【作者】 胡志亮；

【导师】 谭德明；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 目的：原发性肝细胞癌(Hepatocellular carcinoma, HCC)是来源于肝细胞的恶性肿瘤,是世界上最常见的恶性肿瘤之一。流行病学研究表明慢性乙型肝炎病毒感染和原发性肝癌的发生发展密切相关,几乎所有HBV (hepatitis B virus)相关的肝癌组织中可以检测到基因组整合的HBV DNA。在众多病毒基因组整合亚单位中HBx(Hepatitis B virus x gene)基因可以转录并翻译成相应HBx蛋白,其与肝癌的关系被广泛研究。我们前期研究发现在肝癌组织的基因组中广泛存在HBx基因的整合和突变,并筛选出2个缺失突变体HBx-d382(HBx基因nt382-400缺失突变)和HBx-d431,其中HBx-d382在肝癌组织中检出率较高,我们猜想HBx-d382可能与肝细胞癌密切相关,随后我们成功构建了HBx-d382真核表达载体pcDNA3.0/HBx-d382。本研究将建立稳定表达HBx基因缺失突变体(HBx-d382)的L02肝细胞株L02/HBx-d382,探讨HBx-d382对L02细胞增殖,G1期细胞周期调控及microRNA表达谱的影响。方法：含HBx-d382的重组质粒(pcDNA3.0/HBx-d382)经过PCR(Polymerase chain reaction)扩增、双酶切及DNA测序鉴定后,通过脂质体转染和G418筛选获得稳定表达HBx-d382的L02肝细胞株,PCR鉴定L02细胞基因组中HBx-d382基因整合,RT-PCR(Reverse transcription polymerase chain reaction)和western blot鉴定其表达。通过MTT法,软琼脂克隆形成实验检测HBx-d382对L02细胞增殖及非锚定依赖生长能力的影响,用流式细胞仪检测其对细胞周期的影响；通过real-time定量PCR及western-blot鉴定转染HBx-d382后L02细胞cyclin D1,cylinG1,E2F1表达改变；通过microRNA芯片技术检测HBx-d382对L02细胞miRNA (microRNA)表达谱的影响。结果：(1)经PCR、酶切及测序鉴定HBx-d382重组质粒构建正确。(2)稳定转染该质粒的L02细胞基因组存在HBx-d382整合,RT-PCR及Western-blot表明在RNA水平和蛋白水平存在HBx-d382表达。(3) MTT及软琼脂克隆形成实验结果表明稳定表达HBx-d382的L02细胞其增殖能力和非锚定生长能力增强,细胞周期检测证实转染HBx-d382的L02细胞S+G2期细胞比例升高。(4)real-time定量PCR结果表明与转染空质粒pcDNA3.0的L02细胞(L02/pcDNA3.0)相比L02/HBx-d382细胞cyclin D1, cylin G1, E2F1的mRNA表达明显增强(P<0.05);western-blot结果证实与102/pcDNA3.0细胞相比L02/HBx-d382细胞cyclin D1, cylin G1, E2F1蛋白表达明显增强(p<0.05) (5)microRNA芯片发现与L02/pcDNA3.0组细胞相比L02/HBx-d382组细胞mir-1,mir-338-3p, mir-551b, miR-455-3p, miR-200c表达下调,miR-501,miR-595, miR-1307, miR-1180, miR-497, miR-1246, miR-623表达上调。结论：本研究成功的构建了HBx基因缺失突变体(HBx-d382)的真核表达模型,证实HBx-d382能增强L02细胞增殖及非锚定依赖生长能力。HBx-d382促使L02细胞打破G1期阻滞,进入S期,这种效应可能与其增强G1调控相关基因如cyclin D1, cylin G1, E2F1表达有关。HBx基因缺失突变体(HBx-d382)能影响L02肝细胞microRNA的表达谱,这为阐明HBx-d382致肝细胞恶性转化提供新的分子生物学机制。更多还原

【Abstract】 Objective:Hepatocellular carcinoma (HCC) is a malignant tumor arising from hepatocytes; it is one of the most common malignancies worldwide. Epidemiological studies have showed that there is a strong relationship between the chronic hepatitis B virus infection and development of hepatocellular carcinoma, almost all of the HBV-associated HCCs harbor chromosomally integrated HBV DNA. In most integrated subviral HBV genomes, the HBx gene can be transcribed and translated into HBx protein, its significance in the hepatocarcinogenesis is be widely studied. our previous study has found that HBx gene integration and mutation occur frequently in HCC samples, and isolated two HBx deletion mutations naming HBx-d431 and HBx-d382(HBx gene nt 382-400 deletion),among which the HBx-d382 occurs more frequently in HCC samples, we guess it may be implicated in liver carcinogenesis, and then we have successfully constructed the eukaryotic expression vector for the expression of HBx-d382.In this study, we will establish a L02 cell line stably expressing HBx deletion mutation (HBx-d382) and further study the influence of HBx-d382 on cell proliferation,G1 cell cycle control and microRNA expression profile of L02 cells.Methods:The recombinant plasmid encoding the HBx deletion mutation (pcDNA3.0/HBx-d382) was tested by PCR amplification、double digestion and DNA sequencing, and then introduced into L02 cells by liposome transfection. Positive clones were selected by G418. The genome integration of the HBx gene was test by PCR amplification, and the HBx expression was confirmed by reverse transcription PCR (RT-PCR) and western blot. Cell proliferation changes were measured by MTT assay and soft agar clone formation assay, and the cell cycles were tested by flow cytometry; the mRNA and protein expression of cyclin D1, cylin G1, E2F1 were detected by Real-time Quantitative PCR and Western blot respectively; microRNA expression profiles were analyzed by microRNA microarray.Results:(1)The recombinant plasmid pcDNA3.0/HBx-d382 was successfully identified by PCR amplification、double digestion and DNA sequencing. (2)Positive clone that selected by G418 harbored chromosomally integrated HBx-d382; RT-PCR and Western blot confirmed the HBx expression at the mRNA and protein lever respectively.(3)This cell line showed enhanced proliferation and anchorage-independent growth ability when tested by MTT assay and soft agar clone formation assay; and the proportion of S and G2 phrase cells was increased simultaneously. (4)Real-time Quantitative PCR showed that the mRNA expression of cyclin D1, cylin G1, E2F1 were increased in L02/HBx-d382 cells compared with L02 cells transfected with the pcDNA3 empty plasmid(102/pcDNA3.0) (p<0.05); Western blot confirmed that the protein expression of cyclin D1, cylin G1, E2F1 were increased in L02/HBx-d382 cells compared with L02/pcDNA3.0 cells.(5) Based on the microRNA microarray, mir-1, mir-338-3p, mir-551b, miR-455-3p, and miR-200c were down-regulated, miR-501, miR-595, miR-1307, miR-1180, miR-497, miR-1246 and miR-623 were up-regulated in L02/HBx-d382 cells compared with the L02/ pcDNA3.0 cells.Conclusion:L02 cell line stably expressing HBx deletion mutation (HBx-d382) is established successfully. HBx-deletion mutation(HBx-d382) can enhance the proliferation and anchorage-independent growth ability of L02 cells, and promotes cell cycle progression from G1 to the S phase, which may due to the increase gene expression of cyclin D1, cylin G1, E2F1 that are associated with G1 cell cycle control. This virus gene can also influence the microRNA expression profile in the 102 cells, which may be a new molecular biological mechanism of HBx-d382 induced hepatocytes malignant transformation.更多还原