外泌体介导miRNA-155靶向CTHRC1对胃癌细胞腹膜种植转移的影响  

Effect of exosomal transport miRNA-155targeting CTHRC1on peritoneal implantation and metastasis of gastric cancer cells

作　　者：高正兴[1] 赵琳 GAO Zheng-xing;ZHAO Lin(Physical Examination Center,Beijing Daxing District People’s Hospital,Beijing102600,China)

机构地区：[1]北京市大兴区人民医院检验科,北京102600

出　　处：《中国实验诊断学》2022年第10期1521-1527,共7页Chinese Journal of Laboratory Diagnosis

基　　金：北京市希思科恒瑞肿瘤研究基金项目(Y-HR2017-054)。

摘　　要：目的探讨外泌体转运miRNA-155靶向胶原三螺旋重复蛋白1(CTHRC1)对胃癌细胞腹膜种植转移的影响。方法提取胃癌BCG-823细胞外泌体并采用透射电子显微镜鉴定;实时荧光定量PCR检测胃癌BCG-823细胞和外泌体miRNA-155水平;激光共聚焦显微镜观察人腹膜间皮HMrSV5细胞对外泌体吞噬情况;设立阴性对照组、外泌体组和miRNA-155mimic组,通过划痕实验检测外泌体转运miRNA-155对腹膜间皮细胞HMrSV5迁移力的影响;利用生物信息预测软件结合荧光素酶报告基因检测验证miRNA-155的靶基因;Western blotting检测细胞CTHRC1蛋白表达。结果提取的外泌体呈现杯口状,直径40-150nm,外泌体标志性蛋白CD9、CD81呈阳性,而内质网分子伴侣Calnexin不表达;外泌体miRNA-155表达水平显著高于胃癌BCG-823细胞(P<0.05);采用PKH26对外泌体标记后在显微镜下呈红色,其主要分布在人腹膜间皮细胞HMrSV5的细胞质内,且视野下大部分细胞均可见红色荧光。利用生物分析软件(TargetScan,CLIP-Seq,miRDB and miRanda)预测了miRNA-155的下游靶基因为CTHRC1。与阴性对照组比较,外泌体组和miRNA-155mimic组腹膜间皮细胞迁移力、细胞增殖、细胞活力和细胞迁移显著增加,且miRNA-155mimic组高于外泌体组,其中外泌体中miRNA-155表达水平为0.49±0.03,CTHRC1蛋白表达水平显著降低,且miRNA-155mimic组低于外泌体组(P<0.05)。结论胃癌细胞来源的外泌体可促进腹膜间皮细胞迁移,因此增加肿瘤转移风险,其机制可能是外泌体内过表达的miRNA-155靶向抑制CTHRC1蛋白表达水平所引起。Objective To investigate the effect of exosomal transport miRNA-155targeting CTHRC1on peritoneal implantation and metastasis of gastric cancer cells.Methods The exosomes of gastric cancer BCG-823cells were extracted and identified by transmission electron microscopy.Real-time fluorescent quantitative PCR was used to detect gastric cancer BCG-823cells and exosomal miRNA-155levels.Laser confocal microscopy was used to observe the phagocytosis of human peritoneal mesothelial HMrSV5cells situation.The control group,exosomal group and miRNA-155mimic group were established,and the effect of exosomal transport miRNA-155on the migration of peritoneal mesothelial cells HMrSV5was detected by scratch experiment.Western blotting was used to detect the expression of CTHRC1protein in cells.Results The extracted exosomes were cup-shaped,40-150nm in diameter,and positive for exosomal marker proteins CD9and CD81.The expression level of exosomes miRNA-155was significantly higher than that of gastric cancer BCG-823cells(P<0.05).PKH26was red under the microscope after being labeled with exosomes.It is mainly distributed in the cytoplasm of human peritoneal mesothelial cells HMrSV5,and red fluorescence can be seen in most of the cells in the field of view.Compared with the control group,the migration of peritoneal mesothelial cells in the exosome group and the miRNA-155mimic group was significantly increased,and the miRNA-155mimic group was higher than that in the exosome group,the CTHRC1protein expression level was significantly reduced,and the miRNA-155mimic group was lower than that in the exosomes group(P<0.05).Conclusion Exosomes derived from gastric cancer cells can promote the migration of peritoneal mesothelial cells,thereby increasing the risk of tumor metastasis.The mechanism may be caused by the targeted suppression of CTHRC1protein expression by miRNA-155overexpressed in exosomes.

关 键 词：外泌体 胃癌 miRNA-155 胶原三螺旋重复蛋白1 腹膜种植 

分 类 号：R735.2[医药卫生—肿瘤]