【作者】 刘顺芳；

【导师】 易继林；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 博士

【摘要】 第一部分DJ-1在肝细胞癌中的表达及其预后价值目的：DJ-1作为新近发现的癌基因参与多种恶性肿瘤的发病机制。本研究旨在探讨DJ-1在人肝细胞癌(Hepatocellular carcinoma, HCC)临床病理组织标本中的表达,及其与HCC临床病理变量和生存预后的关联性。方法：收集149例未经治疗的HCC组织标本,所有标本均具备完整的临床病理及随访预后资料。采用逆转录聚合酶链反应(Polymerase Chain Reaction, PCR)、免疫组织化学染色和Western Blot方法,检测DJ-1在HCC中的表达,统计分析DJ-1的表达与临床病理变量及生存预后的关联性。结果：与对应的癌旁组织、肝硬化组织和正常肝脏组织比较,DJ-1在HCC组织中显著高表达；Pearson卡方检验分析表明,DJ-1表达与术前AFP水平、肝硬化程度、门静脉侵犯与否、分化程度和Edmondson分期等临床病理变量密切相关；DJ-1高表达组患者的无瘤生存时间和总体生存时间明显短于DJ-1低表达组；多因素Cox比例风险模型分析显示,DJ-1为判断HCC患者总体生存时间的独立预后因素(HR,2.568；P=0.003)；相关性分析表明,HCC组织中DJ-1与抑癌基因PTEN表达显著性负相关(r=-0.836,P<0.001)。结论：DJ-1在HCC组织标本中高表达。DJ-1的表达水平与HCC临床病理资料及患者预后相关,说明DJ-1在HCC的发生、发展中起到重要作用。第二部分DJ-1在人肝癌细胞株的表达及其shRNA稳定转染HepG2细胞系的建立目的：检测DJ-1在人肝癌细胞株的表达,构建针对DJ-1的小发夹RNA (small hairpin RNA, shRNA)真核表达载体,获取干扰DJ-1表达的质粒,转染人肝癌HepG2细胞,稳筛后挑选亚克隆细胞系。方法：在不同的人肝癌细胞株中,应用Western Blot及免疫细胞化学法检测DJ-1的表达水平及细胞定位；设计干扰DJ-1表达的shRNA序列结构,构建RNA干扰(RNA interference, RNAi)真核表达载体pGenesil-1.1质粒,经过酶切、测序鉴定；干扰质粒经脂质体介导转染人肝癌细胞HepG2,经G418持续压力选择和有限稀释法获得稳定转染的细胞系；利用半定量RT-PCR及Western Blot分别检测筛选亚克隆细胞系的mRNA和蛋白表达。结果：DJ-1在人肝癌细胞株：HepG2、MHCC-97L、MHCC-97H、SMMC-7721及Huh-7中高表达,且不同肝癌细胞株中DJ-1表达存在差异(P<0.05),但在对照组人胚胎肝细胞L02中表达缺失。激光共聚焦显示,DJ-1表达主要定位于肝癌细胞胞质；合成并构建DJ-1 shRNA质粒载体,双酶切后电泳测序证实DJ-1干扰序列及读码框完全正确；成功筛选稳定转染干扰质粒的HepG2亚克隆细胞系,荧光显微镜下呈现绿色荧光,半定量RT-PCR检测显示干扰质粒明显抑制DJ-1 mRNA表达,Western Blot检测DJ-1蛋白抑制率为98.78%(P<0.05)。结论：DJ-1在人肝癌细胞株胞质高表达,且其表达程度与肝癌细胞恶性生物学特性相关；成功构建DJ-1 shRNA真核表达载体,建立干扰质粒稳定转染HepG2细胞系亚细胞克隆,为以DJ-1基因为靶点的人肝癌基因治疗的理论研究提供实验基础。第三部分shRNA抑制DJ-1表达在人肝癌细胞HepG2中调控Akt/NF-kappaB信号通路目的：研究癌基因DJ-1在人肝癌细胞株HepG2中对Akt/NF-KB信号通路的调控作用。方法：应用Western Blot方法在不同的人HCC细胞株,HepG2、SMMC-7721、Huh-7、Hep3B、MHCC-97L和MHCC-97H中检测癌基因DJ-1和抑癌基因PTEN的表达。采用基因沉默shRNA技术,在人HCC细胞HepG2中特异性稳定抑制DJ-1表达。离体及在体观察,DJ-1潜在作用靶点：Akt和磷酸化Akt、核因子κB (nuclear factor kappaB, NF-κB)及其抑制因子(inhibitor of NF-κB, IκB)的表达差异性。流式细胞检测法研究细胞周期转换、MTT比色法判断细胞增殖能力、黏附侵袭实验观察细胞黏附转移能力；建立HepG2细胞裸鼠皮下种植瘤模型,评估DJ-1对成瘤能力的影响。结果：DJ-1在不同转移潜能的人肝癌细胞株：HepG2、SMMC-7721、Huh-7、Hep3B、MHCC-97L和MHCC-97H中高表达,且不同肝癌细胞株中DJ-1表达存在差异(P<0.05); PTEN在人肝癌细胞株中低表达；成功构建DJ-1 shRNA真核表达载体,建立干扰质粒稳定转染HepG2细胞系亚细胞克隆；与对照组及HepG2组相比,干扰DJ-1表达组在裸鼠皮下种植瘤模型中肿瘤生长缓慢(P<0.05)；在HepG2细胞及裸鼠动物模型组织标本中,干扰DJ-1表达上调PTEN表达水平、抑制Akt磷酸化及NF-κB的激活；在HepG2细胞中干扰DJ-1表达,抑制细胞分化增殖、降低细胞黏附侵袭、阻滞细胞G1/S周期转换。结论：shRNA介导的RNAi干扰DJ-1的表达,抑制Akt磷酸化及NF-κB激活入核,上调PTEN表达水平；干扰DJ-1表达能够抑制HepG2细胞增殖、黏附及侵袭能力。DJ-1参与对Akt/NF-KB信号通路的调控,在HCC的肿瘤发生机制中发挥着重要作用。更多还原

【Abstract】 Increased DJ-1 and its prognostic significance in hepatocellular carcinomaObjective:To investigate the expression profile of DJ-1 gene and its clinical relevance and prognostic value in hepatocellular carcinoma (HCC).Methods:Specimens from 149 HCC patients were applied for DJ-1 expression through immunohistochemistry. The correlation of DJ-1 levels with clinicopathologic variables and prognosis was analyzed.32 paired HCC and para-carcinomatous liver tissue (PCLT) specimens from 149 HCC patients plus 10 hepatic cirrhosis specimens and 10 normal liver specimens were detected by reverse transcription quantitative