남성 특이적인 B형 간염 바이러스 preS1 W4P 변이주에 의한 간세포암 성별차이 기전 규명

Abstract

Introduction: The mechanism underlying gender disparity in hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) still remains unclear. Recently, I have reported a novel W4P/R mutation in preS1 region of a large hepatitis B surface protein (LHB) associated with gender disparity of HCC patients in Korea via a molecular epidemiologic study. In the present study, I investigated whether a LHB mutant with W4P mutaion in preS1 region of HBV from a HCC patient play a pivotal role in gender disparity of hepatocarcinogenesis in vitro and in vivo mouse xenograft model. Methods: LHB sequences from a HCC patients with wild type (WT) HBV and W4P mutant HBV were cloned and transfected into mouse fibroblast cell line, NIH3T3 and human hepatocellular carcinoma cell line, Huh7. The effect of W4P mutation on the cell proliferation and the tumorigenicity was assessed by trypan blue exlusion assay and colony formation assay, respectively. To confirm in vivo turmorigenecity of WT and W4P mutant-transduced cell lines upon gender disparity, both cell lines were injected into male and female mice. And then, it was additionally confirmed with the injection of estrogen to each group. The serum levels of cytokines in each group of mice and patients with HCC were analyzed by ELISA. Through the inhibitor study with JAK2 inhibitor, I also examined whether IL-6 plays a critical role in the tumorigenecity of W4P mutant. Results: I have found that both WT and W4P mutant showed their enhanced proliferating activity based on the results from cell cycle analysis and colony formation assay, but it was significantly higher in W4P mutant than in WT. In addition, tumor growth in Xenograft model was only observed in the mice injected with W4P mutant LHB-expressing NIH3T3. Interestingly, it was not only definitely increased in mlae mice, but also decreased by the administration of estrogen. IL-6, but not tumor necrosis factor-α, was elevated in male mice harboring W4P-induced tumor, and was reduced by estrogen. IL-6 levels of HCC patients with the W4P mutant LHB were significantly higher than those of patients with WT LHB. W4P mutant LHB induced higher production of IL-6 than WT LHB from its transfeced cell lines, and IL-6 production by it was reducedwith the treatment of estrogen. The ability to reduce cell proliferation and colony formation of W4P mutant-expressing cells was hampered by inhibition of IL-6 signaling. Conclusions: This study suggests that the accumulation of W4P mutant in LHBs during the natural course of chronic hepatitis B virus infection, might be closely involved in the development of HCC in chronic HBV patients. It is particularly observed in male in an IL-6-dependent manner. This is the first report regarding direct relationship between mutation at LHBs of HBV and gender disparity of HBV infection and tumorigenecity. Therefore, this study will provide a novel insight for gender disparity of HBV infection and the process of hepatocarcinogenesis.