慢性乙型肝炎患者肝细胞中调控HBV复制基因的筛选及作用机制研究
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摘要
第一部分慢性乙型肝炎患者宿主肝细胞中调控HBV复制基因的筛选。方法：入选慢性HBV感染患者83例,其中免疫耐受期22例、免疫清除期(HBeAg P日性慢性乙型肝炎)25例、免疫激活期(HBeAg阴性慢性乙型肝炎)25例、非活动免疫控制期11例,并入选6例健康受试者；慢性乙型肝炎诊断标准符合中华医学会感染病学分会及肝病学分会2005年制定的《慢性乙型肝炎防治指南》中的诊断标准。超声引导下经皮肝穿刺活检术获取肝组织,采用Affymetrix RNA芯片,分析不同临床阶段慢性HBV感染患者肝组织的基因表达谱；为了降低免疫因素的影响,我们主要观察免疫耐受期和免疫控制期的不同慢性HBV感染状态下肝组织基因表达差异。结果：与免疫耐受期患者比较,非活动免疫控制期患者肝组织有109个基因的表达存在显著统计学差异(p<0.01),其中54个基因的表达明显上调,55个基因的表达明显下调。结论：慢性HBV感染各临床阶段肝组织基因表达存在差异性。其中,非活动免疫控制期与免疫耐受期相比,有109个基因的表达存在显著差异。
第二部分在不同肝癌细胞系体外模型中利用小RNA干扰技术沉默特异性基因的表达,用southern blot和ELISA验证差异基因沉默后对HBV复制的影响。方法：在稳定表达HBV的肝癌细胞系HepG2.2.15中,分别转染针对109个基因的特异性小干扰RNA后,用Southern blot检测胞内HBV复制中间体、CMIA法检测上清液中HBsAg及HBeAg抗原分泌的水平；同时将具有复制能力的HBV感染性克隆和siRNA瞬时共转染Huh7肝癌细胞系,同法观察这些基因在瞬转模型中对HBV复制的调控作用。结果：沉默部分差异基因的表达后,HepG2.2.15或Huh7瞬转模型中HBV的复制水平出现明显上调或下调。结论：稳定转染和瞬转HBV感染细胞模型证实前述差异表达基因可影响HBV复制,其中大部分宿主肝细胞基因与HBV复制的关系乃首次发现。
第三部分进一步在体外验证前述研究发现的对HBV复制影响显著的4个代表性基因(FAM176A, HIGD1A, MARVELD3和TOMM34)对HBV复制的调控效应和可能机制。方法：分析慢性HBV患者肝组织内FAM176A, HIGD1A, MARVELD3和TOMM34的表达水平与患者血清中HBV DNA载量的相关性；在稳定表达HBV的肝癌细胞系HepG2.2.15中转染不同浓度梯度的小干扰RNA后,用Southern blot检测HBV复制中间体水平、CMIA法检测细胞培养上清HBsAg和HBeAg,用Western blot检测胞内核心抗原的水平和核衣壳形成的变化以了解是否在转录后水平影响HBV的复制。在Huh7细胞系中共转染FAM176A, HIGD1A, MARVELD3和TOMM34表达质粒和可复制的HBV克隆,同样应用上述方法检测过表达该基因后对HBV的复制以及病毒蛋白表达的影响。观察过表达质粒和HBV克隆共转染后对HBV复制影响的剂量依耐性。luciferase荧光素酶活性检测来观察4个基因和HBV的启动子的直接相互作用。结果：FAM176A, HIGD1A和TOMM34的肝内表达水平与病人血清中HBV DNA水平呈负相关,而MARVELD3的肝内表达与患者血清中HBV DNA水平呈正相关。在HepG2.2.15细胞系中转染针对FAM176A, HIGD1A, MARVELD3和TOMM34的特异性siRNA可以促进HBV的复制和抗原HB-sAg及HBeAg的表达；而在Huh7瞬转模型中过表达这些基因可明显抑制HBV复制,并呈明显的剂量依赖性；沉默MARVELD3的表达能促进HBV核心启动子的活性,而过表达FAM176A能显著抑制HBV核心抗原水平和核衣壳的形成。结论：宿主肝细胞基因FAM176A, HIGD1A, MARVELD3和TOMM34可能具有调控HBV复制的功能,其中MARVELD3抑制HBV的复制可能与其对核心启动子直接作用有关。这些结果有助于加深对HBV复制调控分子机制的认识和新的基因治疗方案的开发。
Part Ⅰ Objective The objective of the current study was to identify the differential intrahepatic host genes influence on the regulating of HBV replication in the liver of chronically HBV-infected (CHB) patients. Methods:Affymetrix RNA chip analysis was used to analyse the gene expression profiles of different clinical stages of chronic HBV infection in liver tissue.83CHB patients were included in the present study and divided into5groups:immune tolerant phase (n=22), immune clearance phase (HBeAg-positive chronic hepatitis B)(n=25), immune activation (HBeAg negative chronic hepatitis B)(n=25), inactive HBsAg carrier (n=11) and healthy control(n=6). Diagnostic criteria in accordance with the Chinese Medical Association in2005about "Chinese hepatitis B prevention and treatment guidelines in the diagnosis chronic HBV infection". In order to exclude the influence of immune factors, we mainly concentrate on the gene expression profiles between immune tolerant group and inactive carrier group. Results:There were statistically109differentially expressed genes in inactive carrier group compared with the immune tolerant phase (p<0.01); among them,54genes significantly up-regulated and55genes were down-regulated respectively. Conclusion:109cellular genes expression might correlate with HBV replication and reflect the immune response to HBV infection. The analysis of different clinical stages of CHB with liver RNA chip results showed that the expression of109genes were significantly different.
