The Targets Affected By HBx In P53-dependent And P53-independent Pathway Of Hepatoma Cells

Objective:Chronic infection of hepatitis B virus(HBV) is one of the main reasons that contributed to the carcinogenesis of primary hepatocellular carcinoma(HCC). HBV encoded a multi-function protein, 17KD, named x protein or HBxAg which is necessary for the transcription of virus genome and is considered to be related to the processing from HBV chronic infection to HCC. But the affection on growth of hepatic cell and the molecular mechanisms in the formation and development of HCC are not elucidated. p53 is a very important tumor suppressor gene, and cell signal transduction pathway mediated by p53 play an important role in regulating the normal activities of cells. Recent studies show that HBx can combine with p53 protein and then accumulate p53 protein in cells in HCC of HBV chronic infection. But the affection and the molecular mechanisms on the growth of p53-dependent and p53-independent cells are still not well-known. Therefore, to observe the alterations of hepatoma cell affected by HBx and the relationships between HBx and p21WAF1 or p14ARF which is involved in p53 pathway and to investigate the targets of HBx in p53-dependent and p53-independent apoptosis pathway is very significant in theory that may demonstrate the mechanism of HCC carcinogenesis. Methods:1. HBx and HBx3-40,40 amino acids on C terminal of which was removed, were transfected into hepatoma cell line SMMC-7721, and then Flowcytometry was used todetect the change of apoptosis; CAT-ELISA was applied to analysis the transactivation function of HBx gene; cell growth curve, clone formation in plate and tumor forming in nude mice were used to observe the alterations of SMMC-7721 cell growth affected by HBx and HBx3-40.The levels of the expression of p14ARF, MDM2, p53.. p21WAF1using western blotting was detected in different hepatoma cells.2. The sense and antisense wt-p53 plasmid were constructed and then transfected or cotransfected into SMMC-7721 with HBx or HBx3-40; HBx was also transfected into hepatoma cells with different p53 status; Then, the mechanism the affection HBx on p53 , p21WAF1 and the relation between HBx and the growth of hepatoma cell were analysed through observing the changes of cell apoptosis, cell cycle, activities of luciferase which contain p21WAF1 promoter that can combine p53 and the alteration of the expression of p53 and p21WAF1 using Western blot.3. HBx and p14ARF were transfected and cotransfected into HepG2 cell, which contain wt-p53 but do not show the expression of p14ARF protein by Western blot. Several biological techniques were used to analyse whether HBx can affect the growth of hepatoma cell through p14ARF pathway.Results1.After transfected with HBx and HBx3-40, hepatoma cell line SMMC-7721 is more sensitive to apoptosis induced by the damage of DNA. But the rate of apoptosis of HBx3-40 is less than that of HBx, while the transactivation ability of HBx shows stronger than HBx3-40. Furthermore, we found the groups which was transfected by HBx and HBx3-40 growed far more faster than control group. Plate clone forming test and tumor-forming in null-mice test also show that the rates of clone-forming of HBx and HBx3-40 as well as the rates of tumor-forming in nude mice are enhanced significantly. The expression level of PCNA in tumors of both groups is also increased. These biological markers in HBx3-40 group is more notable.This suggests that the 40 amino acids deletion in the C-terminal of HBx may be correlated with mechanism of promoting the malignant transformation of cancer cell and the ability of HBx to increase the sensitity of cancer cells to DNA damagemay be related with its transactivation ability and combination with p53.2.The sense and antisense wt-p53 plasmids were constructed and then transfected and cotransfected into SMMC-7721 with HBx or HBx3-40. The results show that both HBx and HBx3-40 can reduce the apoptosis effects of cells induced by wt-p53, which suggests that the inhibitory effects of HBx on the apoptosis is indepe...