微囊泡介导的EGFR调控肝脏miR-26-HGF轴促进胃癌肝转移的机制研究

According to the “seed and soil” hypothesis, tumor metastasis has organ specificity instead of irregularity. Liver metastasis is one of the main ways in the advanced gastric cancer; however, the detailed mechanism is still not clear. Microvesicles (MVs) are small particles that are secreted from cells, regulating the signaling pathways of targeted cells, but the function of gastric tumor derived MVs in promoting liver metastasis by reconstructing the microenvironment of liver has not been reported yet. We have found that the EGFR is highly expressed in the serum MVs of gastric cancer patients, as well as in the MVs secreted by gastric cancer cell line. This form of secreted EGFR is proved to be delivered by MVs and finally relocated in the outer surface of liver cell membrane. Subsequently, the up-regulated EGFR in primary liver cells activates HGF expression and paracrine by suppressing miR-26. HGF is well known to promote cancer cell growth and migration, thus gastric cancer MVs play a key role in regulating liver microenvironment. This project is designed to prove that the up-regulation of HGF in liver cells promotes the colonization and proliferation of CTC; and we will evaluate the function of liver microenvironment re-constructed by MV in regulating cancer cells; the molecular mechanism that EGFR suppresses miR-26 will also be explored. Our study will be important to reveal a novel mechanism of tumor metastasis; moreover, it provides a new direction and target for preventing the progress of gastric cancer.

肿瘤转移的“种子和土壤”假说认为，转移的部位不是随机的，而是具有嗜器官性。肝转移是胃癌重要的转移形式之一，但发生机制仍不明朗。微囊泡(MV)由细胞主动分泌，可调控靶细胞的信号通路，但胃癌MV是否可以调控肝脏微环境来促进肝转移尚无报道。我们研究发现胃癌患者血清MV和胃癌细胞MV中均含有EGFR；并证明分泌型EGFR能由MV运输并定位到肝细胞膜上。进一步研究发现，运输到肝细胞的EGFR通过抑制miR-26，激活肝细胞生长因子（HGF）；而HGF是促进肿瘤增殖转移的重要因子，因而胃癌MV可以改造肝脏微环境。本课题拟证明肝细胞中上调的HGF具有促进血液中循环肿瘤细胞（CTC）在肝脏的“着陆”和生长；在体内和体外实验，证明被MV改造的肝脏微环境可以调节癌细胞生物学特征；并尝试探讨EGFR抑制miR-26表达的分子机理。本课题不仅揭示了胃癌肝转移的新机制，更为临床胃癌治疗提供了新方向。

肿瘤转移的“种子和土壤”假说认为，转移的部位不是随机的，而是具有嗜器官性的。肝转移是胃癌重要的转移形式之一，但其具体发生机制还未被阐明。外泌体（exosome）由细胞主动分泌,可调控靶细胞的信号通路，但目前尚无报道胃癌外泌体是否可以调控肝脏微环境来促进肝转移。我们的研究发现胃癌患者血清外泌体和胃癌细胞外泌体中均含有EGFR，且发现EGFR的表达水平与胃癌严重程度成正相关；通过蛋白印迹和免疫荧光检测几个肝细胞和基质细胞的典型指标的表达情况，证明EGFR可被外泌体运输并定位到肝细胞膜上。由于HGF主要在肝脏中表达，是已知的肿瘤促进因素。因此检测SGC外泌体对肝HGF的作用，发现胃癌细胞的外泌体显着促进了混合肝细胞中HGF和EGFR的表达，而当去除外泌体中的EGFR时，HGF的表达被抑制。这说明是外泌体中的EGFR促进HGF的表达，表明胃癌外泌体可以改造肝脏微环境。进一步结合生物信息学分析发现，运输到肝细胞的EGFR通过抑制miR-26a/b来激活肝细胞生长因子（HGF）。体外实验通过直接在SGC7901细胞中转染过表达和降表达HGF后进行EDU细胞增殖实验，Transwell细胞侵袭实验来检测细胞功能的改变，发现上调肝细胞分泌的HGF可明显促进胃癌细胞的增殖和侵袭能力，表明被外泌体改造的肝脏微环境可以调节肿瘤细胞的生物学特征。在体内水平通过多点注射HGF shRNA或HGF过表达的慢病毒对小鼠肝脏进行预处理，然后在进行原位肿瘤的植入，检测不同处理组中肝组织中HGF的表达水平和miR-26a/b的表达情况，发现外泌体EGFR通过抑制miR-26a/b激活肝脏HGF，且发现上调HGF可明显促进肝转移。总体而言，本课题证明了通过重塑肝脏微环境，肿瘤来源的外泌体在决定肝脏特异性转移中起着重要作用，肝细胞中上调的HGF具有促进血液中循环肿瘤细胞（CTC）在肝脏的“着陆”和生长的作用。 我们的结果支持“种子和土壤”假说，揭示了胃癌肝向性转移的新机制，同时也为临床上胃癌的治疗提供了新方向。