Part Ⅱ The differentially expressed genes effect on HBV replication and gene expression were validated in vitro in hepatoma cell lines by specific siRNAs knock down. Methods:HepG2.2.15in hepatoma cell lines stably expressing HBV were transfected with109gene-specific small interfering RNA, and southern blot detect the intracellular HBV replication intermediates, the CMIA assay detect the HBV antigen secretion in the supernatant of cells. Moreover, Huh7hepatoma cell line were transiently contransfected with specific siRNA and replication competent HBV clone, and southern blot test the HBV relication, CMIA assay detect the HBsAg and HBeAg antigen secretion. Results:HBV replication in HepG2.2.15or Huh7were significantly enhanced or inhibited after silencing some of these genes. Conclusion:Knock down some of these109genes with specific small interfering RNA could influence the HBV replication markedly in hepatoma cell lines in vitro. More importantly, some of selected genes effect on HBV replication were identified for the first time.
Part Ⅲ We selected4genes named FAM176A, HIGD1A, MARVELD3and TOMM34which showed strong effect on the HBV replication for further study and verified their effect on the HBV replication or antigen secretion and the possible mechanism on the control of HBV replication in details. Methods:The siRNA targeting the FAM176A, HIGD1A, MARVELD3and TOMM34were transfected to HepG2.2.15cells. Additionally, over-expression of these genes with plasmids cotranfected with HBV replication competent clone in Huh7cells. Intracellular HBV replication intermediates were detected by southern blot. The expression of HBsAg and HBeAg antigens were detected by CMIA test. Intracellular HBcAg protein expression after siRNA transfection was detected by western blot. Knock down of these genes effect on HBV promoter activities were evaluated by luciferse reporter assay. Results: The intrahepatic FAM176A、 HIGD1A and TOMM34level in CHB patients had a negative correlation with the patients HBV DNA titers, but the MARVELD3level in the liver was positive correlated with HBV DNA titers. It was noted that these genes knocking down resulted in a marked increase of HBV replication, accompanied with up-regulated antigen expression, and progeny secretion in HepG2.2.15cells. Consistently, Overexpression of these genes could significantly inhibit the HBV replication or antigen secretion in HBV plasmid co-transfected Huh7cells. Knocking down of MARVELD3by specific siRNA could enhance the HBV core and X promoter activity in HepG2.2.15hepatoma cells. Conclusion:We found that the4genes intrahepatic expression level in CHB patients had a significant correlation with the serum HBV DNA titers. Our results demonstrated that these differentially expressed intrahepatic genes might control HBV replication and their expression level might reflect the different outcomes of HBV infection. It will be useful for studying host-virus interactions in the pathogenesis of HBV infection and providing novel perspectives on development of therapeutic strategies in counteracting chronic HBV infection.
