RLN2 regulates in vitro invasion and viability of osteosarcoma MG-63 cells via S100A4/MMP-9 signal.

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.