ClC-3氯通道蛋白在肿瘤转移中的功能研究

项目名称： ClC-3氯通道蛋白在肿瘤转移中的功能研究

项目编号： No.30800435

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 毛建文

作者单位： 广东药学院

项目金额： 20万元

中文摘要： 本研究利用病理学、细胞生物学、分子生物学和电生理学等方法探讨ClC-3氯通道蛋白在肿瘤转移中的作用及其调控机理。结果显示：①ClC-3在转移癌组织和高转移潜能的肿瘤细胞中高表达。②ClC-3表达沉默抑制肿瘤细胞静脉注射以及皮下移植瘤的肺转移，ClC-3过表达则促进肿瘤细胞的肺转移。③过表达和敲除ClC-3的HeLa细胞形态发生变化，ClC-3高表达的细胞呈长条形纤维细胞状，敲除ClC-3的HeLa细胞则成圆形。④过表达ClC-3的细胞迁移和侵袭能力增强，敲除ClC-3的细胞胞迁移和侵袭能力明显下降。⑤ClC-3聚集于迁移肿瘤细胞前端的皱褶处，高表达ClC-3的细胞与对照组比较皱褶明显增加，ClC-3表达沉默的肿瘤细胞皱褶消除。⑥ClC-3与整合素b1共定位于细胞的膜皱褶处， ClC-3表达的沉默对抑制整合素b1的再循环。⑦整合素b1被刺激再循环时与ClC-3共定位运输且与骨架蛋白共定位于胞浆和膜皱褶处，免疫共沉淀结果提示三者相互结合。本研究确证了ClC-3氯通道蛋白在肿瘤转移过程中发挥了重要作用，其调控机理是介导细胞骨架蛋白和整合素b1依赖性的肿瘤细胞迁移时膜皱褶的形成。

中文关键词： ClC-3;氯通道；肿瘤转移；细胞迁移；膜皱褶；整合素

英文摘要： In this study, methods of pathology, cell biology, molecular biology and electrophysiology were used to investigate the role of ClC-3 chloride channel protein in tumor metastasis and the underlying mechanism. The results showed that: ①ClC-3 were highly expressed in metastatic tumors and tumor cells with high metastatic potential. ②ClC-3 silencing inhibited the lung metastases of tumor cell injection and subcutaneous tumors. However, the overexpression of ClC-3 promoted tumor metastasis. ③Overexpression and knockdown of ClC-3 changed morphology of HeLa cell. ClC-3 overexpression induced cells to be elongated strip-like. HeLa cells with ClC-3 knockout were changed to be cycloid. ④Overexpression of ClC-3 enhanced cell migration and invasion, but knockout of ClC-3 decreased significantly cell migration and invasion. ⑤ClC-3 gathered in ruffles of the migrated tumor cells. Compared with the control group ruffles significantly increased in cells with high expression of ClC-3. Silencing of ClC-3 expression eliminated the ruffles of tumor cells. ⑥ClC-3 and integrin b1 were co-localizated in ruffles of migrated cells . ClC-3 expression silencing inhibited the recycling of integrin b1. ⑦ClC-3, a kind of cytoskeleton and integrin b1 were co-localizated in the cytoplasm and membrane ruffles when integrin b1 recycling was induced by growth factor stimulation. Co-immunoprecipitation results suggested that the three was combined. This study confirmed that the ClC-3 chloride channel protein play important roles in tumor metastasis through mediating cytoskeletal proteins and integrin b1-dependent membrane ruffling of migrated tumor cells.

英文关键词： ClC-3; chloride channel; tumor metastasis;cell migration; membrane ruffle; integrin