Inhibition effect of silencing CXCR4 on the invasion of hepatocellular carcinoma cell lines in vitro

OBJECTIVE:To investigate the effect of CXCR4 knockdown by RNA interference on the proliferation and invasion in hepatocellular carcinoma cells.METHODS:The CXCR4 expression level of different hepatocellular carcinoma cell lines was detected by western blotting at protein level and Real time RT-PCR at mRNA level.The shRNA vector targeting to CXCR4 was transfected into the HepG2 cells,and the stable transfected cells were selected by G418.Western blotting and Real time RT-PCR were performed to observe the inhibitory effect of RNAi on CXCR4 expression.MTT assay was used to assess the influence of CXCR4 shRNA on cell proliferation.Transwell assay was applied to observe the cell invasion ability.The expressions of MMP-2 and MMP-9 protein were determined by Western blotting and immunofluorescence.RESULTS:Stable transfection of CXCR4shRNA into HepG2 cells resulted in efficiently declined expression of CXCR4.CXCR4 protein expression in HepG2,HepG2-Vector and HepG2-CXCR4-cells were 0.56±0.07,0.54±0.04 and 0.14±0.05respectively(F=57.42,P0.001).CXCR4 mRNA expression in HepG2,HepG2-Vector and HepG2CXCR4-cells were 1.04±0.05,1.05±0.11and 0.19±0.03respectively(P0.001).MTT experiments showed that silencing CXCR4 significantly decreased proliferation of HepG2 cells.In 72h,the proliferation of HepG2,HepG2-Vector and HepG2-CXCR4-cells were 1.34±0.05,1.32±0.03and 1.14±0.03.The number of migrated cells which attracted by FBS were 85±13,89±17and 23±6 in HepG2,HepG2-Vector and HepG2-CXCR4-group respectively(F=63.91,P 0.001).Furthermore,the number of migrated cells which attracted by SDF-1were 168±20,171±24 and 30±9.Using Western blot,we found that the relative expression of MMP-2was obviously declined in HepG2-CXCR4-cells(0.31± 0.06)comparing with normal HepG2 cells(0.83±0.04)(F=78.34,P0.001),and the relative expression of MMP-9 were no significant difference in HepG2(0.35±0.04),HepG2-Vector(0.33±0.07)and HepG2-CXCR4-cells(0.32± 0.06)(F=0.23,P=0.79).CONCLUSION:CXCR4 knockdown is successfully performed by CXCR4 shRNA transfection.CXCR4 knockdown inhibits the proliferation and invasion in hepatocellular carcinoma cell.