| Background Hepatocellular carcinoma (HCC) is a common malignancy and one of the major reasons of death induced by cancer. Recent epidemiological data indicate that, not only in Asia and Africa, but also in North America and Europe, the incidence of liver cancer has shown an upward trend, and will be so far for the next decade. HBV infection is an important factor in many risk factors associated with hepatocellular carcinoma. Although safe and effective HBV vaccine has been widely used, but chronic hepatitis B virus (HBV) infection remains a major HCC risk factors, more than half of the HCC patients were HBV chronic carriers .In Asia and North America, studies have shown that HBsAg carriers increased risk of liver cancer 25-37 times compared to non-HBV infected individuals. Liver cancer prognosis is generally poor and often diagnosed at advanced stage, surgery and local therapy are considerable difficulty, and liver cancer has considerable resistance to chemotherapy. X protein encoded by HBV plays an important role in the carcinogenic mechanism of HBV, that has been further confirmed in many relative research, however, whether HBx promotes the further development of HCC, and the relevant mechanism is not very clear , therefore, in-depth research of the molecular mechanism of the development of HCC, is expected to find more critical elements, in order to provide a theoretical basis for the clinical targeted therapy. Objective To observe the change of expressions of MKK3 and p38MAPK in HCC induced by hepatitis B virus X protein,and the correlative mechanisms of the effect in signal transduction,to investigate the effect of hepatitis B virus X protein to HCC.Methods The human hepatoma carcinoma cells HepG2 were transfected with pCDNA3.1 and pCDNA-3.1-HBx recombinant plasmids by lipofectamine 2000.Then the stable cell lines expressing constantly HBx protein and the negative control cell lines were established by adding G418 to select single cell G418-resistant clones and identification with reverse transcriptase-PCR and Western blot assays,named respectively HepG2-HBx and HepG2-pCDNA3.1 cells,meanwhile with the HepG2 as blank.To detect the change on mRNA and protein level of MKK3 and p38MAPK in above three species cells by RT-PCR and Western blot assays,changes of phosphorylation-p38MAPK protein in nucleus and cytoplasm were observed with cell immunofluorescence method;To measure the proliferation,cell cycle and apoptosis of these cells by MTT assays.Dates were analysised by SPSS12 system.Result The results of RT-PCR and Western blot showed that expressing of MKK3 on mRNA and protein was higher in HepG2-HBx than that in other two species cells. There is no evident change on the mRNA and total protein level of p38MAPK for all various cell models,but the phosphorylation of p38 and the quantity of it in nucleus were up-regulated in HepG2-HBx. Immunofluorescence experiments shows that phosphorylation of p38MAPK is rich in HepG2-HBx cells than that in the other two cells, p38MAPK was significantly more In the nucleus of HepG2-HBx cells than that in the other two groups cells.HepG2-HBx's cell was stronger in proliferation than other two species cells, especially in the 72 hours after transfection.Conclusions HBx can up-regulate the expression of MKK3 in hepatoma carcinoma cell and promote the phosphorylation of p38 and precipitate p38 into nucleus,further activate the downstream molecule.p38MAPK signal transductio- nal path plays important role in the changes of proliferation hepatoma carcinoma cell induced by HBx.This may be one of the mechanisms that make an tumor biological behaviour different in clinic character between the HBV-correlated and no HBV-correlated hepatocellular carcinoma. |
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