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.
Materials and 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.