Cheng-Gong Wang1, Zhan Liao1, Han Xiao1, Hua Liu1, Yi-He Hu1, Qian-De Liao1, Da Zhong2. 1. Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha 410008, PR China. 2. Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha 410008, PR China. Electronic address: Dr_Zhongda@126.com.
Abstract
BACKGROUND: 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. METHOD: 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. RESULTS: 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. CONCLUSIONS: KCNQ1OT1 positively regulated osteogenic differentiation of BMSCs by acting as a ceRNA to regulate BMP2 expression through sponging miR-214.
BACKGROUND: 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. METHOD: 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. RESULTS: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. CONCLUSIONS:KCNQ1OT1 positively regulated osteogenic differentiation of BMSCs by acting as a ceRNA to regulate BMP2 expression through sponging miR-214.