polymerase chain reaction (RT-PCR) and Western Blot.Results:DJ-1 was up-regulated significantly in HCC by semi-quantitative/Real-Time PCR and Western blot. DJ-1 expression closely correlated with preoperative AFP, liver cirrhosis, vein invasion, differentiation and Edmondson grade in HCCs by Pearson Chi-square test. Both of tumor-free survival time and overall survival time in the DJ-1 high expression group were shorter than those in the low expression group. DJ-1 was adopted as an independent prognostic factor for overall survival of HCC patients through multivariate Cox proportional hazard model analysis (HR,2.568; P=0.003). Additionally, immunohistochemistry analysis revealed that expression of DJ-1 negatively correlated with expression of tumor suppressor gene phosphatase and tensin homolog deleted on chromosome ten (PTEN) in HCC (r=-0.836; P<0.001).Conclusion:DJ-1 expression is significantly up-regulated in HCC, and its expression level correlates with clinicopathological variables and prognosis of HCC patients, which suggests that DJ-1 maybe a candidate prognostic biomarker of HCC. Prognosis.The expression of oncogene DJ-1 in human hepatoma cell lines and DJ-1 small hairpin RNA stably transfected HepG2 cellsObjective:To detect the expression of oncogene DJ-1 in human hepatoma cell lines, construct the DJ-1 small hairpin RNA (short hair RNA, shRNA) eukaryotic expression vector, obtain RNA interference DJ-1 expression plasmid and the stably transfected HepG2 cell lines.Methods:Adopt Western Blot and immunocytochemistry for exploring the protein expression level and subcellular localization of oncogene DJ-1 in different human hepatoma cell lines, respectively. Design interference sequence structures targeted oncogene DJ-1, and inserted into RNA interference (RNAi) eukaryotic expression vector plasmid pGenesil-1.1, digested with restriction enzymes and DNA sequencing. The recombinant RNAi vectors were transfected into human hepatoma HepG2 cells with Lipofectamine 2000. The transfected cells were persistently screened under G418, and isolated with a limited dilution. The mRNA expression and protein expression of DJ-1 in the selected clones were evaluated by semi-RT PCR and Western Blot, respectively.Results:DJ-1 was highly expressed in human hepatoma cell lines, including HepG2, MHCC-97L, MHCC-97H, SMMC-7721 and Huh-7, in which the expressions have evidently differences (P<0.05). DJ-1 expression was mainly localized in the cytoplasm of human hepatoma cells detected by Confocal. However, the expression of DJ-1 was negative in human fetal liver cell line L02 (control). Synthesis and contruct recombinant DJ-1 shRNA plasmid vectors, and DNA sequencing indicated that RNAi eukaryotic expression vectors targeting DJ-1 with correct reading frame and nucleotide sequence. Green fluorescence of the stably transfected HepG2 cell lines were observed under the fluorescence microscope. semi-RT PCR revealed that DJ-1 shRNA effectively silenced mRNA expression