Rab3D蛋白促进肿瘤转移和Hsp90α质膜转运信号的研究

发布时间：2018-05-01 03:28

本文选题：外泌体 + Rab3D　；参考：《清华大学》2015年博士论文

【摘要】：真核生物中广泛存在着一类分子量为20-27 kDa之间的小分子Rab蛋白。它们作为分子开关调节了细胞囊泡运输的许多进程,包括外泌体exosome的释放。而最新针对肿瘤细胞分泌exosome的研究报道也指出,Rab27A和Rab27B存在于外泌体中,参与了exosome的分泌调控,并推动了肿瘤进展。作为参与蛋白分泌的一个重要小分子GTP酶,Rab3D的功能多见于分泌组织中,而在癌症中的作用从没被系统的被报道过。本论文的目的旨在于研究蛋白Rab3D在肿瘤细胞中的功能和机制,包括肿瘤转移中的重要作用。首先,我们在多种类型的癌症细胞及多种常见癌症组织中检测了该蛋白的蛋白水平表达情况,发现其含量较正常组织会呈现不正常的高表达,并和肿瘤恶性程度有相关性。我们进一步发现Rab3D会影响肿瘤细胞的细胞形态,并参与肿瘤细胞的侵袭和迁移过程,在裸鼠原位移植模型体内实验中也证实该蛋白在动物水平上会增加肿瘤转移的能力。分子机制实验揭示了Rab3D会通过激活信号通路如AKT/GSK3β促进细胞的上皮间质化转变过程,并调节外泌体的释放和重要的分子伴侣热休克蛋白90α的分泌,这些过程共同参与了肿瘤细胞的侵袭和迁移,充分说明了Rab3D作为调节肿瘤转移的一个重要分子,将会成为潜在的检测和治疗靶点。另一方面,为了确证Rab3D不仅仅在细胞内存在,我们通过免疫印迹、质谱和电子显微镜等技术检测到在肿瘤细胞分泌的外泌体及其培养基中,Rab3D是高表达的。更重要的是,在不同分期的恶性乳腺癌病人血浆中,Rab3D的表达与恶性程度之间呈现良好的相关性,说明了其未来开发为肿瘤标志物的可能性。同时,针对几乎参与肿瘤所有进程的分子伴侣热休克蛋白90α,我们也开展了一些基础研究工作。胞内热休克蛋白90α目前已被报道可以被分泌到细胞外,在肿瘤细胞膜表面的该蛋白还会参与肿瘤的侵袭和转移,但是该蛋白在肿瘤侵袭过程中的质膜转运调控机理并未阐明。我们发现表皮生长因子会特异性地增加该蛋白在肿瘤细胞表面的表达,且定位于细胞突触的前沿。进一步筛选得到促进肿瘤伪足形成的信号通路PLCγ1-PKCγ参与了热休克蛋白90α的质膜转运过程,且该过程依赖于外泌体的运输。该分子机理为细胞膜表面的热休克蛋白90α促进肿瘤伪足形成和侵袭转移提供了新的理论依据。
[Abstract]:A class of small Rab proteins with molecular weight of 20-27 kDa exist widely in eukaryotes. They act as molecular switches to regulate many processes of vesicular transport, including the release of exosome from the exocrine. Recent studies on exosome secretion by tumor cells also show that Rab27A and Rab27B exist in the exocrine body, participate in the regulation of exosome secretion, and promote tumor progression. The function of Rab3D, an important small molecule involved in protein secretion, is found in secretory tissues, but its role in cancer has never been systematically reported. The purpose of this thesis is to investigate the function and mechanism of protein Rab3D in tumor cells, including the important role of tumor metastasis. Firstly, we detected the expression of the protein in various types of cancer cells and various common cancer tissues, and found that the expression of the protein was higher than that in normal tissues, which was correlated with the malignancy of tumor. We further found that Rab3D can affect the morphology of tumor cells and participate in the invasion and migration of tumor cells. In vivo experiments in nude mice model of orthotopic transplantation also confirmed that the protein can increase the ability of tumor metastasis at the animal level. The molecular mechanism suggests that Rab3D promotes the transition of epithelial stroma by activating signaling pathways such as AKT/GSK3 尾, and regulates the release of exocrine and the secretion of important molecular chaperone heat shock protein 90 伪. These processes are involved in the invasion and migration of tumor cells, indicating that Rab3D, as an important molecule regulating tumor metastasis, will become a potential target for detection and treatment. On the other hand, in order to confirm the existence of Rab3D not only in the cells, we detected the overexpression of Rab3D in the exocrine secreted by tumor cells and its medium by Western blotting, mass spectrometry and electron microscopy. More importantly, there is a good correlation between the expression of Rab3D and the degree of malignancy in patients with malignant breast cancer at different stages, which indicates the possibility of its development as a tumor marker in the future. At the same time, we have also carried out some basic research on the molecular chaperone heat shock protein 90 伪, which is involved in almost all tumor processes. The intracellular heat shock protein 90 伪 has been reported to be secreted out of cells, and the protein on the surface of tumor cell membrane is involved in the invasion and metastasis of tumor. However, the mechanism of plasma membrane transport regulation of the protein during tumor invasion has not been elucidated. We found that epidermal growth factor (EGF) specifically increased the expression of EGF on the surface of tumor cells and was located at the front of cell synapses. Furthermore, the signal pathway PLC 纬 1-PKC 纬 which promotes the formation of pseudopodia was selected to participate in the plasma membrane transport of heat shock protein 90 伪, which is dependent on the transport of exocrine. The molecular mechanism provides a new theoretical basis for the promotion of pseudopodia formation and invasion and metastasis by heat shock protein 90 伪 on the surface of cell membrane.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R73-37

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