DANCR 过表达在很大程度上消除了 miR-320a 抑制剂对 BMSCs 成骨分化过程中 CTNNB1 表达和 Wnt/β-catenin 信号通路的影响。我们在骨质疏松症动物模型来源的 BMSCs 中进一步证实了上述结果。总之，我们的研究结果表明，DANCR 和 miR-320a 通过抑制 CTNNB1 调节骨质疏松症中成骨分化过程中的 Wnt/β-catenin 信号通路。我们的研究结果强调了 DANCR 和 miR-320a 作为骨质疏松症治疗有前途的治疗靶点的潜在价值。