level of DJ-1. Western Blot discovered that DJ-1 shRNA obviously suppressed protein expression level of DJ-1, of which the inhibition rate was 98.78% (P< 0.05).Conclusion:DJ-1 was highly expression in the cytoplasm of human hepatoma cell lines, of which the expression levels could correlate with the malignant biological characteristics in different cell lines. DJ-1 shRNA eukaryotic expression vectors were constructed successfully and the establishment of stably transfected HepG2 cell lines provided an original route for exploring the mechanism of DJ-1 in human hepatocellular carcinoma cells further.Role of DJ-1 down regulation by shRNA on the Akt/NF-kappaB signaling pathway in human hepatocellular carcinoma HepG2 cellsObjective:To explore the expression profile and potential downstream effectors of the oncogene DJ-1 in hepatocellular carcinoma (HCC) cell lines and its role in the Akt/nuclear factor (NF)-kappaB signaling pathway.Methods:Expressions of DJ-1 and PTEN proteins were analyzed by Western Blot in several human HCC cell lines, including HepG2, SMMC-7721, Huh-7, Hep3B, MHCC-97L and MHCC-97H. Knock-down of DJ-1 was achieved by transfecting DJ-1-specific small hairpin RNAs into HepG2 cells. Potential targets of DJ-1, including Akt, phosphorylated Akt, nuclear factor kappaB (NF-κB) and inhibitor of NF-κB (IκB), were observed in vitro and in vivo. Cell cycle, proliferation, adhesion and invasion were analyzed via flow cytometry, MTT assay, adhesion and invasion assays in vitro, respectively. Tumor growth was evaluated in vivo.Results:DJ-1 was profoundly up-regulated in different HCC cell lines with different hepatoma cell metastasis potential including HepG2, SMMC-7721, Huh-7, Hep3B, MHCC-97L and MHCC-97H, in which the expressions have evidently differences (P< 0.05). PTEN was constitutively low expression in HCC cell lines. A DJ-1 shRNA eukaryotic expression vector was successfully obtained and transfected stably into HepG2 cells. Tumor formation in the nude mouse model was successfully established, the tumor growth velocity of silencing DJ-1 expression was significantly slow (P<0.05). The down-regulation of DJ-1 in HepG2 cells increased PTEN expression, decreased phosphorylation of Akt and inhibited activation of NF-κB both in hepatoma cells (in vitro) and in nude mouse tumor samples (in vivo). Furthermore, suppression of DJ-1 expression resulted in decreased proliferation, adhesion and invasion of HepG2 cells in vitro, inhibited tumor formation in vivo and blocked the G1/S transition in the cell cycle.Conclusion:DJ-1 shRNA expression vector-mediated RNAi could downregulate DJ-1 expression, phosphorylation of Akt and activation of NF-κB, and upregulate PTEN expression in human HCC HepG2 cells. Meanwhile, DJ-1 downregulation suppressed the proliferation, adhesion and invasion of HepG2 cells. Our findings provide a clue that DJ-1 plays a crucial role in the oncogenesis of HCC and regulates the activation of the Akt/NF-κB signaling pathway.更多还原