肝癌转移消失蛋白MIM-B与Caveolin-1相互作用调节EGF受体内吞循环的分子机制及干预

Palliative liver resection of hepatocellular carcinoma (HCC) accelerated residual tumor metastasis. Our study showed that human gene Metastasis Suppressor 1 (MTSS1) was situated in the central position of the gene function net of postoperative residual HCC, and the protein of Missing in Metastasis B (MIM-B) encoded by MTSS1 enhanced malignant potential of HCC. Further study revealved the interaction effect and a colocation of MIM-B and Caveolin-1 in HCC cells, which stimulated endocytosis of epidermal growth factor receptor (EGFR) and activated downstream signaling molecules. Therefore, we will construct co-expression of RFP-MIM-B and GFP-Caveolin-1 in HCC cells based on our previous research. Iodine-125 labeled EGF will be applied to examine alteration of EGFR and activation of downstream key signaling molecules. Dynamic observation by laser scanning confocal microscopy will be done for description spatial location changes of MIM-B and Caveolin-1. Then, the data mining technology of gene silencing will be performed to find the key domain of MIM-B and Caveolin-1 interaction and to demonstrate the molecular mechanism of EGFR endocytic cycle underlying the interaction effect between MIM-B and Caveolin-1. Finally, with the using of the domain mutagenesis and the designed polypeptide, respectively, to block the key domain, we will evaluate the inhibitory effects on proliferation, invasion and metastasis of HCC in vitro and in vivo, which indicates its potential application in patients with HCC.

姑息性肝切除加速肝细胞癌(肝癌)转移。我们研究提示转移抑制基因MTSS1为术后肝脏残存肝癌基因功能网络枢纽基因，其编码的转移消失蛋白MIM-B增强肝癌恶性潜能；进一步研究提示MIM-B与细胞膜脂筏标志性蛋白Caveolin-1空间共定位，并相互作用促进表皮生长因子EGF刺激下细胞膜EGF受体内吞及下游信号分子磷酸化。在此基础上本项目构建RFP-MIM-B、GFP-Caveolin-1共表达肝癌细胞，采用放射性碘125标记EGF，检测EGF刺激下不同时间点EGF受体在细胞膜上量和活性变化及下游关键信号分子活性水平；借助共聚焦显微镜动态观察MIM-B和Caveolin-1空间位置改变，应用分段基因沉默技术挖掘二者相互作用的关键结构域，研究二者相互作用调节EGF受体内吞循环的分子机制；分别采用突变关键结构域和设计特异性多肽阻断关键结构域方法，体内、外观察其抑制肝癌的效果，具有潜在的临床应用价值。

肝细胞癌(HCC)占全球肝恶性肿瘤的70%-85%，肝切除中约34%为姑息性切除，术后残癌转移潜能增加与转移抑制基因MTSS1编码的转移消失蛋白MIM-B相关。本研究成功构建携带MIM-B与FLAG融合基因的pcDNA3.1载体，pcDNA3.1-MIM-B-FLAG转染效率90.0%，以Hep3B、SMMC7721及MHCC97H等HCC细胞株为研究模型，脂质体法转染HCC细胞显著表达MIM-B，通过细胞免疫荧光以及激光共聚焦显微镜实时观察MIM-B和Caveolin-1的细胞内空间定位关系，应用内、外源免疫共沉淀确认二者相互作用；共聚焦显微镜结果显示MIM-B沉淀Caveolin-1；MIM-B与Caveolin-1共定位，明确了MIM-B的生物学作用依赖Caveolin-1。进一步通过分别缺失结构域的方法研究发现，IRSp53/MIM domain motif (IMD) 和Wiskott-Aldrich syndrome protein homology 2 motif (WH2)结构域是MIM-B和Caveolin-1相互作用的关键基础。我们借助慢病毒转染系统干扰高表达MIM-B的肝癌细胞株，发现抑制MIM-B和Caveolin-1显著降低EGFR信号通路中EGFR、STAT3、AKT和 ERK磷酸化水平，说明MIM-B和Caveolin-1通过IMD和WH2结构域相互作用调节EGFR信号通路促进肝癌侵袭转移。通过免疫荧光的方法检测到EGFR和Rab5、Rab7、Rab11以及Lamp2共定位情况，揭示了EGFR通过内吞为主。在低表达MIM-B的Hep3B细胞中过表达MIM-B和Caveolin-1促进肝癌细胞迁移能力，在高表达MIM-B的MHCC97H细胞中敲低MIM-B和Caveolin-1抑制肝癌细胞迁移侵袭能力。接下来，在临床肝癌标本中验证MIM-B和Caveolin-1蛋白表达情况。结果显示，同癌旁组织相比，癌组织高表达MIM-B和Caveolin-1，肝癌肺转移患者高表达MIM-B，同时高表达MIM-B和Caveolin-1的肝癌患者更易出现肺转移，中位总体生存期明显降低，差异存在统计学意义。成功构建荷肝癌的裸鼠模型，联合应用索拉非尼并抑制MIM-B的体内实验结果显示，有效抑制MHCC97H肺转移，延长荷瘤裸鼠生存期，具有转化医学临床应用价值。