HBx drives alpha fetoprotein expression to promote initiation of liver cancer stem cells through activating PI3K/AKT signal pathway

Hepatitis B virus (HBV)‐X protein (HBx) plays critical role in inducing the malignant transformation of liver cells. Alpha fetoprotein (AFP) expression is closely related to hepatocarcinogenesis. We report that Oct4, Klf4, Sox2 and c‐myc expression positively associated with AFP(+)/HBV(+) hepatocellular carcinoma(HCC) tissues, and the expression of the stemness markers CD44, CD133 and EpCAM was significantly higher in AFP(+)/HBV(+) HCC tissues compared to normal liver tissues or AFP (?)/HBV(?) HCC tissues. AFP expression turned on prior to expression of Oct4, Klf4, Sox2 and c‐myc, and the stemness markers CD44, CD133 and EpCAM in the normal human liver L‐02 cell line or CHL cell lines upon transfection with MCV‐HBx vectors. Stem‐like cells generated more tumour colonies compared to primary cells, and xenografts induced tumourigenesis in nude mice. Expression of reprogramming‐related proteins was significantly enhanced in HLE cells while transfected with pcDNA3.1‐afp vectors. The specific PI3K inhibitor Ly294002 inhibited the effects of pcDNA3.1‐afp vectors. AFP‐siRNA vectors were able to inhibit tumour colony formation and reprogramming‐related gene expression. Altogether, HBx stimulates AFP expression to induce natural reprogramming of liver cells, and AFP plays a critical role in promoting the initiation of HCC progenitor/stem cells. AFP may be a potential novel biotarget for combating HBV‐induced hepatocarcinogenesis.     

乙型肝炎病毒X蛋白对肝细胞L02增殖及GSK3β表达的影响

目的:通过研究乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)对人肝细胞株L02细胞增殖、细胞周期以及细胞中糖原合成酶激酶3β(glycogen synthase kinase 3β,GSK3β)表达的影响,探讨乙型肝炎病毒(hepatitis B virus,HBV)相关性原发性肝细胞癌(hepatoccellular carcinoma,HCC)的发生机制。方法:用HBx腺病毒(Ad-HBx)感染人肝细胞株L02细胞后,RT-PCR法检测L02细胞中HBx和GSK3β mRNA表达情况;MTT法检测L02细胞的增殖率变化;FCM法检测细胞周期中各时相所占比例;蛋白质印迹法检测HBx、总GSK3β(total-GSK3β,t-GSK3β)、磷酸化GSK3β(phospho-GSK3β,p-GSK3β)、β-连环素(β-catenin)以及细胞用期蛋白(cyclinD1)等蛋白的表达水平。结果:Ad-HBx感染L02细胞后,HBx的mRNA和蛋白均出现表达,细胞增殖率随着时间的延长而增加;G1期细胞所占比例较对照组减少,S期和G2期细胞比例较对照组增加(P<0.05)。感染Ad-HBx后,t-GSK3β在mRNA和蛋白水平上均无明显变化,而p-GSK3β、β-catenin以及cyclinD1蛋白的表达量增加(P<0.05)。结论:HBx可能通过促进人肝细胞株L02细胞中GSK3β的磷酸化,激活Wnt/β-catenin下游信号通路,从而促进细胞的增殖。     

A mutant of hepatitis B virus X protein （HBx△127） enhances hepatoma cell migration via osteopontin involving 5-1ipoxygenase

Aim:

To explore a novel function of a mutant of the hepatitis B virus X protein (HBxΔ127) in the promotion of hepatoma cell migration.

Methods:

The effect of HBxΔ127 and wild type HBx on the migration ability of hepatoblastoma HepG2 cells were examined using wound healing assays in stable transfection systems. The full-length osteopontin(OPN) promoter sequence was cloned into the pGL3-Basic plasmid. The promoter activities of OPN in stably HBxΔ127-transfected hepatoblastoma HepG2 (HepG2-XΔ127) and hepatocellular carcinoma H7402 (H7402-XΔ127) cells were determined using luciferase reporter gene assays. The mRNA expression levels of OPN were detected by RT-PCR. And the effect of MK886, a specific inhibitor of 5-lipoxygenase (5-LOX), on OPN promoter activity and mRNA expression in HepG2-XΔ127 and H7402-XΔ127 cells were examined using luciferase reporter gene assays and RT-PCR, respectively. Finally, the migration ability of HepG2-XΔ127 was observed after treatment with siRNA targeting OPN mRNA and HBx mRNA using wound healing assays.

Results:

HepG2-XΔ127 cells exhibited a greater capacity for wound repair compared to HepG2-X cells. The promoter activity and mRNA expression levels of OPN were also increased in HepG2-XΔ127 and H7402-XΔ127 cells. Moreover, MK886 abolished the HBxΔ127-mediated upregulation of OPN. Wound healing assays demonstrated that the migration ability of HepG2-XΔ127 cells can be suppressed by treatment with siRNA targeting OPN mRNA and siRNA targeting HBx mRNA.

Conclusion:

HBxΔ127 strongly promotes hepatoma cell migration via activation of OPN involving 5-LOX.     

