外泌体穿梭介导KLF4-miR-106a-Smad7促胃癌腹膜转移的分子机制

Peritoneal metastasis is the main lethal factor for advanced gastric carcinoma; however, the pathogenesis remains unclear. We have previously reported that the increased expression of miR-106a regulated peritoneal metastasis of gastric cancer through targeting TIMP2. However, the deregulation of miR-106a and the concrete molecular mechanism for regulating gastric cancer peritoneal metastasis are still unclear. Subsequent study disclosed that the transcriptional factor KLF4 could mediate the transcription of miR-106a. At the same time, miR-106a can regulate the key target Smad7 expression. It was also found that gastric cancer cell exosomes (TDEs) had the high expression of miR-106a; exo-miR-106a can be transported to the recipient cell peritoneal mesothelium. These data suggests that exosomes shuttle may be the key link for the acquisition of high concentration expression of miR-106a and the regulation of peritoneal metastasis of gastric cancer. Based on these, we will use in vitro assays, animal models and human specimens through report gene, electrophoretic mobility shift assay, laser scanning confocal microscope and tissue microarray to elucidate the over-expression of miR-106a and the effect of TDEs on acquisition of high metastatic ability of gastric cancer, as well as the influence of TDEs-miR-106a transportation on peritoneum in order to reveal the molecular mechanism of KLF4-miR-106a-Smad7 mediated by exosomes on peritoneal metastasis of gastric carcinoma and to provide a new idea for gastric cancer prevention and treatment.

腹膜转移是进展期胃癌最主要致死因素，发病机制不明。本项目组已报道过表达miR-106a靶向TIMP2调控胃癌腹膜转移。然而，miR-106a表达失调及调控胃癌腹膜转移的具体分子机制仍不清楚。后续发现转录因子KLF4调控miR-106a转录，同时miR-106a调控关键靶点Smad7表达，胃癌细胞外泌体（exosomes，TDEs）miR-106a显著高表达，并转运至腹膜间皮。提示：外泌体穿梭可能是miR-106a获得高浓度表达并调控胃癌腹膜转移的关键环节。本课题拟在细胞、动物模型及临床样本中通过报告基因、EMSA、共聚焦、组织芯片等阐明miR-106a过表达原因，TDEs促胃癌细胞高转移能力获得及转运miR-106a对腹膜的影响，以期揭示外泌体穿梭介导KLF4-miR-106a-Smad7促胃癌腹膜转移的机制，为胃癌防治提供新思路。

项目背景：本项目研究胃癌腹膜转移的分子机制，探索外泌体穿梭介导KLF4-miR-106a-Smad7在胃癌腹膜转移中的作用。.项目内容：一、阐述 KLF4 调控 miR-106a 表达的分子机制；二、阐明 TDEs 对胃癌细胞侵袭转移 EMT 的影响；三、证实 TDEs-miR-106a 对胃癌腹膜转移的作用；四、阐明 TDEs-miR-106a-RC 促胃癌腹膜转移的分子机制。.重要结果：一、完成生物信息学鉴定miR-106a上游直接转录因子KLF4；二、完成外泌体介导miR-106a传递对腹膜间皮细形态、功能和MMT的作用；三、完成外泌体介导下miR-106a-Smad7作用的进一步分析；四、完成外泌体miR-106a靶向Smad7对裸鼠移植瘤的影响；五、进一步确定miR-106a与下游靶基因Smad7的负相关关系验证外泌体对腹膜间皮细胞的作用；六、验证TGF-β/Smad通路对腹膜间皮细胞的影响；七、阐明外泌体通过转运miR-106a至受体细胞介导胃癌细胞转移，检测TDEs-miR-106a对靶基因作用。.关键数据：一、构建miR-106a启动子报告质粒和KLF4过表达质粒使用双荧光素酶系统、染色质免疫沉淀、组织标本证实KLF4与miR-106a呈负相关关系。二、胃癌细胞迁移能力和分化程度的考察得出AGS最高，BGC-823次之。AGS外泌体抽提和鉴定。处理间皮细胞，发现外泌体miR-106a影响间皮细胞增殖、凋亡和迁移，并靶向调节间皮细胞Smad7表达。三、外泌体miR-106a-Smad7对间皮细胞表型和功能均有影响，使用胃癌细胞检测KLF4-miR-106a-Smad7在胃癌中的表达，miR-106a与Smad7具有负相关关系。四、动物实验表明外泌体miR-106a调控Smad7影响移植瘤生长和基因表达。五、定量PCR和形态学检测进一步确定miR-106a与Smad7的负相关关系。六、采用外泌体分泌和抑制策略阐明外泌体对腹膜间皮细胞存在影响。七、根据Smad7，进一步证实TGF-β/Smad通路对腹膜间皮细胞的影响。八、根据TGF-β，证实外泌体转运miR-106a靶向Smad7激活TGF-β作用于腹膜间皮细胞，影响胃癌腹膜转移。.科学意义：本项目从细胞、动物、组织、分子四个层面展开，为针对外泌体miRNA探索胃癌诊疗新靶点提供关键理论基础。