Effects of SH3BP4 gene on the migration and proliferation of hepatocarcinoma cells

Abstract:

Objective：To investigate the effect of SH3BP4 on the migration and proliferation of hepatocarcinoma cells by the construc－tion of SH3BP4 recombinant adenovirus vector and lentiviral vector. Methods：The SH3BP4 cDNA sequence was amplified and then the recombinant Adplasmid pAdTrack-SH3BP4 was con－structed. The recombinant adenovirus Ad-SH3BP4 was packaged and amplified by using HEK293 cells. The short hairpin RNA recombinant lentiviral vector of SH3BP4 was constructed and transfected into HEK293T cells，then the recombinant lentivi-ruses shSH3BP4 was packaged and amplified. The endogenous expression of SH3BP4 in normal human hepatocytes and hepa－tocarcinoma cells was examined by the Western blot. The SH3BP4 overexpression experiment was divided into three groups：mock group（the blank control group），AdGFP group（the control group of those were infected with adenovirus AdGFP） and AdSH3BP4 group（the experimental group of those were infected with adenovirus AdSH3BP4）；the SH3BP4 knockdown experiment was divided into two groups，shControl group，the control group of those were in－fected with lentiviruses PLL3.7 vector，and shSH3BP4 group，the experimental group of those were infected with lentiviruses shSH3BP4. The effect on the migration of hepatocarcinoma cells was observed by the Transwell and wound healing assay，and the effect on the proliferation was observed by the MTS and colony formation assay. Results：A recombinant adenovirus AdSH3BP4 and a recombinant lentiviral shSH3BP4 were successfully constructed，verified by the restriction endonuclease and DNA sequencing. The Transwell assay showed that the number of cell migration in AdSH3BP4 group（74.800±0.544） significantly decreased compared with that in mock group（219.467±2.640） and AdGFP group（210.533±8.498）（P=0.000）. In contrast，the number of cell migration in shSH3BP4 group（191.467±3.906） increased significantly compared with that in shControl group（91.733±2.763）（P=0.000）. The wound healing assay showed that the cell repair rate in AdSH3BP4 group[（10.400±0.036）%] was lower than that in mock group[（37.500±0.063）%] and AdGFP group[（50.000±0.063）%]（P<0.000），but it was significantly higher in shSH3BP4 group[（55.00±0.05）%] compared with that in shControl group[（23.300±0.029）%]（P=0.000）. The MTS assay showed that the absorbance value at 96 h in AdSH3BP4 group（0.921±0.053） decreased significantly compared with that in black control group（1.091±0.043） and AdGFP group（1.062±0.024），while it was significantly higher in shSH3BP4 group（1.497±0.020） compared with that in shControl group（1.142±0.103）（P=0.004）. The colony formation assay showed that the number of clone forming cells in AdSH3BP4 group（34.333±2.082） significantly decreased compared with that in mock group（86.333±4.726） and AdGFP control group（87.333±1.528）（P=0.000），but it was significantly higher in shSH3BP4 group（65.000±6.557） compared with that in shControl（31.000±2.000）（P=0.001）. Conclusion：SH3BP4 can inhibit the migration，invasion and proliferation of hepatocarcinoma cells，and it may provide a new target for the diagnosis and treatment of liver cancers.