The Effect Of C-terminally Truncated HBX On The Biological Activity Of HepG2 Cells

ObjectiveTo construct human Hepatocellular HepG2 cell model that can express C-terminally truncated HBX gene and protein,and to explore its effects on proliferation,invasion,migration and apoptosis of HepG2 cells.Methods1.HBX frequently integrated fragment HBXΔ32(HBX lost 32 amino acids at its C-terminus,369 bp in length),HBXΔ4(HBX lost 4 amino acids at its C-terminus,453 bp in length)and wild type HBX(465 bp in length)were used as the target segment,adenovirus vectors were constructed and then transfected into HepG2 cells.The transfected LV5-HBXΔ32,LV5-HBXΔ4,and LV5-HBX cells were used as experimental groups,and LV5-NC(empty virus)and HepG2 cells were used as control groups,to explore the effect of C-terminally truncated HBX on the biological activity of HepG2 cells.2.qPCR and WB were used to detect the expression of HBX gene and protein in each group after transfection,and judge whether the transfection was successful.3.CCK8 was added to the culture medium and the OD values of the cells were detected and compared at 1 hour,2 hours,3 hours,and 4 hours.The effect of the C-terminally truncated HBX on HepG2 cell proliferation was investigated.4.Transwell assay was used to detect the invasion and migration ability of the cells after transfection,and the effect of C-terminally truncated HBX on the invasion and migration of HepG2 cells was explored.5.Flow cytometry was used to detect the apoptosis of the cells after transfection,and the effect of C-terminally truncated HBX on the apoptosis of HepG2 cells was investigated.Results1.This study successfully constructed HepG2 cells that expressed C-terminally truncated HBX genes and proteins.GFP green fluorescence signal was observed under microscope,and HBX mRNA and protein expression was verified by qPCR and WB.2.The proliferation ability of HBXΔ32,HBXΔ4,HBX,NC,HepG2 cells in each group was different.There was a statistically significant difference in OD values at each time point(P<0.05).After comparison,the OD values of cell proliferation in HBXΔ32 and HBXΔ4 groups at each detection time point were higher than those in other groups,and the difference was statistically significant(P<0.05).There was no statistically significant difference in cell proliferation between the HBXΔ32 and HBXΔ4 groups(P > 0.05).3.The number of invasive cells in HBXΔ32,HBXΔ4,HBX,NC,and HepG2 groups were 277.89±20.64,266.78±22.09,163.44±21.02,157.78±22.02,and 160.56±21.51 respectively.The difference in the number of invading cells between these groups was statistically significant(P = 0.000).After comparison,it was found that the number of invasive cells in HBXΔ32 and HBXΔ4 groups were higher than that in other groups,and the difference was statistically significant(P<0.05).There was no statistically significant difference in the number of invasive cells between HBXΔ32 and HBXΔ4 groups.P = 0.856).4.The number of migrating cells in HBXΔ32,HBXΔ4,HBX,NC and HepG2 groups were 95.22±16.84,94.44±12.99,66.56±9.50,71.00±8.79 and 69.78±9.71 respectively.The difference in the number of migrating cells between these groups was statistically significant(P = 0.000).The comparison showed that the number of migrating cells in the HBXΔ32 and HBXΔ4 groups were higher than that in other groups,and the difference was statistically significant(P<0.05).There was no statistically significant difference in the number of migrating cells between the HBXΔ32 and HBXΔ4 groups(P = 0.588).5.The apoptosis rates of HBXΔ32,HBXΔ4,HBX,NC and HepG2 were 1.43±0.522%,1.14±0.236%,1.06±0.033%,1.26±0.466%,and 1.24±0.653%,respectively.There was no statistical significance between the apoptotic rates in these groups(P = 0.505).ConclusionC-terminally truncated HBXΔ32 and HBXΔ4 have potential promoting effects on the proliferation,invasion,and migration of human hepatoma HepG2 cells,and have not been found to affect the apoptosis of HepG2 cells.The rear 4-32 amino acids of the C-terminus of HBX may not be the major functional domains affecting the proliferation,invasion and migration of hepatoma cells.This study preliminarily explained the role of the integration of the C-terminally truncated HBX gene in the pathogenesis of hepatocellular carcinoma in the high-incidence region of Guangxi,and provided a certain experimental basis and theoretical reference for the etiological study and comprehensive prevention of HBV-related HCC.