THE MALIGNANT BEHAVIOR OF HEPATOCARCINOMA CELLS PROMOTED BY HEPATITIS B VIRUS X PROTEIN BY ACTIVATING NF-KB TO PROMOTE P65 INTO THE NUCLEAR UPREGULATED CALMODULIN SMALL SUBUNIT

Objective: To study the mechanism of hepatocarcinoma cell migration promoted by Hepatitis B virus X protein by activating NF-kappa B to promote p65 into the nuclear-upregulated calpain small subunit (Capn4). Method: The expression change of Capn4 in HBx HepG2-X was detected using real-time PCR and Western blot; The expression of HBx in a HepG2-X cell line was silenced by siRNA and the change of Capn4 in pSilencer-HBx was detected using real-time PCR and Western blot; The construction of HepG2-X (Capn4 siRNA) cell line by siRNA technique, HepG2, HepG2-P, HepG2-X, HepG2-X (Control siRNA), HepG2-X (HBx siRNA) and HepG2-X (Capn4 siRNA) migration distance of each cell line were compared using a monolayer cell repair test; Immunofluorescence staining was used to observe the effect of the HepG2-X cell line HBx on NF-xli active subunit p65; The effect of HepG2-X (p65 siRNA) constructed using a siRNA technique on expression of Capn4 in HepG2-X cell was analyzed by Western blot; Western blots were used to detect the effect of NF-kappa B p65 specific inhibitor PDTC on Capn4 expression in HepG2-X cells. Result: Compared with HepG2 and HepG2-P, Capn4 expression levels were significantly increased at both mRNA and protein levels in HepG2-X; PSilencer-HBx inhibited the gene transcription of Capn4 in a dose-dependent manner compared with the blank control group and pSilencer control group at mRNA and protein levels; The migration ability of HepG2-X cell line was significantly higher than that of HepG2 and HepG2-P cell lines and the migration ability of HepG2-X (HBx siRNA) and HepG2-X (Capn4 siRNA) cell lines was significantly lower than that of HepG2-X cell line at 48h after scratching. The NF-kappa B active subunit p65 was mostly concentrated in the cytoplasm in the HepG2 and HepG2-P cell lines, while p65 was concentrated in the nucleus mostly in the HepG2-X cell line, and the nuclear and cytosolic phosphorylation of p65 was significantly increased; NF-kappa B p65 specific siRNA p65 inhibited Capn4 expression in HepG2-X cells in a dose-dependent manner; NF-kappa B p65 specific inhibitor PDTC inhibited Capn4 expression in HepG2-X cells in a dose-dependent manner. Conclusion: Hepatitis B virus X protein promotes the migration of hepatoma cells by activating NF-kappa B to promote p65 into the nuclear-upregulated calmodulin small subunit.