| 题名 | Symplekin 在紧密连接中的功能及其在肿瘤中的表达调控机制 |
| 作者 | 常宏 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-05 |
| 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 | 紧密连接 上皮屏障 Symplekin 启动子 microRNA-124 |
| 其他题名 | Functions of Symplekin in Tight Junctions and Its Regulation Mechanisms in Cancers |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 细胞紧密连接构成细胞间不通透性连接，其在调节上皮屏障功能、细胞极性形成及维持、上皮细胞间相互黏附及细胞信号传导等方面，有重要作用。紧密连接复合体由跨膜蛋白及膜下相关蛋白共同组成，当紧密连接蛋白发生功能异常时，常伴随疾病的发生。Symplekin是细胞紧密连接膜下相关蛋白，其在上皮细胞内具有细胞膜及细胞核双重定位。以往的研究发现，Symplekin参与调节mRNA腺苷酸化、上皮细胞增殖、肠上皮细胞分化及肿瘤细胞有丝分裂。我们之前的研究发现，Symplekin在癌前病变及癌变的肝细胞中，其在紧密连接处表达下调。然而，Symplekin在细胞紧密连接中的功能及其在肿瘤中的表达下调机制，目前尚未有系统研究。 本研究发现，Symplekin在上皮细胞中的表达随细胞紧密连接的形成而发生上调；通过RNAi干扰Symplekin的表达，发现Symplekin下调可以导致上皮细胞屏障通透性的增加及细胞极性的丧失；进一步的研究发现，下调Symplekin可以抑制ZO-1蛋白在紧密连接处的定位并下调ZO-1的表达（ZO-1蛋白在细胞膜处的定位预示紧密连接结构形成），且免疫共沉淀实验显示，Symplekin与ZO-1可以形成蛋白复合体。这些研究结果提示，Symplekin参与紧密连接的形成，其通过与ZO-1相互作用或调控ZO-1的表达进而调节上皮细胞屏障通透性及上皮细胞极性。 我们亦研究了Symplekin在肿瘤中的表达调控机制。研究结果发现，在肿瘤中，Symplekin启动子区域未发生DNA甲基化修饰；在细胞系中，microRNA-124与Symplekin呈现表达相反趋势，以microRNA-124 mimics处理HepG2细胞，Symplekin表达下调；通过测序，发现Symplekin启动子区域有18 bp AT碱基富含序列，其在肝癌组织及相关细胞系中，与Symplekin表达下调具有相关性，进一步体外报告基因实验结果显示，该18 bp AT碱基富含序列可以降低Symplekin启动子转录活性。这些结果提示，Symplekin在肝癌中的表达下调，可能受到microRNA-124及其启动子区18 bp AT碱基富含序列的影响。通过研究Symplekin在紧密连接中的功能及其表达调控机制，有助于深入了解其病理及生理功能。 |
| 英文摘要 | The tight junction is a fundamental intercellular adhesion that regulates tissue structure. Tight junctions control the diffusion of paracellular molecules and particles across epithelial monolayer barriers and seal the plasma membrane of neighbor epithelial cells. In addition, tight junctions regulate the formation and maintenance of epithelial cell polarity. Moreover, the tight junction is a trafficking and signaling platform that regulates the cell cycle, proliferation, and differentiation. Symplekin is multifunctional protein localized to both the tight junction and the nucleus with known roles in mRNA polyadenylation, proliferation, differentiation and tumorigenesis. Our previous studies showed deceased expression of symplekin in premalignant and malignant hepatocytes. However, functions of symplekin at tight junctions and its regulation mechanisms in carcinogenesis have not been systematically investigated. In this study, increased expression of symplekin was observed during the formation of tight junctions in cultured HepG2 and HT-29 epithelial cells. Repression of symplekin by RNAi increased the permeability of epithelial monolayers, disrupted cellular polarity, and decreased the expression of the tight junction protein ZO-1. Moreover, symplekin was co-localized with ZO-1 at tight junctions and co-immunoprecipitated with ZO-1, indicating that ZO-1 and symplekin form complexes. In conclusion, symplekin expression regulates the assembly of tight junctions, thus helps determine the epithelial monolayers permeability and cellular polarity. The regulation mechanisms of symplekin were also investigated. No methylation modification was observed in symplekin promoter in tumors. The opposite expression pattern of symplekin and miR-124 was observed. The treatment of miR-124 repressed the expression of symplekin in vitro. In symplekin promoter, an 18 bp AT-rich sequence was newly found, and its heterozygous or homozygous status was related to the expressions of symplekin in cultured cells and hepatocellular carcinomas. Dual luciferase analysis showed that the transcriptional activity was reduced by the promoter with the 18 bp AT-rich sequence. To study functions of symplekin in tight junction and its regulation mechanisms during carcinogenesis can help to fully understand its roles in physiology and pathology. |
| 公开日期 | 2013-04-24 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7390]  |
| 专题 | 昆明动物研究所_分子病理学 |
| 推荐引用方式 GB/T 7714 | 常宏. Symplekin 在紧密连接中的功能及其在肿瘤中的表达调控机制[D]. 北京. 中国科学院研究生院. 2011. |
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