GOLPH2调节CD44泛素化修饰促进内吞再循环的分子机制及其对肝癌转移潜能的影响

The poor prognosis of hepatoma carcinoma(HCC) was associated with character of cell high metastasis, which was regulated by ubiquitination-mediated signal transduction.GOLPH2 was a biomarker for diagnosis and prognosis in HCC. Our previous study revealed that high expression of GOLPH2 was associated with poor prognosis in HCC. Ectopic expression of GOLPH2 could promote hepatocellular carcinoma invasion. Further studies found that GOLPH2 inhibited the ubiquitination level of CD44 and promoted CD44 recycle back to plasma membrane in HCC. However, the mechanism of CD44 ubiquitination regulated by GOLPH2 was unknown. Moreover, GOLPH2 could interact with March8, which was E3 ligases for CD44. Theses results suggested that GOLPH2 possibly promote CD44 recycle after endocytosis via interacted with March8 and inhibited CD44 ubiquitination. Hence, using cell endocytosis, ubiquitination, and mice metastasis models, we will study the mechanism of CD44 ubiquitination regulated by GOLPH2 in the process of CD44 endocytosis, in order to reveal the mechanism of hepatocellular carcinoma metastasis regulated by GOLPH2. The study provides one new opportunity for effective intervention of hepatocellular carcinoma metastasis.

肝癌细胞具有的高转移特性一直是影响肝癌预后改善的重要因素。近年来研究发现，信号分子的修饰能够通过调节相关信号传递影响肝癌的发生和发展。GOLPH2是与肝癌诊断及预后密切相关的肿瘤标志物，前期研究也证实GOLPH2高表达与肝癌患者预后差相关，且能够显著促进肝癌细胞侵袭和转移，抑制CD44泛素化，进而促进CD44分子再循环利用，但GOLPH2抑制CD44泛素化的分子机制却不明确。进一步研究发现，GOLPH2能够特异性结合CD44的E3泛素连接酶March8，提示GOLPH2可能通过结合March8抑制CD44泛素化，从而促进CD44内吞后再循环，进而“放大”CD44信号，促进肝癌细胞侵袭。为此，本项目将应用细胞内吞模型、泛素化模型以及裸鼠转移模型，研究GOLPH2调节CD44內吞循环过程中，GOLPH2对CD44泛素化调节的作用及其内在的分子机制，从而阐明GOLPH2促进肝癌转移的机理。

本课题旨在研究肝癌细胞中，GOLPH2对信号分子CD44内吞循环的调节作用，以及阐明GOLPH2如何调节CD44内吞循环。首先，本课题研究证实GOLPH2高表达与肝癌患者预后差相关，应用基因过表达和敲除手段研究了GOLPH2对肝癌转移的调节作用，通过侵袭和伤口愈合实验等多种实验方法证明了GOLPH2能够有效促进肝癌细胞转移。然后，用生物素标记膜蛋白纯化技术分离细胞膜成分，检测GOLPH2对CD44内吞循环的调节，发现过表达能够增加CD44膜含量，促进CD44定位到细胞膜上，加速CD44在膜上的再循环速度；而敲除GOLPH2能够降低CD44膜含量，CD44微囊泡分布细胞核周围，CD44和LAMP1共定位明显增加，进入晚期内涵体或溶酶体增多，提示GOLPH2 抑制CD44內吞后进入晚期内涵体-溶酶体内吞循环途径；可见，GOLPH2 能够抑制CD44 内吞后的降解，有效增强CD44膜上循环速度。进而，为了研究GOLPH2如何调节CD44内吞循环，通过数据库分析及文献调阅，我们构建了CD44可能的E3泛素化连接酶March8 野生型及其活性突变型（W115A）的稳定细胞株，检测March8 野生型及活性突变型细胞系中CD44泛素化修饰情况差异，结果显示March8 野生型能够明显促进CD44泛素化，而W115A突变株却不能促进CD44的泛素化；应用免疫共沉淀的方法检测了GOLPH2与CD44和March8的结合情况，结果显示GOLPH2抑制CD44与March8的结合；并应用泛素化的实验方法检测GOLPH2对March8对CD44泛素化的调节情况，结果显示GOLPH2明显抑制March8对CD44的泛素化作用；可见GOLPH2是通过抑制March8与CD44的结合，抑制March8对CD44泛素化修饰作用，进而增强CD44内吞循环速度。最后，用基因过表达和敲除手段证明GOLPH2是通过FAK/Src信号通路发挥作用的。.总之，本课题阐明了GOLPH2是通过结合并抑制March8而抑制CD44与March8结合，从而March8使CD44泛素化水平受到抑制，促使含有CD44的内涵体小泡在分选过程中，完全进入再循环体，引起膜表面CD44信号分子的增加，从而促进