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.     

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.     

转基因细胞模型L02/HBx的构建及HBx对细胞周期的影响

目的:构建L02/HBx转基因细胞模型并研究HBx对肝细胞周期的影响.方法:运用脂质体转染和G418筛选获得L02/ HBx阳性克隆,并分别用RT-PCR和Western blot鉴定HBx mRNA与蛋白的表达.进一步用四唑蓝(MTT)比色试验、流式细胞仪检测L02/HBx的增殖、凋亡和细胞周期.结果:RT-PCR和Western blot分别检测到L02/ HBx细胞中HBx mRNA和蛋白的表达.MTT比色试验显示L02/HBx生长速度加快,流式细胞仪检测发现L02/HBx凋亡率低(0.09%±0.13% vs 3.74%±1.29%,P<0.05),G1期细胞比例减少(61.35%±0.82% vs 67.80±6.84%,P<0.05),S期细胞比例相应增加(36.59%±2.54% vs 22.37%±2.17%,P<0.05).经阿霉素(ADM)培养后,L02/HBx的凋亡率显著增加(34.91%±5.85% vs 0.09%±0.13%,P<0.05),G1期细胞比例明显增加但低于对照组(82.81%±6.48% vs 61.35%±0.82%,P<0.05;82.81%±6.48% vs 87.19%±1.92%,P<0.05),S期细胞比例降低但较对照组高(13.84%±6.16% vs 36.59%±2.54%,P<0.05;13.84%±6.16% vs 2.22%±1.26%,P<0.05).结论:L02/HBx构建成功,HBx能促进细胞周期进程,加快细胞的生长并抑制细胞的凋亡;转染HBx基因的肝细胞凋亡更易受凋亡因子所触发,表明HBx可能会增加正常肝细胞对诱导凋亡因素的敏感性.     

HBx在乙型肝炎病毒相关性肝癌形成与发展中的分子机理

目的 了解HBx基因在乙肝病毒相关性肝癌形成与发展中的分子机理。方法 综合国外近5年的文献进行分析。结果 HBx具有促进细胞恶性转化、抑制受损DNA的修复、反式激活、抑制wtp53功能和抑制细胞凋亡等生物学功能。HBx可能通过直接致癌作用、抑制DNA的修复、抑制wtp53、干扰Fas/CD95系统和抑制Caspase-3活性等分子机理,诱发肝癌形成和促进肝癌发展。结论 HBx及其编码的蛋白HBxAg具有广泛的生物学功能,从多方面、多途径促进肝癌的形成和发展。     

乙型肝炎病毒x蛋白对肝细胞FasL表达的激活作用

目的探讨ＨＢｘ蛋白对肝细胞Ｆａｓ配基（ＦａｓＬ）表达的调控作用。方法构建ＨＢｘ基因的真核表达载体ｐＣＥＰ４ｘ,电击法转染ＨｅｐＧ２细胞,潮霉素筛选阳性克隆细胞ＨｅＰＧ２Ｘ。用免疫细胞化学染色和免疫蛋白印迹法检测转染ＨＢｘ基因的ＨｅｎＧ２ｘ细胞ＦａｓＬ蛋白的表达。结果逆转录ＰＣＲ祛分析表明,ＨＢｘ基因在转染细胞内可有效转录;ＨｅｐＧ２ｘ细胞出现新的ＦａｓＬ蛋白表达。结论乙肝病毒ｘ蛋白可激活肝细胞表达ＦａｓＬ。     

迷迭香酸干预下HBX基因对人肝癌细胞凋亡的影响

目的研究迷迭香酸(Rosmarinic acid)对人肝癌HepG2细胞及稳定转染HBx基因的HepG2细胞(HepG2-X)增殖、侵袭力的影响,并探讨其可能的机制。方法体外培养HepG2及HepG2-X细胞,加入不同浓度用迷迭香酸处理后,用MTT实验检测细胞增殖抑制率,用细胞划痕和Transwell小室法测定对细胞迁移力的影响。结果不同浓度迷迭香酸均能够抑制HepG2、HepG2-X细胞的增殖,且呈时间和剂量依赖性,各HepG2-X组细胞A值均明显高于HepG2细胞(P<0.05),显示HBX对HepG2细胞具有促进增殖作用。与对照组相比,实验组细胞划痕愈合减缓(P<0.05),Transwell穿膜细胞数明显减少(P<0.05)。且呈浓度依赖性。结论迷迭香酸能够抑制肝癌HepG2细胞的增殖、迁移及侵袭力,HBx在迷迭香酸的干预下能促进HepG2细胞凋亡的作用。