title: [miR-93-5P SUPPRESSES OSTEOGENIC DIFFERENTIATION OF MOUSE C3H10T1/2 CELLS BY TARGETING Smad5].
abstract: OBJECTIVE
  To investigate whether miR-93-5p suppresses osteogenic differentiation of mouse mesenchymal stem cells (C3H10T1/2) by targeting Smad5, a predicted target in silicon.
  
  
  METHODS
  Smad5 3'-UTR-luciferase vector (pmiR-RB-REPORT) was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-93-5p on Smad5 3'-UTR-luciferase activity to identify whether Smad5 was the target gene of miR-93-5p. miR-93-5p mimics (group M), miR-93-5p inhibitor (group In), miR-93-5p mimics negative control (group MC), and miR-93-5p inhibitor negative control (group InC) were transfected into the C3H10T1/2 cells, respectively, and followed by induction of osteogenic differentiation. After 48 hours, the real-time fluorescent quantitative PCR (qRT-PCR) and Western blot assays were performed to detect the relative expressions of Smad5 mRNA and protein. At 14 days, to realize the regulation role of miR-93-5p in osteogenic differentiation, the extracellular calcium deposition during the osteogenesis of C3H10T1/2 cells was tested by Alizarin red staining.
  
  
  RESULTS
  Dual-luciferase reporter gene assay showed that miR-93-5p could combine with Smad5 mRNA 3'-UTR specificity, and inhibited its luciferase activity (P < 0.05). After 48 hours, no significant difference was shown in the relative expression of Smad5 mRNA between group M and group MC as well as between group In and group InC by qRT-PCR assay (P > 0.05); however, the results of Western blot assay showed that the relative expression of Smad5 protein was significantly decreased in group M and increased in group In when compared with groups MC and InC (P < 0.05). At 14 days after osteogenic induction, Alizarin red staining showed that the extracellular calcium deposition of group M was obviously less than that of group MC, and it was obviously more in group In than in group InC.
  
  
  CONCLUSIONS
  Smad5 may be the target gene of miR-93-5p. And miR-93-5p can suppress osteogenic differentiation of C3H10T1/2 cells by directly targeting Smad5.
authors.forename: Lian
authors.initials: L
authors.lastname: Xu
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Li
authors.forename: Yin
authors.initials: Y
authors.lastname: Liu
authors.forename: Qingquan
authors.initials: Q
authors.lastname: Kong
authors.forename: Dan
authors.initials: D
authors.lastname: Long
authors.forename: Shengfu
authors.initials: S
authors.lastname: Li
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D059630:Mesenchymal Stem Cells; D051379:Mice; D035683:MicroRNAs / Q000502:physiology*; D010006:Osteoblasts / Q000166:cytology*; D010012:Osteogenesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051902:Smad5 Protein / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D035683:MicroRNAs; C551470:Mirn93 microRNA, mouse; D012333:RNA, Messenger; D051902:Smad5 Protein; C497798:Smad5 protein, mouse