HBx蛋白激活肝细胞表达的FasL的生理效应

目的：观察HepG2细胞表达的FasL蛋白的生理效应，期望能进一步阐明乙型病毒性肝炎的分子发病机制。方法：(1)建立转染HBx基因的HepG2细胞系。(2)HtepC2x细胞表达的FasL生理效应的分析：采用细胞混合培养后，测定LDH释放、DNA Ladder及流式细胞仪方法检测细胞凋亡情况。结果：HepC2x细胞可表达FasL，HepC2x细胞与HepC2o、Jurkat细胞混合培养48小时后，HepG2o及Jurkat细胞特异性LDH释放率均等于零oDNAladder及流式细胞仪检测APO2.7抗体标记细胞结果表明，与HepC2x混合培养后，HepG2o、Jurkat细胞发生凋亡；增强HepG2o细胞表达Fas，细胞凋亡程度也增加。结论：HepC2x细胞表达的FasL具有致表达Fas的肝细胞或Jurkat细胞凋亡的作用。     

HBx Downregulates miR-422a Expression via Activation of FOXG1/Q1/E1 in HepG2 Cells

microRNA-422a (miR-422a) is downregulated in many hematopoietic tumors and solid tumors including hepatocellular carcinoma. We previously demonstrated that hepatitis B virus X protein (HBx) downregulated expression of miR-422a in HCC cell line HepG2 in vitro. However, we explore the mechanisms underlying this action. Forkhead box proteins (FOX) G1/Q1/E1 are known to negatively regulate miR-422a expression, and this prompted us to determine whether HBx suppresses miR-422a expression via activation of FOXG1/Q1/E1. The relationship between FOXG1/Q1/E1 and miR-422a in HepG2 cells stably expressing HBx was assessed with qRT-PCR. The correlation between HBx and FOXG1/Q1/E1 was determined with qRT-PCR and western blot in vitro. The cell cycle and CCK-8 assays were used to elucidate the consequence of miR-422a transfection in HepG2-hbx cells. FOXG1/Q1/E1 activated by HBx was found to be responsible, at least in part, for the downregulation of miR-422a in HepG2 cells. miR-422a transfection hampered the growth of HepG2-hbx cells by arresting cells in G1 phase. Both FOXG1/Q1/E1 and miR-422a may be suitable molecular targets for treatment of HBV-infected HCC.     

乙型肝炎患者血清抗-HBx抗体的检测及临床意义研究

目的 获得重组HBx蛋白，检测乙型肝炎患者血清抗-HBx抗体并探讨其临床意义。方法 利用分子克隆技术克隆HBVX基因构建PET32a-HBX原核表达载体，表达和纯化HBx蛋白，用该重组蛋白建立检测抗-HBx抗体的间接ELISA方法；采用该ELISA分别检测正常人组、急性肝炎组、慢性肝炎组、肝硬化组和肝细胞癌组患者血清中的抗-HBx抗体。结果 获得具有免疫原性的HBx融合蛋白；ELISA检测表明，慢性肝炎组、肝硬化组和肝细胞癌组的抗-HBx抗体的水平均高于急性肝炎组，差异具有显著性；在这三组之间，慢性肝炎组高于肝硬化组和肝细胞癌组，差异具有显著性，肝硬化组和肝细胞癌组的抗-HBx抗体水平无显著性差异。结论 HBV患者血清中抗-HBX抗体是乙型肝炎病毒感染的一种特异性抗体，是HBV感染的血清学指标之一，可以反映乙型肝炎肝炎患者病情的变化。     

Two oral HBx vaccines delivered by live attenuated Salmonella: both eliciting effective anti-tumor immunity

Live attenuated bacteria have great potential for use in vaccine development due to several unique advantages, including stable antigen expression, effective antigen presentation, convenient and inexpensive delivery, and low cost of vaccine production. In this study, we expressed hepatitis B virus x gene (HBx) on mouse melanoma cells as the target antigen and constructed Salmonella-based HBx vaccines by two strategies, i.e., recombinant eukaryotic plasmid encoding HBx and a recombinant prokaryotic plasmid encoding Type III secretion system effector-HBx fusion protein. Both HBx constructs elicited significant levels of CTL reaction and IFN-gamma secreting T cells. When mice were challenged with melanoma cells expressing HBx, tumor growth rates in immunized animals were significantly slower than controls. Tumor sizes and tumor weight indices of immunized mice were also significantly lower than controls. We conclude that both strategies described in this study may lead to novel approaches of tumor vaccines.     

HBx genotype D represses GSTP1 expression and increases the oxidative level and apoptosis in HepG2 cells

Epigenetics has been implicated in human cancer development. Epigenetic factors include HBx protein, which is able to induce hypermethylation and suppresses tumor suppressor genes. One of such tumor suppressor genes, GSTP1, shows reduced expression in many human cancers. Hypermethylation of GSTP1 is the most studied mechanism of its silence. In the present study, we reported that GSTP1 expression was completely depleted in HBV integrated HepG2.2.15 cells due to the hypermethylation in its promoter region. And it was HBx, especially HBx genotype D, that played the key role in repressing GSTP1 expression. Further functional studies like ROS assay and apoptosis detection were also used to confirm this repression. Our findings should facilitate the understanding of HBV and their influences on the epigenetic modulations for epigenetic tumorigenesis during HBV‐mediated hepatocellular carcinogenesis.