The Effects Of DRAM1Knockdown On Migration And Invasion Ability In HepG2Cells And Its Relation With Autophagy

Objective:To investigate the effects of migration and invasion ability in hepatic carcinoma cell line in vitro after DRAM1knockdown and its underlay mechanisms related to autophagy.Methods:Human hepatic carcinoma HepG2cells were cultured and transfected with two small interfering RNA (DRAM1siRNA1/2) chemosynthesized by company. The interfering efficacy, EMT related protein and autophagy related protein were tested by Western blot analysis after HepG2cells were transfected with DRAM1siRNA1/248h later. We also analyzed the migratory and invasive ability of hepatocellular carcinoma cell by Transwell chamber migratory and invasive culture system. Immunefluorescence was used to detect the expression of autophagic proteins in HepG2cells transfected with DRAM1siRNA. To further study whether the effect of DRAM1knockdown on HepG2cell migratory and invasive ability, ATG5siRNA targeting to interfere with ATG5mRNA was used to inhibitor cell autophagy. Interfering efficacy of ATG5siRNA and autophagy related protein was analyzed by Western blot. Immunefluorescence was used to detect the expression of autophagic proteins in HepG2cells treated with ATG5siRNA described as above. Then we also analyzed the migratory and invasive ability of hepatocellular carcinoma cell by Transwell chamber migratory and invasive culture system.Results:Two small interfering RNA (DRAM1siRNA1/2) were well transfected into the cells with Lipofectamine2000and had the higher transfection efficiency was detected by Western blot, respectively. Western blot analysis showed that the expression of epithelial specific protein E-Cadherin was significantly higher and the expression of mesenchymal specific protein Vimentin and transcription factor snail was significantly lower than the control group. Transwell chamber cell culture system indicated that the migratory and invasive ability of hepatocellular carcinoma cells was significantly decreased after DRAM1knockdown. Western blot and immunefluorescence were used to detect the expression of autophagic proteins which showed the decreased LC3(LC3-II) protein as well as the increased p62protein. One small interfering RNA (ATG5siRNA) was used to inhibitor cell autophagy with lower LC3(LC3-II) protein and higher p62protein detected by Western blot and immunefluorescence. Transwell chamber cell culture system was also used to investigate the migratory and invasive ability of hepatocellular carcinoma cells and results showed a significantly decreased after DRAM1knockdown. The above results were statistically significant by statistical analysis.Conclusion:Knockdown of DRAM1reduced epithelia·mesenchymal transition, inhibited cell autophagy as well as the ability of migration and invasion in hepatocellular carcinoma cells. The ability of migration and invasion also descreased after autophagy was inhibited by ATG5knockdown. Our results revealed that knockdown of DRAM1in HepG2cells inhibited invasion and migration that may through the pathways by inhibiting cell autophagy.