Bcl-2通过Vimentin促进喉癌侵袭和转移分子机制研究

Vimentin has a role in promoting tumor invasion and metastasis. The research was reported that high expression of Bcl-2 promoted the occurrence and development of laryngeal cancer, however, the mechanism remained unclear. We preliminary confirmed that Bcl-2 could promote invasion and metastasis of laryngeal cancer and induce the expression of Vimentin. Therefor, we propose the following scientific hypothesis: "high expression of Bcl-2 can promote invasion and metastasis of laryngeal cancer by inducing the expression of Vimentin". The project intends to use the overexpression or silencing expression of Bcl-2 to observe the expression of Vimentin and the invasion and metastasis of laryngeal carcinoma in vivo and in vitro,and to explore the mechanism of induction from the two aspects of EMT-inducible transcription factors and promoter activity.We continue to observe invasion and metastasis of laryngeal carcinoma in vivo and in vitro by the overexpression or silencing Vimentin, to separate and identify the Vimentin-interacting protein in the laryngeal cancer cells by the tandem affinity purification-coupled mass spectrometry-based targeted proteomics technology, to detect whether Vimentin protein and its interacting protein have role in change of the dynamic molecular network to effect on signaling pathway of Src and PI3K-AKT and invasion and metastasis of laryngeal cancer, and ultimately to elucidate the molecular mechanism which Bcl-2 promoted invasion and metastasis in laryngeal cancer by the induction of Vimentin. We hope to provide new experimental basis for the targeted and individual therapy.

Vimentin具有促进肿瘤侵袭和转移的作用。研究发现Bcl-2高表达促进喉癌的发生发展，但机制未明。前期证实Bcl-2能促进喉癌的侵袭和转移，并诱导Vimentin表达。为此，我们提出如下科学假说："Bcl-2通过诱导Vimentin表达，促进喉癌侵袭和转移"。本项目拟采用过表达或沉默Bcl-2观察对Vimentin表达和体内/外侵袭和转移的影响，从EMT相关转录因子和启动子等两个方面探讨诱导机制；观察过表达或沉默Vimentin对喉癌体内/外侵袭和转移的影响，并用串联亲和纯化耦联质谱技术为主的靶向蛋白质组学技术分离、鉴定喉癌细胞中Vimentin相互作用蛋白，研究Vimentin与其相互作用蛋白作用时动态分子网络的改变对Src和PI3K-AKT等信号通路和喉癌侵袭转移的影响，从而阐明Bcl-2通过Vimentin促进喉癌侵袭和转移的分子机理，为喉癌的靶向和个体化治疗提供新的实验依据。

目的：探讨高表达的Bcl-2对喉癌细胞侵袭迁移能力的影响及分子机制。方法：本项目拟采用过表达或沉默Bcl-2观察对Vimentin表达和体内/外侵袭和转移的影响；并用串联亲和纯化耦联质谱技术为主的靶向蛋白质组学技术分离、鉴定喉癌细胞中Bcl-2相互作用蛋白，研究Bcl-2与其相互作用蛋白作用时动态分子网络的改变对喉癌细胞凋亡的影响；观察过表达或沉默Vimentin对喉癌体内/外侵袭和转移的影响；观察Bcl-2与EMT相关蛋白之间的临床关系；采用粘附实验检测Bcl-2高表达对细胞的粘附能力的影响；应用流式细胞仪和Western blotting实验分析Bcl-2对细胞表面integrin表达的变化；研究低氧对喉癌细胞侵袭迁移的影响，应用免疫组化技术和统计学分析低氧与EMT之间的临床和预后的关系。结果：成功构建高表达Bcl-2细胞株，高表达的Bcl-2能够诱导Vimentin表达并促进喉癌细胞的侵袭与迁移；采用串联亲和纯化耦联质谱技术为主的靶向蛋白质组学技术研究显示，Bcl-2能够与28个蛋白相互作用，其中与Hsp90 β相互作用能够增强喉癌细胞的抗凋亡的能力，临床样本研究显示，Bcl-2与Hsp90 β呈正相关，并且Bcl-2与Hsp90β均与喉癌的分化、分期和侵袭转移明显相关；Bcl-2下调了细胞的粘附能力，并降低了细胞表面integrin α2β1的表达，而未见对其他integrin的表达的影响；低氧能诱导喉癌细胞发生EMT并促进细胞的侵袭与迁移能力；低氧与肿瘤的分期和侵袭转移相关。结论：高表达Bcl-2诱导Vimentin表达并促进喉癌细胞的体内/外侵袭和转移；Bcl-2与Hsp90 β相互作用能够增强喉癌细胞的抗凋亡的能力；高表达Bcl-2可能通过下调了细胞表面integrin α2β1的表达从而减低细胞的粘附能力而促进喉癌细胞的侵袭与迁移。