紧密连接分子Symplekin 在肝脏疾病中表达及调控机制的研究
文献类型：学位论文
| 作者 | 易濒 |
| 学位类别 | 硕士 |
| 答辩日期 | 2009-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 曹毅 |
| 关键词 | 肝细胞癌 Symplekin 表达 调控机制 |
| 中文摘要 | 肝细胞癌是世界上多发的肿瘤之一，在中国及东南亚地区尤为多见，其死亡率高且预后差。肝癌具有多种发病原因且伴有多种肿瘤相关基因的分子突变。细胞连接分子（紧密连接、粘着连接、桥粒）在维护细胞极性及上皮细胞屏障方面起着重要作用，其表达异常与恶性肿瘤发生、发展有很大相关性。Symplekin 是新近发现的紧密连接相关分子，紧密连接分子 Symplekin 是多定位与多功能的蛋白，除参与上皮细胞紧密连接的形成外，Symplekin 还参与RNA 3’端腺苷酸化的过程，并且具有调节细胞增殖的作用。我们前期工作发现Symplekin 在癌前病变、恶性病变的肝细胞中明显降低，可能参与肝细胞的恶性转化。研究紧密连接分子Symplekin 在肝脏疾病中表达及调控机制对于阐明肝癌发生的机理及对于肝癌的预防和治疗具有十分重要的意义。多种分子调控机制导致基因表达水平的降低，如：基因启动子区域的超甲基化现象，基因核心启动子区域的碱基缺失，炎症相关因子TNF-alpha 和/或 INF-gamma导致基因表达水平的下降以及microRNAs对于靶基因的下调作用。因此，本研究利用Bisulfite restriction PCR、半定量PCR、q-RT-PCR、Western-blot等方法检测Symplekin在肝硬化、肝癌及多种癌细胞系中表达水平改变，及其在肝癌及肝癌细胞系中表达降低的机理——启动子区域发生 CpG岛的甲基化；启动子区域缺失；细胞因子TNF-alpha 和 IFN-gamma 对Symplekin 表达水平的影响；MicroRNAs在癌细胞系中与Symplekin的相对表达情况。实验结果显示（1）Symplekin 在肝硬化和肝癌组织中mRNA 表达水平呈下降趋势， Symplekin 在癌细胞系如肝癌细胞系（ HepG2 、HuH-7 ）、肺癌细胞系（GLC,Spca-1,Ncih446,801D）、宫颈癌细胞系（Hela）、乳腺癌细胞系（Mcf-7）中表达均下降。（2）利用细胞因子TNF-alpha、INF-gamma 同时处理HepG2 细胞系，Symplekin mRNA、蛋白均表达下降。（3）应用q-RT-PCR 检测5 个细胞系中Symplekin、Mir-124 的相对表达量，发现Mir-124 和Symplekin 表达量变化有相反趋势。（4）应用bisulfite restriction PCR 对13 例肝癌组织、10 例肝硬化组织、4 例正常肝组织以及肝癌细胞系HepG2 、Huh7 启动子区域甲基化状态进行检测，发现Symplekin 启动子区域都无甲基化现象；（5）同时，对8 例肝癌组织、10 例正常肝组织、5 例上皮细胞系及6 例白血病细胞系启动子区域缺失进行检测，发现Symplekin 启动子区域确实有碱基缺失，但其在肝癌组织、肝硬化组织、正常肝组织间没有统计学意义。实验结果提示Symplekin 很可能在肝细胞的恶性转化中起着重要的作用，此外 Symplekin 表达下降可能不仅参与肝癌发生且与其它肿瘤的发生具有相关性。推测在肝炎、肝硬化中，Symplekin 的下降可能会导致紧密连接功能下降，肝胆管上皮屏障功能降低， CB(结合胆红素)返流入血中，可能也是造成黄疸形成的原因之一。在肝脏疾病炎症反应过程中，细胞因子可能会协同作用影响Symplekin 的表达。Mir-124 有可能直接负调控Symplekin 的表达从而导致其表达降低。而Symplekin 启动子区域甲基化或缺失与肝癌发生无相关性。结论：（1）Symplekin 在大部分肝炎、肝硬化、肝癌组织中mRNA 表达水平呈下降趋势，这表明Symplekin 很可能在肝细胞的恶性转化中起着重要的作用。（2）Symplekin 在癌细胞系如肝癌细胞系（HepG2,HuH-7）肺癌细胞系（GLC,Spca-1,Ncih446,801D）、宫颈癌细胞系（Hela）、乳腺癌细胞系（Mcf-7）中表达均下降，这提示Symplekin 表达下降可能不仅参与肝癌发生而且参与其它肿瘤的发生。（3）Symplekin 启动子区域甲基化或缺失在肝癌、肝硬化及正常肝组织之间无显著性差异，表明在肝癌发生时Symplekin 的表达下降可能与启动子DNA 甲基化和缺失无关。（4）体外实验表明炎症细胞因子TNF-alpha 与INF-gamma 的协同参可能是体内Symplekin 表达及调控的机制之一。（5）Mir-124 对于Symplekin 的负调控作用也可能是体内Symplekin 表达及调控的机制之一。炎症细胞因子TNF-alpha 与INF-gamma 及Mir-124 可能在肝脏疾病及肝癌发生过程中起着重要作用。 |
| 英文摘要 | Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, which is of high mortality and poor prognosis, and it usually occurs in China and Southeast Asia. There are many etiological factors that could result in hepatocarcinogenesis and HCC is associated with multi-mutations of tumor associated genes. Cell junction molecule (tight junction, adherens junction, desmosome) are vital to maintenance of cell polarity and epithelial barrier. Dysfunction of expression of cell junction molecule is greatly associated with tumorigenesis of malignant tumor. Symplekin, as a tight junction associated protein, is multi-localization and multifunction protein. Besides tight junction formation of epithelial cells, Symplekin plays vital and multiple roles in mRNA polyadenylation and cell cycle regulation. Our early works showed that Symplekin was greatly decreased in premalignant lesions and malignant lesions of hepatocyte. Symplekin may be involved in transformation of malignant tumor. Thus, investigating the decreased mechanisms of Symplekin is vital to the elucidation of the mechanisism of hepatocarcinogenesis and the precaution and treatment of HCC. Multi-mechanisms could result in decreased expression of genes. Hypermethylation and promoter deletion had been studied in gene silencing. Inflammatory cytokines such as TNF-alpha and INF-gamma and micor-RNAs were suggested to be involved in down-regulation of tumor associated genes. In this research, bisulfite restriction PCR, semiquantitative PCR, q-RT-PCR and western-blot were employed to detect the expression alteration of Symplekin in liver cirrhosis, HCC and a variety of cancer cell