title: RLN2 regulates in vitro invasion and viability of osteosarcoma MG-63 cells via S100A4/MMP-9 signal.
abstract: OBJECTIVE
  Relaxin-2 (RLN2) increases cell migration, invasiveness and proliferation in vitro of osteosarcoma cells, but the molecular mechanisms of this action are still unknown. In the present study, we identified S100A4 /MMP-9 signaling as a major mediator of the actions of RLN2 in osteosarcoma cells in vitro.
  
  
  METHODS
  We have established stable transfectants of osteosarcoma MG-63 cells using small interfering RNA (siRNA) targeting RLN2. The stable transfectants (MG-63/RLN2 siRNA cells) were treated with 20 mM BB94 (a kind of MMP-9 activator) or 100 mM recombinant Human RLN2 (B-29/A-24) for 24 hs or transfected with S100A4 cDNA plasmid for 48 hrs or MMP-9 siRNA for 48 hrs then treated 100 mM recombinant Human RLN2 (B-29/A-24). Western blot assay was used to detect RLN2, S100A4 and MMP-9 expression. Matrigel invasion assay and wound healing assay was used to detect invasion in vitro. MTT was used to detect cell viability.
  
  
  RESULTS
  Knockdown of RLN2 using small interfering RNA decreases S100A4 and MMP-9 expression and inhibits invasion and cell viability in vitro. in MG-63 cells. Treatment with 100 mM recombinant Human RLN2 (B-29/A-24) for 24 hrs in MG-63/ RLN2 siRNA cells increases S100A4 and MMP-9 expression, and increases the invasion and cell viability in vitro in MG-63 cells. Transfection with S100A4 cDNA plasmid in MG-63/RLN2 siRNA cells for 48 hrs increases MMP-9 expression, and increase the invasion and cell viability of MG-63/RLN2 siRNA cells. Treatment with 20 mM BB94 (MMP-9 activator) for 24 hrs in MG-63/RLN2 siRNA cells increases MMP-9 expression, and increases the invasion and cell viability in vitro. in MG-63 cells.
  
  
  CONCLUSIONS
  Our results indicate that RLN2 regulats cell migration, invasiveness and proliferation of osteosarcoma cells in vitro, which may be mediated through S100A4/MMP-9 signaling.
authors: Department of Spine, the Affiliated Hospital of Qingdao University, Qingdao, China. renxuanfengqd@126.com.
authors: X-F
authors: XF
authors: Ren
authors: H
authors: H
authors: Zhao
authors: X-C
authors: XC
authors: Gong
authors: L-N
authors: LN
authors: Wang
authors: J-F
authors: JF
authors: Ma
mesh_terms: D001859:Bone Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis*; D009361:Neoplasm Invasiveness / Q000473:pathology / Q000517:prevention & control; D012516:Osteosarcoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D012065:Relaxin / Q000008:administration & dosage*; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000096:biosynthesis*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: C095068:RLN2 protein, human; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C077591:S100A4 protein, human; D012065:Relaxin; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9