第二部分在不同肝癌细胞系体外模型中利用小RNA干扰技术沉默特异性基因的表达,用southern blot和ELISA验证差异基因沉默后对HBV复制的影响。方法：在稳定表达HBV的肝癌细胞系HepG2.2.15中,分别转染针对109个基因的特异性小干扰RNA后,用Southern blot检测胞内HBV复制中间体、CMIA法检测上清液中HBsAg及HBeAg抗原分泌的水平；同时将具有复制能力的HBV感染性克隆和siRNA瞬时共转染Huh7肝癌细胞系,同法观察这些基因在瞬转模型中对HBV复制的调控作用。结果：沉默部分差异基因的表达后,HepG2.2.15或Huh7瞬转模型中HBV的复制水平出现明显上调或下调。结论：稳定转染和瞬转HBV感染细胞模型证实前述差异表达基因可影响HBV复制,其中大部分宿主肝细胞基因与HBV复制的关系乃首次发现。
第三部分进一步在体外验证前述研究发现的对HBV复制影响显著的4个代表性基因(FAM176A, HIGD1A, MARVELD3和TOMM34)对HBV复制的调控效应和可能机制。方法：分析慢性HBV患者肝组织内FAM176A, HIGD1A, MARVELD3和TOMM34的表达水平与患者血清中HBV DNA载量的相关性；在稳定表达HBV的肝癌细胞系HepG2.2.15中转染不同浓度梯度的小干扰RNA后,用Southern blot检测HBV复制中间体水平、CMIA法检测细胞培养上清HBsAg和HBeAg,用Western blot检测胞内核心抗原的水平和核衣壳形成的变化以了解是否在转录后水平影响HBV的复制。在Huh7细胞系中共转染FAM176A, HIGD1A, MARVELD3和TOMM34表达质粒和可复制的HBV克隆,同样应用上述方法检测过表达该基因后对HBV的复制以及病毒蛋白表达的影响。观察过表达质粒和HBV克隆共转染后对HBV复制影响的剂量依耐性。luciferase荧光素酶活性检测来观察4个基因和HBV的启动子的直接相互作用。结果：FAM176A, HIGD1A和TOMM34的肝内表达水平与病人血清中HBV DNA水平呈负相关,而MARVELD3的肝内表达与患者血清中HBV DNA水平呈正相关。在HepG2.2.15细胞系中转染针对FAM176A, HIGD1A, MARVELD3和TOMM34的特异性siRNA可以促进HBV的复制和抗原HB-sAg及HBeAg的表达；而在Huh7瞬转模型中过表达这些基因可明显抑制HBV复制,并呈明显的剂量依赖性；沉默MARVELD3的表达能促进HBV核心启动子的活性,而过表达FAM176A能显著抑制HBV核心抗原水平和核衣壳的形成。结论：宿主肝细胞基因FAM176A, HIGD1A, MARVELD3和TOMM34可能具有调控HBV复制的功能,其中MARVELD3抑制HBV的复制可能与其对核心启动子直接作用有关。这些结果有助于加深对HBV复制调控分子机制的认识和新的基因治疗方案的开发。
Part Ⅰ Objective The objective of the current study was to identify the differential intrahepatic host genes influence on the regulating of HBV replication in the liver of chronically HBV-infected (CHB) patients. Methods:Affymetrix RNA chip analysis was used to analyse the gene expression profiles of different clinical stages of chronic HBV infection in liver tissue.83CHB patients were included in the present study and divided into5groups:immune tolerant phase (n=22), immune clearance phase (HBeAg-positive chronic hepatitis B)(n=25), immune activation (HBeAg negative chronic hepatitis B)(n=25), inactive HBsAg carrier (n=11) and healthy control(n=6). Diagnostic criteria in accordance with the Chinese Medical Association in2005about "Chinese hepatitis B prevention and treatment guidelines in the diagnosis chronic HBV infection". In order to exclude the influence of immune factors, we mainly concentrate on the gene expression profiles between immune tolerant group and inactive carrier group. Results:There were statistically109differentially expressed genes in inactive carrier group compared with the immune tolerant phase (p<0.01); among them,54genes significantly up-regulated and55genes were down-regulated respectively. Conclusion:109cellular genes expression might correlate with HBV replication and reflect the immune response to HBV infection. The analysis of different clinical stages of CHB with liver RNA chip results showed that the expression of109genes were significantly different.
