Luo-Dan Zhao1, Wei-Cheng Xu2, Jian Cui2, Yan-Can Liang1, Wei-Qi Cheng1, Bing-Chang Xin3, Jia Song4. 1. Department of Oral and Maxillofacial Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, PR China; Key Laboratory of Malignant Tumor Gene Regulation and Target Therapy of Guangzhou Higher Education Institutes, Sun Yat-Sen University, Guangzhou, 510120, PR China. 2. Department of Dental Implantology, Yantai Stomatological Hospital, Yantai, 264001, PR China. 3. Department of Cariology and Endodontology, Qingdao Stomatological Hospital, Qingdao, 266001, PR China. Electronic address: xinbingchang@163.com. 4. Department of Cariology and Endodontology, Qingdao Stomatological Hospital, Qingdao, 266001, PR China. Electronic address: songjia7751@163.com.
Abstract
OBJECTIVE: The aim of the present study was to investigate the biological roles and underlying mechanism of the long non-coding RNA maternally expressed gene 3 (MEG3) on osteogenic differentiation of human dental pulp stem cells (hDPSCs). METHODS: The expression levels of MEG3, microRNA-543 (miR-543), osterix, osteopontin, osteocalcin and runt-related transcription factor 2 (RUNX2) were measured by quantitative real-time PCR (qRT-PCR). Alkaline phosphatase (ALP) activity assay and alizarin red S staining (ARS) were used to measure the impacts exerted by MEG3, miR-543 on osteogenic differentiation. Cell proliferation was measured by MTT assay. In addition, the targeted relationships between miR-543, MEG3, and Smad ubiquitin regulatory factor 1 (SMURF1) were assessed through dual luciferase reporter assay. RESULTS: During osteogenic induction, the expression of MEG3 was gradually reduced, whereas the expression of miR-543, osterix, osteopontin, osteocalcin and RUNX2 were gradually increased. Functional analysis implied that MEG3 overexpression or miR-543 inhibition reduced the cell proliferation, ALP activity, ARS levels, and decreased the expression of osteoblast-related proteins. Moreover, MEG3 promoted SMURF1 expression by directly targeting miR-543 as a competing endogenous RNA. Furthermore, overexpression of miR-543 or silencing SMURF1 could reverse the inhibitory effects of MEG3 on the osteogenic differentiation of hDPSCs. CONCLUSIONS: In conclusion, our study revealed that overexpression of MEG3 inhibited hDPSCs osteogenic differentiation via miR-543/SMURF1/RUNX2 regulatory network, which may contribute to the functional regulation and clinical applications of hDPSCs.
OBJECTIVE: The aim of the present study was to investigate the biological roles and underlying mechanism of the long non-coding RNA maternally expressed gene 3 (MEG3) on osteogenic differentiation of human dental pulp stem cells (hDPSCs). METHODS: The expression levels of MEG3, microRNA-543 (miR-543), osterix, osteopontin, osteocalcin and runt-related transcription factor 2 (RUNX2) were measured by quantitative real-time PCR (qRT-PCR). Alkaline phosphatase (ALP) activity assay and alizarin red S staining (ARS) were used to measure the impacts exerted by MEG3, miR-543 on osteogenic differentiation. Cell proliferation was measured by MTT assay. In addition, the targeted relationships between miR-543, MEG3, and Smad ubiquitin regulatory factor 1 (SMURF1) were assessed through dual luciferase reporter assay. RESULTS: During osteogenic induction, the expression of MEG3 was gradually reduced, whereas the expression of miR-543, osterix, osteopontin, osteocalcin and RUNX2 were gradually increased. Functional analysis implied that MEG3 overexpression or miR-543 inhibition reduced the cell proliferation, ALP activity, ARS levels, and decreased the expression of osteoblast-related proteins. Moreover, MEG3 promoted SMURF1 expression by directly targeting miR-543 as a competing endogenous RNA. Furthermore, overexpression of miR-543 or silencing SMURF1 could reverse the inhibitory effects of MEG3 on the osteogenic differentiation of hDPSCs. CONCLUSIONS: In conclusion, our study revealed that overexpression of MEG3 inhibited hDPSCs osteogenic differentiation via miR-543/SMURF1/RUNX2 regulatory network, which may contribute to the functional regulation and clinical applications of hDPSCs.