Xiang-Bei Qi1, Bei Jia2, Wei Wang1, Guo-Hui Xu1, Ji-Chao Guo1, Xu Li1, Jian-Ning Liu3. 1. Department of Orthopaedics, The Third Hospital of Hebei Medical University, Shijiazhuang, Hebei 050035, China. 2. Department of Infectious Diseases, The First Hospital of Hebei Medical University, Shijiazhuang, Hebei 050031, China. 3. Department of Orthopaedics, The Third Hospital of Hebei Medical University, Shijiazhuang, Hebei 050035, China. Electronic address: ljnljn_77458@sina.com.
Abstract
OBJECTIVE: miR-199a-5p was increased during osteoblast differentiation, which may target and regulate TET2, a gene attracted a lot of attention in the osteoblast differentiation in the past few years. However, the role of miR-199a-5p in osteoblast differentiation by targeting TET2 is not established. METHODS: The correlation between miR-199a-5p and TET2 was verified through dual luciferase reporter assay, and their expressions in human bone marrow stromal cells (hBMSCs) during the osteoblast differentiation were detected. hBMSCs were transfected with TET2 siRNA, miR-199a-5p mimic or/and TET2 CRISPR activation plasmid., and then prepared for the induction of osteoblast differentiation, followed by alkaline phosphatase (ALP) and alizarin red staining, qRT-PCR and Western blotting. In vivo, ovariectomized (OVX) mice were injected with agomir-miR-199a-5p, antagomiR-199a-5p or/and TET2 siRNA to calculate the BMD and BV/TV ratio of mice, as well as to measure the expressions of osteogenesis-related genes in bone tissues. RESULTS: A gradual increase of miR-199a-5p was observed in hBMSCs during the induction of osteoblast differentiation, while TET2 expression was decreased. Besides, miR-199a-5p was reduced in the bone tissue of OVX mice, while TET2 was up-regulated. In addition, overexpression of miR-199a-5p and inhibition of TET2 augmented ALP activity in hBMSCs, with the enhanced calcification and the up-regulated expressions of Runx2, OSX and OCN, which also increased the quality of bone in OVX mice accompanying the enhancement BV/TV ratio, BMD and osteogenesis-related genes. CONCLUSION: MiR-199a-5p may promote the osteoblast differentiation and prevent OVX-induced osteoporosis by targeting TET2.
OBJECTIVE: miR-199a-5p was increased during osteoblast differentiation, which may target and regulate TET2, a gene attracted a lot of attention in the osteoblast differentiation in the past few years. However, the role of miR-199a-5p in osteoblast differentiation by targeting TET2 is not established. METHODS: The correlation between miR-199a-5p and TET2 was verified through dual luciferase reporter assay, and their expressions in human bone marrow stromal cells (hBMSCs) during the osteoblast differentiation were detected. hBMSCs were transfected with TET2 siRNA, miR-199a-5p mimic or/and TET2 CRISPR activation plasmid., and then prepared for the induction of osteoblast differentiation, followed by alkaline phosphatase (ALP) and alizarin red staining, qRT-PCR and Western blotting. In vivo, ovariectomized (OVX) mice were injected with agomir-miR-199a-5p, antagomiR-199a-5p or/and TET2 siRNA to calculate the BMD and BV/TV ratio of mice, as well as to measure the expressions of osteogenesis-related genes in bone tissues. RESULTS: A gradual increase of miR-199a-5p was observed in hBMSCs during the induction of osteoblast differentiation, while TET2 expression was decreased. Besides, miR-199a-5p was reduced in the bone tissue of OVX mice, while TET2 was up-regulated. In addition, overexpression of miR-199a-5p and inhibition of TET2 augmented ALP activity in hBMSCs, with the enhanced calcification and the up-regulated expressions of Runx2, OSX and OCN, which also increased the quality of bone in OVX mice accompanying the enhancement BV/TV ratio, BMD and osteogenesis-related genes. CONCLUSION: MiR-199a-5p may promote the osteoblast differentiation and prevent OVX-induced osteoporosis by targeting TET2.
Authors: Cecilia Catellani; Gloria Ravegnini; Chiara Sartori; Beatrice Righi; Pietro Lazzeroni; Laura Bonvicini; Silvia Poluzzi; Francesca Cirillo; Barbara Predieri; Lorenzo Iughetti; Paolo Giorgi Rossi; Sabrina Angelini; Maria Elisabeth Street Journal: Front Endocrinol (Lausanne) Date: 2022-06-22 Impact factor: 6.055