title: MiR-9 promotes osteoblast differentiation of mesenchymal stem cells by inhibiting DKK1 gene expression.
abstract: The aim of this study is to investigate the role of miR-9 and its mechanism on the osteoblast differentiation of mesenchymal stem cells. Real-time PCR and western blotting were used to study gene expression. Assay of Alkaline phosphatase activity and alizarin red staining were used to examine osteoblast differentiation. Transfection of miR-9 mimics or lent-shmiR-9 was used to modulate the level of miR-9 in C2C12. Overexpression of miR-9 in C2C12 cells stimulated alkaline phosphatase activity and osteoblast mineralization, as well as the expression of osteoblast marker genes Col I, Ocn and Bsp. Gene silencing of miR-9 in C2C12 resulted in the suppression of alkaline phosphatase activity and osteoblast mineralization, as well as the expression of Col I, Ocn and Bsp. DKK1 mRNA was not affected by miR-9 overexpression, however, DKK1 protein was significantly decreased. Moreover, DKK1 3'-UTR mediated transcriptional luciferase activity was also significantly suppressed by miR-9 overexpression. DKK1 mRNA was not affected by miR-9 gene silencing, however, DKK1 protein was significantly stimulated. Moreover, DKK1 3'-UTR mediated transcriptional luciferase activity was significantly stimulated by miR-9 gene silencing, and suppressed by miR-9 overexpression, however, DKK1 3'-UTR mutant mediated luciferase activity was unaffected. The siRNA derived gene silencing of DKK1 blocked the inhibiting effect of shmiR-9 on the expression of alkaline phosphatase; and blocked the inhibiting effect of shmiR-9 on the expression of ColI, Ocn and Bsp. MiR-9 promotes osteoblast differentiation of mesenchymal cell C2C12 by suppressing the gene expression of DKK1.
authors.affiliation: Department of Spinal Surgery, The Affiliated Hospital of Qingdao University, Qingdao, 266003, China.
authors.forename: Xiangyun
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Joint Surgery, The Affiliated Hospital of Qingdao University, No. 16 Jiangsu Road, Qingdao, 266003, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Spinal Surgery, The Affiliated Hospital of Qingdao University, Qingdao, 266003, China.
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Kou
authors.affiliation: Qingdao Central Blood Station, Qingdao, 266003, China.
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Spinal Surgery, The Affiliated Hospital of Qingdao University, Qingdao, 266003, China.
authors.forename: Xiujun
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Department of Joint Surgery, The Affiliated Hospital of Qingdao University, No. 16 Jiangsu Road, Qingdao, 266003, China. yutengbo1234@163.com.
authors.forename: Tengbo
authors.initials: T
authors.lastname: Yu
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D010006:Osteoblasts / Q000502:physiology*; D034622:RNA Interference
chemical_list: C501127:Dkk1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; C551804:MIRN9 microRNA, mouse; D035683:MicroRNAs; D000469:Alkaline Phosphatase
keywords: C2C12; DKK1; Mesenchymal stem cells; MiR-9; Osteoblast differentiation