Part Ⅱ The differentially expressed genes effect on HBV replication and gene expression were validated in vitro in hepatoma cell lines by specific siRNAs knock down. Methods:HepG2.2.15in hepatoma cell lines stably expressing HBV were transfected with109gene-specific small interfering RNA, and southern blot detect the intracellular HBV replication intermediates, the CMIA assay detect the HBV antigen secretion in the supernatant of cells. Moreover, Huh7hepatoma cell line were transiently contransfected with specific siRNA and replication competent HBV clone, and southern blot test the HBV relication, CMIA assay detect the HBsAg and HBeAg antigen secretion. Results:HBV replication in HepG2.2.15or Huh7were significantly enhanced or inhibited after silencing some of these genes. Conclusion:Knock down some of these109genes with specific small interfering RNA could influence the HBV replication markedly in hepatoma cell lines in vitro. More importantly, some of selected genes effect on HBV replication were identified for the first time.
Part Ⅲ We selected4genes named FAM176A, HIGD1A, MARVELD3and TOMM34which showed strong effect on the HBV replication for further study and verified their effect on the HBV replication or antigen secretion and the possible mechanism on the control of HBV replication in details. Methods:The siRNA targeting the FAM176A, HIGD1A, MARVELD3and TOMM34were transfected to HepG2.2.15cells. Additionally, over-expression of these genes with plasmids cotranfected with HBV replication competent clone in Huh7cells. Intracellular HBV replication intermediates were detected by southern blot. The expression of HBsAg and HBeAg antigens were detected by CMIA test. Intracellular HBcAg protein expression after siRNA transfection was detected by western blot. Knock down of these genes effect on HBV promoter activities were evaluated by luciferse reporter assay. Results: The intrahepatic FAM176A、 HIGD1A and TOMM34level in CHB patients had a negative correlation with the patients HBV DNA titers, but the MARVELD3level in the liver was positive correlated with HBV DNA titers. It was noted that these genes knocking down resulted in a marked increase of HBV replication, accompanied with up-regulated antigen expression, and progeny secretion in HepG2.2.15cells. Consistently, Overexpression of these genes could significantly inhibit the HBV replication or antigen secretion in HBV plasmid co-transfected Huh7cells. Knocking down of MARVELD3by specific siRNA could enhance the HBV core and X promoter activity in HepG2.2.15hepatoma cells. Conclusion:We found that the4genes intrahepatic expression level in CHB patients had a significant correlation with the serum HBV DNA titers. Our results demonstrated that these differentially expressed intrahepatic genes might control HBV replication and their expression level might reflect the different outcomes of HBV infection. It will be useful for studying host-virus interactions in the pathogenesis of HBV infection and providing novel perspectives on development of therapeutic strategies in counteracting chronic HBV infection.