lines and to elucidate the decreased mechanisms of Symplekin in HCC and cancer cell lines which included hypermethylation and deletion of Symplekin’s promoter, effect of TNF-alpha and IFN-gamma on expression of Symplekin, and relative expression of MicroRNAs and Symplekin in cancer cell lines. The results showed that (1) the mRNA of Symplekin in liver cirrhosis and HCC as well as in cancer cell lines such as HepG2, HuH-7, GLC, Spca-1, Ncih446, 801D, Hela and Mcf-7 all showed decreased expression. (2) With both TNF-alpha and INF-gamma treatment of HepG2, both mRNA and protein of Symplekin were down-regulated. (3) Q-RT-PCR was employed to detect the relative expression of Symplekin and Mir-124 in five cell lines. Our results showed that alteration of Mir-124 and Symplekin expression have reverse tendency. (4) Bisulfite restriction PCR was carried out to check the methylation differences among 13 cases of hepatocellular carcinoma, 10 cases of liver cirrhosis and 4 cases of normal liver tissues as well as hepatocellular carcinoma cell lines, HepG2 and Huh7. The results showed that there were no methylation alternations either in liver diseases group, in normal liver tissues, or in hepatocellular carcinoma cell lines. (5) Deletion of core promoters of Symplekin from 8 cases of hepatocellular carcinoma, 10 cases of normal liver tissues, 4 cases of epithelial cell lines and 7 cases of leukemia cell lines had also been investigated in this study. Our data showed that a deletion did exsist in Symplekin promoter. However, the deletion in promoter of Symplekin had no statistical significance among HCC, liver cirrhosis and normal liver groups. The results give a hint that Symplekin may have an important role in malignant transformation and the decreased expression of Symplekin may not only be involved in hepatocarcinogenesis but also participate in the development of other tumors. We presume that decreased Symplekin may degrade the function of tight junction, epithelial barrier of bile duct of liver and led to back flow of conjugated bilirubin. Decreased Symplekin may be one of the reason producing jaundice. It indicates that proinflammation cytokines may have a synergetic effect on expression of Symplekin during liver disease inflammation. Mir-124 may affect expression of Symplekin, and then result in decreased expression of Symplekin. However, methylation and deletion of Symplekin promoter had no association with hepatocarcinogenesis. Our conclusions: (1) the mRNA of Symplekin in liver cirrhosis and HCC mostly showed decreased expression. It gives a hint that Symplekin may be vital to malignant transformation. (2) The mRNA of Symplekin in cancer cell lines such as HepG2, HuH-7, GLC, Spca-1, Ncih446, 801D, Hela and Mcf-7 all showed decreased expression. It gives a cue that the decreased expression of Symplekin may not only be involved in hepatocarcinogenesis but also participate in the development of other tumors. (3) Methylation and deletion of Symplekin promoter had no association with the expression of symplekin and hepatocarcinogenesis. (4) Synergy of proinflammation cytokines TNF-alpha and INF-gamma on the expression of Symplekin may be one of the mechanisms of Symplekin expression and regulation in vivo. (5) The negative regulation role of Mir-124 on the expression of Symplekin also may be one of the mechanisms of Symplekin expression and regulation in vivo. Proinflammation cytokines TNF-alpha, INF-gamma and Mir-124 may be involved in liver disease and hepatocarcinogenesis. |
| 语种 | 中文 |
| 公开日期 | 2010-10-22 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6299]  |
| 专题 | 昆明动物研究所_分子病理学 |
| 推荐引用方式 GB/T 7714 | 易濒. 紧密连接分子Symplekin 在肝脏疾病中表达及调控机制的研究[D]. 北京. 中国科学院研究生院. 2009. |
入库方式： OAI收割
来源：昆明动物研究所
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