Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha 410008, PR China.
Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha 410008, PR China.
Exp Mol Pathol. 2019 Apr;107:77-84. doi: 10.1016/j.yexmp.2019.01.012. Epub 2019 Jan 28.
Osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is of much significance for bone formation, the imbalance of it would result in osteoporosis and other pathological bone defects. Increasing evidences showed that long non-coding RNAs (lncRNAs) and miRNAs played vital roles in the regulation of osteogenic differentiation. LncRNA KCNQ1OT1 was often regarded as an imprinted lncRNA and was related to tumor progression, while its function in osteogenic differentiation remained unclear.
qRT-PCR was performed to detect the expression of KCNQ1OT1, miR-214 and osteogenesis-related genes BMP2, Runx2, OPN, and OCN. Western blotting was carried out to detect osteogenesis-related markers. The osteoblastic phenotype was evidenced by alkaline phosphatase (ALP) activity and Alizarin Red S accumulation detection. Bioinformatics and luciferase assays were used to predict and validate the interaction between KCNQ1OT1 and miR-214 as well as BMP2 and miR-214.
KCNQ1OT1 was significantly up-regulated during the process of osteogenic induction while miR-214 was contrarily down-regulated. Knockdown of KCNQ1OT1 inhibited osteogenic differentiation and down-regulated BMP2 and osteogenesis-related genes. It was also confirmed that KCNQ1OT1 directly interacted with miR-214. Meanwhile, miR-214 could bind to 3'UTR of BMP2 and therefore inhibited its expression. Furthermore, co-transfection of miR-214 inhibitor could rescue the down-regulation of BMP2 and osteogenesis-related genes and osteogenic differentiation suppression induced by KCNQ1OT1 knockdown. Moreover, miR-214 inhibitor significantly reversed the decreased protein levels of p-Smad1/5/8, Runx2 and Osterix induced by shKCNQ1OT1.
KCNQ1OT1 positively regulated osteogenic differentiation of BMSCs by acting as a ceRNA to regulate BMP2 expression through sponging miR-214.
骨髓间充质干细胞（BMSCs）的成骨分化对于骨形成至关重要，其失衡会导致骨质疏松症和其他病理性骨缺损。越来越多的证据表明，长非编码 RNA（lncRNA）和 microRNA（miRNA）在成骨分化的调控中发挥着重要作用。lncRNA KCNQ1OT1 通常被认为是一种印迹 lncRNA，与肿瘤进展有关，但其在成骨分化中的功能尚不清楚。
采用 qRT-PCR 检测 KCNQ1OT1、miR-214 及成骨相关基因 BMP2、Runx2、OPN、OCN 的表达。采用 Western blot 检测成骨相关标志物。通过碱性磷酸酶（ALP）活性和茜素红 S 积累检测来证实成骨表型。通过生物信息学和荧光素酶报告基因实验预测并验证 KCNQ1OT1 与 miR-214 以及 BMP2 与 miR-214 之间的相互作用。
在成骨诱导过程中，KCNQ1OT1 明显上调，而 miR-214 则相反下调。敲低 KCNQ1OT1 抑制成骨分化并下调 BMP2 和骨形成相关基因。也证实了 KCNQ1OT1 与 miR-214 直接相互作用。同时，miR-214 可以与 BMP2 的 3'UTR 结合，从而抑制其表达。此外，共转染 miR-214 抑制剂可挽救由 KCNQ1OT1 敲低引起的 BMP2 和骨形成相关基因下调以及成骨分化抑制。此外，miR-214 抑制剂显著逆转了 shKCNQ1OT1 引起的 p-Smad1/5/8、Runx2 和 Osterix 蛋白水平的降低。
KCNQ1OT1 通过作为 ceRNA 调节 BMP2 表达，从而正向调节 BMSCs 的成骨分化，通过海绵吸附 miR-214。
