阳性表达E-钙粘蛋白介导的细胞—细胞粘附对PTEN的调控及其分子机制研究

发布时间：2018-01-08 20:21

本文关键词：阳性表达E-钙粘蛋白介导的细胞—细胞粘附对PTEN的调控及其分子机制研究　出处：《复旦大学》2007年博士论文　论文类型：学位论文

【摘要】： E-钙粘蛋白是表皮细胞中一类重要的细胞粘附分子。它的主要功能是介导细胞-细胞间的粘附连接,同时也参与传递细胞间的通讯。E-钙粘蛋白在肿瘤的发生进展中也起重要作用,它的失表达会导致肿瘤的侵袭、转移等恶性转化及早期增殖速度的加快等,因此被认为是个重要的“抑癌因子”。细胞膜上的E-钙粘蛋白二聚体在相互识别结合时,会传递信号给胞内的信号通路比如PI3K/Akt信号通路。PTEN是PI3K/Akt通路中的负性调控因子,它可以拮抗PI3K的活性,在抑制细胞增殖、迁移、侵袭以及诱导凋亡等方面都发挥了重要作用,但是PTEN的调控研究还很不充分。因此,本课题旨在研究E-钙粘蛋白介导的细胞粘附所激活的信号通路与PI3K/Akt信号通路的“串话”,寻找其对PTEN是否存在调控,并探讨其分子机制。在实验中,我们首先在完全缺失E-钙粘蛋白表达的乳腺癌MDA-MB-435细胞中建立了E-钙粘蛋白表达的模型,并证明了E-钙粘蛋白介导的细胞-细胞间粘附在此模型中得到了部分的恢复。MTT增殖实验发现表达E-钙粘蛋白的乳腺癌细胞的增殖速度减慢。探究其原因,证实并非与β-catenin的核内转录活性有关,而是由于细胞周期蛋白cyclin D1及其抑制因子p27受调控引起的细胞周期阻滞。我们还发现,在E-钙粘蛋白表达的乳腺癌细胞中,PI3K/Akt信号通路亦受到了调控:磷脂酶PTEN的蛋白表达水平明显升高,且Akt的磷酸化水平大大降低。为了探讨PTEN蛋白升高的原因,E-钙粘蛋白表达株与对照株中的PTEN的转录水平首先进行了对比,发现PTEN的mRNA与启动子活性在这两株细胞中均并没有显著的统计学意义的差异,说明此E-钙粘蛋白的表达对PTEN的转录并没有太大影响。接着,对PTEN的蛋白稳定性的研究发现,E-钙粘蛋白表达可以延长PTEN的半衰期,增加PTEN的稳定性,表明E-钙粘蛋白表达可以在翻译后水平上调PTEN的表达。最后,进一步证实了是蛋白酶体途径而非溶酶体途径参与了MDA-MB-435细胞中PTEN的降解,E-钙粘蛋白表达延迟了蛋白酶体依赖的PTEN降解,PTEN的泛素化水平降低。进一步的研究证实,E-钙粘蛋白和PTEN在表达了E-钙粘蛋白的MDA-MB-435细胞内存在共定位和相互作用。缺失与β-catenin结合能力的E-钙粘蛋白缺失株也能上调PTEN的表达,但是利用RNA干扰技术降低细胞中的β-catenin表达后,PTEN的上调也有部分减弱,说明β-catenin参与了E-钙粘蛋白对PTEN的调控,但此调控可能不是通过E-钙粘蛋白与β-catenin的直接结合实现的。另外,我们还证实了,此调控是细胞-细胞粘附依赖的,且并非存在于所有的细胞类型中。综上所述,E-钙粘蛋白的表达在MDA-MB-435细胞中上调了PTEN蛋白的表达,且这种上调并非是发生在转录水平,而是细胞-细胞粘附依赖的,并通过E-钙粘蛋白与PTEN的间接的蛋白-蛋白相互作用,影响了蛋白酶体介导的PTEN蛋白的降解途径,提高了PTEN的稳定性实现的。
[Abstract]:E- cadherin is an important class of cell adhesion molecules in epithelial cells. Its main function is to mediate cell-cell adhesion, and is also involved in the transfer of intercellular communication.E- cadherin also plays an important role in tumor development and progression, expression may lead to tumor invasion and its loss, metastasis of malignant transformation and in the early stage of cell proliferation, so it is considered to be an important tumor suppressor factor. The cell membrane of E- cadherin two dimer in the binding, will send signals to the cytoplasmic signaling pathways such as PI3K/Akt pathway.PTEN is a negative regulatory factor in PI3K/Akt pathway, it can antagonize PI3K the activity in inhibiting cell proliferation, migration, invasion and apoptosis plays an important role, but the research on the regulation of PTEN is still not sufficient. Therefore, the purpose of this paper is to study Bai Jiedao E- calcium sticky eggs Cell adhesion activated signaling pathway and PI3K/Akt signaling pathway, to find the "crosstalk" the existence of PTEN regulation, and explore its molecular mechanism.
In the experiment, we first breast cancer cell MDA-MB-435 expression in the complete absence of E- cadherin in established E- cadherin expression model, and prove that E- cadherin mediated cell-cell adhesion in this model has been part of the recovery of.MTT proliferation were found the expression of E- cadherin in breast cancer cell proliferation rate. Investigate its reason, confirmed that the nuclear transcription activity is not with beta -catenin, but because of cyclin cyclin D1 and its inhibitor p27 regulated by cell cycle arrest. We also found that the expression of E- calcium sticky protein in breast cancer cells, PI3K/Akt signaling pathway has been regulated expression of phospholipase PTEN protein increased significantly, and the level of Akt phosphorylation decreased greatly.
In order to investigate the cause of PTEN protein increased, the transcription level of E- cadherin expression lines and the control lines of PTEN were first compared, found that the difference of PTEN and mRNA promoter activity in these two cell lines were not statistically significant, indicating the transcriptional expression of E- cadherin on PTEN and not too big effect. Then, the research on the stability of PTEN protein found that E- cadherin expression can prolong the half-life of PTEN, increase the stability of PTEN, showed that E- can up regulate the expression of E-cadherin expression in PTEN translation. Finally, further proved that the proteasome pathway and non lysosomal pathway involved in the degradation of PTEN in MDA-MB-435 cells E-, E-cadherin expression delayed PTEN proteasome dependent degradation and ubiquitination level of PTEN decreased.
Further studies showed that E- and PTEN expression in the presence of cadherin co localization and interaction of E- cadherin MDA-MB-435 cells. Deletion and beta -catenin binding ability of E- cadherin mutant can upregulate the expression of PTEN, but using RNA interference technology to reduce the expression of beta -catenin, upregulation of PTEN also attenuated and that -catenin is involved in the regulation of cadherin E- beta on PTEN, but this regulation may be not the E- cadherin and beta -catenin binding directly. In addition, we also confirmed that this is the regulation of cell-cell adhesion dependent, and is not found in all cell types.
In summary, the expression of E- cadherin upregulated the expression of PTEN protein in MDA-MB-435 cells, which was not occurred at the transcriptional level, but cell-cell adhesion dependent, and through E- and PTEN cadherin indirect protein-protein interaction, the effect of proteasome degradation pathways mediated by PTEN protein, improve the realization of the stability of the PTEN.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341

【共引文献】