引文
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1. McMahon, B.J., The natural history of chronic hepatitis B virus infection. HEPATOLOGY,2009.49(5 Suppl):p. S45-55.
2. Fung, J., et al., Profiles of HBV DNA in a large population of Chinese patients with chronic hepatitis B:implications for antiviral therapy. Journal of Hepatology,2011.54(2):p.195-200.
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5. Bertoletti, A. and C. Ferrari, Innate and adaptive immune responses in chronic hepatitis B virus infections:towards restoration of immune control of viral infection. Gut,2011.
6. Nguyen, D.H. and J. Hu, Reverse transcriptase-and RNA packaging signal-dependent incorporation of APOBEC3G into hepatitis B virus nucleocapsids. Journal of Virology,2008.82(14):p.6852-61.
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2. Fung, J., et al., Profiles of HBV DNA in a large population of Chinese patients with chronic hepatitis B:implications for antiviral therapy. Journal of Hepatology,2011.54(2):p.195-200.
3. Vassilopoulos, D., et al., Cellular immune responses in hepatitis B virus e antigen negative chronic hepatitis B. Journal of Viral Hepatitis,2008.15(11):p. 817-26.
4. Tran, T.T., Immune tolerant hepatitis B:a clinical dilemma. Gastroenterol Hepatol (N Y),2011.7(8):p.511-6.
5. Bertoletti, A. and C. Ferrari, Innate and adaptive immune responses in chronic hepatitis B virus infections:towards restoration of immune control of viral infection. Gut,2011.
6. Nguyen, D.H. and J. Hu, Reverse transcriptase-and RNA packaging signal-dependent incorporation of APOBEC3G into hepatitis B virus nucleocapsids. Journal of Virology,2008.82(14):p.6852-61.
7. Li, J., et al., Subversion of cellular autophagy machinery by hepatitis B virus for viral envelopment. Journal of Virology,2011.85(13):p.6319-33.
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15. Sir, D., D.K. Ann, and J.H. Ou, Autophagy by hepatitis B virus and for hepatitis B virus. Autophagy,2010.6(4).
16. Sir, D., et al., The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication. Proceedings of the National Academy of Sciences of the United States of America,2010.107(9):p.4383-8.
17. Tian, Y, et al., Autophagy required for hepatitis B virus replication in transgenic mice. Journal of Virology,2011.85(24):p.13453-6.
18. Nuttall, S.D., et al., hTom34:a novel translocase for the import of proteins into human mitochondria. DNA and Cell Biology,1997.16(9):p.1067-74.
19. Shimokawa, T., et al., Identification of TOMM34, which shows elevated expression in the majority of human colon cancers, as a novel drug target. International Journal of Oncology,2006.29(2):p.381-6.
20. Chewawiwat, N., et al., Characterization of the novel mitochondrial protein import component, Tom34, in mammalian cells. J Biochem,1999.125(4):p. 721-7.
21. Yang, C.S. and H. Weiner, Yeast two-hybrid screening identifies binding partners of human Tom34 that have ATPase activity and form a complex with Tom34 in the cytosol. Archives of Biochemistry and Biophysics,2002.400(1): p.105-10.
22. Blesa, J.R., et al., NRF-1 is the major transcription factor regulating the expression of the human TOMM34 gene. Biochemistry and Cell Biology,2008. 86(1):p.46-56.
23. Raleigh, D.R., et al., Tight junction-associated MARVEL proteins marveld3, tricellulin, and occludin have distinct but overlapping functions. Molecular Biology of the Cell,2010.21(7):p.1200-13.
24. Steed, E., et al., Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family. BMC Cell Biol,2009.10:p.95.
25. Kojima, T., et al., Downregulation of tight junction-associated MARVEL protein marvelD3 during epithelial-mesenchymal transition in human pancreatic cancer cells. Experimental Cell Research,2011.317(16):p.2288-98.