宿主肝细胞膜蛋白基因HIGD1A对乙型肝炎病毒复制的抑制作用及其机制的研究

The immune tolerance and inactive carrier CHB patients have one million times difference in viral load.The current study did not find specific immune response obviously different, suggesting that there are other possible mechanism on control of HBV replication such as host genes.In our previous study,we analyzed 83 chronic hepatitis B infected patients with different infected status by liver tissue transcriptome microarray analysis to indentify the possible host gene regulating the HBV replication. We observed that the expression level of HIGD1A in inactive carrier patients was significantly upregulated than in immune tolerance patients, with the patient serum HBV DNA levels were negatively correlated. Moreover,Silence of HIGD1A with small interfering RNA can increase viral replication in vitro cell line model. Thus, we speculated that HIGD1A can inhibit HBV replication. It may play a role by the following ways: 1) directly interact with HBV promoters or regulate the HBV-associated liver-specific transcription factor activity.2)The possible basic function of this gene associated with stress granule. This project intends to verify the HIGD1A inhibition of HBV replication in vitro and in vivo models by small interfering RNA, Southern blot, luciferase and other experimental skills and possible mechanisms of these gene's regulation on the HBV replication. This study will help to clarify the new intrahepatic host gene influence on HBV replication.

慢性HBV感染免疫耐受期和控制期患者的病毒载量差别高达百万倍，但目前的研究并未发现两者的特异性免疫存在明显的不同,提示存在其他可能控制HBV复制的宿主分子。为了寻找相关宿主基因，我们课题组采用芯片技术分析了83例不同临床阶段慢性HBV感染患者肝组织表达谱，发现宿主肝细胞基因HIGD1A的表达水平在免疫控制组明显高于免疫耐受组，且与患者血清中HBV DNA水平呈负相关。体外实验发现沉默HIGD1A后可上调病毒复制，我们推测，HIGD1A能抑制HBV复制,可能通过以下环节发挥作用：1）直接影响HBV转录（包括HBV RNA、启动子活性、特异性转录因子）和转录后水平（病毒蛋白、核衣壳）2）可能与应激颗粒形成有关。本课题拟采用小干扰RNA、核酸杂交、荧光素酶等方法在HBV复制体外和体内模型中初步阐明HIGD1A对HBV复制的抑制作用及可能的作用机制。本研究有助于阐明影响HBV复制的新的宿主分子。

慢乙肝患者感染肝细胞内乙肝病毒（HBV）的复制受到宿主基因及其相关的胞内信号通路的控制。通过对慢乙肝免疫耐受期和非活动性携带者肝组织进行转录组基因芯片筛选，我们发现缺氧诱导因子家族成员蛋白1A（HIGD1A）在非活动性携带者中高表达，可能参与患者肝内控制HBV感染。本研究探讨了HIGD1A影响HBV复制的效应及其机制。结果显示，沉默肝癌细胞系HepG2.2.15细胞内HIGD1A的表达可以导致HBV复制、转录、抗原表达的上调。反之，Huh7细胞中过表达HIGD1A显著抑制HBV复制。此外，HIGD1A过表达可以显著抑制HBV高压尾静脉注射小鼠模型肝内和血清中HBV DNA和抗原的表达。利用RNA-seq分析发现，HIGD1A通过调节转录因子NR2F1的表达从而抑制HBV核心启动子活性，从而在转录水平影响HBV复制。最后，HIGD1A在肝癌组织中的表达显著高于癌旁组织，其过表达可以促进细胞增殖、细胞周期转化、自噬小体形成。总结来说，我们的结果发现HIGD1A通过影响NR2F1的表达及细胞表型，从而在转录水平抑制HBV复制和基因表达。此外，HIGD1A还有可能参与乙肝相关肝癌的发生发展。