Li Yuling, Chen Shudong, Shan Zhengfei, Bi Liyan, Yu Shengqiang, Li Yongwei, Xu Sen
Department of Pathophysiology, Binzhou Medical University, Yantai 264003, Shandong, China.
Department of Gastrointestinal Surgery Ward II, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai 264000, Shandong, China.
Biosci Rep. 2017 Jun 27;37(3). doi: 10.1042/BSR20170136. Print 2017 Jun 30.
We investigated the effect of miR-182-5p on the viability, proliferation, invasion, and migration ability of human gastric cells by regulating the expression of RAB27A. Real-time PCR assay was used to detect the expression of miR-182-5 and RAB27A in human gastric carcinoma tissues, para-carcinoma tissues, and different cell lines. Western blotting was also used to determine the RAB27A expression in both tissues and cell lines. We chose the HGC-27 cell line as experiment subject as it demonstrated the highest miR-182-5p level. HGC-27 cells were transfected with different vectors and the cell viability, mitosis, invasion, and migration ability were measured through MTT assay, flow cytometry (FCM) analysis, Transwell assay, and wound healing assay. In comparison with the normal tissues, miR-182-5p is expressed at a higher level in gastric cancer (GC) tissues, while RAB27A is expressed at a lower level in cancerous tissues. The down-regulation of miR-182-5p and up-regulation of RAB27A can significantly decrease the viability, migration, invasion, and mitosis of HGC-27 cells. The target relationship between miR-182-5p and RAb27A was confirmed through a dual-luciferase reporter gene assay and Western blot assay. miR-182-5p enhances the viability, mitosis, migration, and invasion of human GC cells by down-regulating RAB27A.
我们通过调节RAB27A的表达,研究了miR-182-5p对人胃细胞活力、增殖、侵袭和迁移能力的影响。采用实时定量聚合酶链反应(Real-time PCR)检测miR-182-5和RAB27A在人胃癌组织、癌旁组织及不同细胞系中的表达。蛋白质免疫印迹法(Western blotting)也用于检测组织和细胞系中RAB27A的表达。我们选择HGC-27细胞系作为实验对象,因为它的miR-182-5p水平最高。用不同载体转染HGC-27细胞,通过MTT法、流式细胞术(FCM)分析、Transwell实验和伤口愈合实验检测细胞活力、有丝分裂、侵袭和迁移能力。与正常组织相比,miR-182-5p在胃癌(GC)组织中表达较高,而RAB27A在癌组织中表达较低。下调miR-182-5p和上调RAB27A可显著降低HGC-27细胞的活力、迁移、侵袭和有丝分裂能力。通过双荧光素酶报告基因实验和蛋白质免疫印迹法证实了miR-182-5p与RAb27A之间的靶向关系。miR-182-5p通过下调RAB27A增强人胃癌细胞的活力、有丝分裂、迁移和侵袭能力。