胃癌细胞中MYO5B调控Met受体转运及降解的研究

Met is a receptor tyrosine kinase (RTK) and plays an important role in the process of carcinogenesis. Upon ligand (HGF) binding, Met becomes catalytically active and tyrosine phosphorylated, enabling the recruitment of signaling proteins to initiate downstream signaling cascades. This process is balanced by Met internalization, allowing for their removal from the cell surface and subsequent signal termination. The internalized Met is diverted toward one of two fates to be routed to late endosomes/lysosomes for degradation, or to be recycled back to the plasma membrane. MYO5B is a member of the class V of unconventional, dimeric nonfilamentous myosins, which has been shown to regulate membrane trafficking along the recycling pathway in epithelial cells. Our previous study showed for the first time that MYO5B is epigenetically silenced in gastric cancer cells and played an important role in gastric carcinogenesis, suggesting that MYO5B may be a potential tumor suppressor gene. Our result also found that suppression of MYO5B expression in gastric cancer cells expressing endogenous MYO5B inhibits HGF-stimulated MET degradation, suggesting that MYO5B may implicated in the process of Met trafficking and degradation in gastric cancer. On the basis of our previous studies, we will investigate the trafficking and degradation pathway of Met by CLSM (confocal laser scanning microscope) in gastric cancer cell in which the expression of MYO5B was upregulated or downregulated. We will figure out the Rabs with which MYO5B is associated in the pathway of Met trafficking. Taken together, we will identify a novel role for MYO5B in Met trafficking and HGF-induced downstream signaling in the following study.

Met是酪氨酸激酶受体，当与配体HGF结合后，Met发生自身磷酸化并激发下游蛋白活化发挥作用。为防止Met过度活化，HGF会诱导Met发生内化，Met或被降解或重新被转运至细胞膜。 MYO5B是一种肌球蛋白，参与细胞内多种蛋白包括部分受体的转运，MYO5B是否参与Met的转运目前尚无文献报道。我们前期试验结果证明MYO5B在胃癌发生发展中发挥重要作用，表观遗传学调控胃癌细胞MYO5B的表达；同时发现MYO5B表达受抑制后，HGF诱导的Met蛋白水平降低被延迟，提示MYO5B与Met的转运及降解可能有关。本项目拟在此研究基础上，利用激光共聚焦等技术，研究MYO5B过表达或受抑制后，Met在细胞内转运及降解途径的变化；Met及其下游信号蛋白磷酸化情况；通过免疫共沉淀等技术鉴定出协同MYO5B参与Met转运及降解的蛋白，阐明MYO5B在Met转运、降解以及HGF诱导的信号通路中发挥的作用。

最新的研究发现，MYO5B调控不同通路参与蛋白在细胞内转运发挥重要作用，我们前期工作发现表观遗传学机制抑制MYO5B 在胃癌细胞中的表达，抑制MYO5B表达可降低HGF诱导的Met受体降解，促进Met受体信号通路发挥作用，但调控这一过程的机制目前不明。我们转染MYO5B的 dominant-negative mutant 形式MYO5B-T抑制MYO5B的功能后发现，Met受体从初级内体转运至次级内体的量降低，抑制了Met受体的降解过程。另外我们发现MYO5B-T可促进胃癌细胞骨架蛋白重构，促进细胞的迁徙转移能力，同时促进Rac1的活化，促进胃癌细胞裸鼠成瘤及裸鼠体内转移。结果提示MYO5B蛋白在胃癌细胞内参与Met受体转运，调控Rac1参与胃癌细胞转移。