外泌体介导miR-106a转运对胃癌细胞腹膜种植转移的影响  

Effect of exosomes-mediated transfer of miR-106a on the peritoneal implantation of gastric cancer cells

作　　者：朱萌 张宁[3] 李潇[1] 李雅睿[1] 郭丹 和水祥 ZHU Meng;ZHANG Ning;LI Xiao;LI Ya-rui;GUO Dan;HE Shui-xiang(Department of Gastroenterology,First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061,China;Department of Gastroenterology,General Hospital of Ningxia Medical University, Yinchuan 750004,China;Department of Pathology,General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]西安交通大学医学部第一附属医院消化内科,西安710061 [2]宁夏医科大学总医院消化内科,银川750004 [3]宁夏医科大学总医院病理科,银川750004

出　　处：《临床与实验病理学杂志》2019年第6期631-636,共6页Chinese Journal of Clinical and Experimental Pathology

基　　金：国家自然科学基金(81802416);中国博士后科学基金(2018M633529);宁夏自然科学基金(NZ16148、NZ16276)

摘　　要：探讨肿瘤源性外泌体(tumor-derived exosomes, TDEs)介导miR-106a转运对胃癌细胞腹膜种植的影响及可能机制。选择人低分化胃癌细胞系AGS进行培养,从细胞培养上清液中提取外泌体,透射电镜及蛋白质印迹法对外泌体进行鉴定;采用Real-time PCR检测外泌体与细胞内miR-106a的表达差异;将外泌体标记荧光染料PKH26与人腹膜间皮细胞HMrSV5共培养,采用激光共聚焦显微镜观察外泌体的吞噬;增设阴性对照组,划痕实验检测外泌体介导下miR-106a转运对间皮细胞迁移能力的影响,Real-time PCR和Western blot检测miR-106a靶基因Smad7及间质标志物α-SMA的表达。结果 透射电镜显示AGS细胞培养上清液中所提取的微囊泡直径30~150 nm,Western blot显示外泌体标记蛋白CD9、CD81均为阳性。Real-time PCR结果显示外泌体miR-106a表达量显著高于细胞内( P <0.05)。携带PKH26红色荧光信号的外泌体可被间皮细胞摄取。划痕实验结果显示外泌体处理组间皮细胞迁移能力明显增强( P <0.001),Real-time PCR和Western blot检测结果显示靶基因Smad7表达下调,α-SMA表达上调( P <0.001)。结论 胃癌细胞来源的外泌体可促进腹膜间皮细胞迁移,参与转移前微环境形成,其机制可能与外泌体介导miR-106a转运,转录后抑制Smad7表达并诱导间质转化相关。Purpose To investigate the effect of tumor-derived exosomes (TDEs) mediated transfer of miR-106a on the peritoneal implantation of gastric cancer cells and the potential mechanism. Methods The poorly-differentiated gastric cancer cell line AGS was selected and exosomes were isolated from cell culture supernatant. The whole-mount exosomes were identified by transmission electron microscopy and immunoblotting. The expression of miR-106a in exosomes and in cells was compared by real-time PCR. The exosomes were labeled with fluorescent dye PKH26 and co-cultured with peritoneal mesothelial cell HMrSV5;the phagocytosis was observed under laser confocal microscope. The negative control group was added and the effect of exosomes-mediated transfer of miR-106a on the migration of mesothelial cells was examined by wound healing assay. The expression of target gene Smad7 and mesenchymal marker α-SMA were detected by real-time PCR and Western blot. Results Transmission electron microscopy showed that a size range of vesicles isolated from AGS cell culture supernatant was 30~150 nm and immunoblotting showed the positive expression of exosomes markers CD9 and CD81. Real-time PCR obtained that the expression of miR-106a in exosomes was significantly higher than that in cells ( P <0.05). Exosomes that carried with PKH26 red fluorescent signals were ingested by mesothelial cells and promoted its migration when compared with the negative control group ( P <0.001), and the expression of target gene Smad7 decreased, whereas α-SMA increased ( P <0.001). Conclusion Gastric cancer cells produce exosomes that promote the mesothelial cells migration, and the mechanism may be related to the exosomes-mediated transfer of miR-106a and the inhibition of Smad7 at post-transcriptional level, followed by the induction of mesenchymal transition.

关 键 词：胃肿瘤 腹膜种植 外泌体 miR-106a 迁移 

分 类 号：R735.2[医药卫生—肿瘤]