Lihua Cao1,2,3, Wei Liu4, Yancheng Zhong1,2, Yanru Zhang1,2, Dan Gao1,2, Tiantian He1,2, Ying Liu1,2, Zi Zou1,2, Yuqing Mo1,2, Shuping Peng1,2,3, Cijun Shuai5,6. 1. NHC Key Laboratory of Carcinogenesis, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, China. 2. School of basic Medical Science, Central South University, Changsha, China. 3. The Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute, Central South University, Changsha, China. 4. Institute of Metabolism and Endocrinology, Nation Clinical Research Center for Metabolic Diseases, The Second XiangYa Hospital, Central South University, Changsha, China. 5. Institute of Bioadditive Manufacturing, Jiangxi University of Science and Technology, Nanchang, China. 6. State Key Laboratory of High Performance Complex Manufacturing, Central South University, Changsha, China.
Abstract
OBJECTIVES: Increasing evidences suggest that inducing mesenchymal stem cells to differentiate into osteoblasts has been as an especially important component in the prevention and therapy for degenerative bone disease. Here, we identify a novel lncRNA, linc02349, which increases significantly during osteogenic differentiation. MATERIALS AND METHODS: Human umbilical cord-derived stem cells (hUC-MSCs) and dental pulp mesenchymal stem cells were used. Overexpression and knockdown of linc02349 in cell lines were generated using lentiviral-mediated gene delivery method. Bioinformatics prediction, Ago2-RIP assay and dual-luciferase reporter system were employed to examine miRNA which interacts with linc02349. The RNA FISH assay was performed to identify the subcelluar location of linc02349. Alizarin Red S staining, ALP staining and qPCR were applied to identify the osteogenic differentiation. The potential linc02349-regulated genes, miR-25-3p and miR-33b-5p, were explored by ChIP, RIP and Western blotting assays. Micro-CT was used to measure the osteogenic content in bone formation assay in vivo. RESULTS: Linc02349 overexpression improves osteogenic differentiation by in vitro and in vivo analysis. Mechanistically, linc02349 acts as a molecular sponge for miR-25-3p and miR-33b-5p to control expression abundance of SMAD5 and Wnt10b, respectively, which eventually activated Dlx5/OSX pathway and hence promoted osteogenic differentiation. In addition, we revealed that STAT3 interacts with linc02349 promoter region and positively regulates the linc02349 transcriptional activity. CONCLUSION: These findings identify that linc02349 modulates the osteogenic differentiation through acting as a sponge RNA of miR-25-3p and miR-33b-5p and regulating SMAD5 and Wnt10b, and proposed a new interaction between STAT3 and linc02349, which could be a potential target in the process the osteogenesis of hUC-MSCs for future clinical application.
OBJECTIVES: Increasing evidences suggest that inducing mesenchymal stem cells to differentiate into osteoblasts has been as an especially important component in the prevention and therapy for degenerative bone disease. Here, we identify a novel lncRNA, linc02349, which increases significantly during osteogenic differentiation. MATERIALS AND METHODS:Human umbilical cord-derived stem cells (hUC-MSCs) and dental pulp mesenchymal stem cells were used. Overexpression and knockdown of linc02349 in cell lines were generated using lentiviral-mediated gene delivery method. Bioinformatics prediction, Ago2-RIP assay and dual-luciferase reporter system were employed to examine miRNA which interacts with linc02349. The RNA FISH assay was performed to identify the subcelluar location of linc02349. Alizarin Red S staining, ALP staining and qPCR were applied to identify the osteogenic differentiation. The potential linc02349-regulated genes, miR-25-3p and miR-33b-5p, were explored by ChIP, RIP and Western blotting assays. Micro-CT was used to measure the osteogenic content in bone formation assay in vivo. RESULTS:Linc02349 overexpression improves osteogenic differentiation by in vitro and in vivo analysis. Mechanistically, linc02349 acts as a molecular sponge for miR-25-3p and miR-33b-5p to control expression abundance of SMAD5 and Wnt10b, respectively, which eventually activated Dlx5/OSX pathway and hence promoted osteogenic differentiation. In addition, we revealed that STAT3 interacts with linc02349 promoter region and positively regulates the linc02349 transcriptional activity. CONCLUSION: These findings identify that linc02349 modulates the osteogenic differentiation through acting as a sponge RNA of miR-25-3p and miR-33b-5p and regulating SMAD5 and Wnt10b, and proposed a new interaction between STAT3 and linc02349, which could be a potential target in the process the osteogenesis of hUC-MSCs for future clinical application.
Authors: Carmen Lanzillotti; Monica De Mattei; Chiara Mazziotta; Francesca Taraballi; John Charles Rotondo; Mauro Tognon; Fernanda Martini Journal: Front Cell Dev Biol Date: 2021-04-09