Chen Hong, Cheng Zheng-Yuan, Pan Ying, Wang Zhi, Liu Yang, Zhang Jin-Qi
Department of Gastroenterology, Zhongda Hospital, Southeast University, Nanjing, 210009, Jiangsu, People's Republic of China,
Hum Cell. 2014 Jul;27(3):103-10. doi: 10.1007/s13577-014-0090-2. Epub 2014 Feb 15.
The aim of this study was to investigate the biological characteristics of the RASAL1 gene in a well-differentiated gastric cancer cell line MKN-28 and a poorly differentiated gastric cancer cell line BGC-823 cells, using RNA interference and gene transfection technology, respectively. MKN-28 cells were transfected with the shRNA of RASAL1 and BGC-823 cells were transfected with the pcDNA 3.1 plasmid vector containing RASAL1. RT-PCR and western blotting were then used to detect the expression of RASAL1 mRNA and protein. The activities of RAS and extracellular signal-regulated kinase 1/2 were analyzed by the pull-down method and western blotting. The proliferate capacity, apoptosis rate, invasive and migratory potentials of MKN-28 or BGC-823 cells were also measured by Cell Counting Kit-8 cell proliferation assay, propidium iodide/Annexin V staining coupled with flow cytometry, and transwell chamber assays, respectively. Measurement of RASAL1 mRNA and protein expression in two cells revealed successful transfection of the shRNA of RASAL1 and RASAL1-pcDNA3.1 plasmid into these two cells. Moreover, decreased expression of RASAL1 in MKN-28 cells resulted in increased expression of RAS-GTP and p-ERK1/2. Interestingly, decreased expression of RASAL1 inhibited apoptosis and facilitated cell proliferation, invasion and migration. The increased expression of RASAL1 in BGC-823 cells caused declined expression of RAS-GTP and p-ERK1/2, as well as promoted apoptosis and restrained cell proliferation, invasion and migration. The down-regulation of RASAL1 promoted the proliferation, invasion and migration of gastric cancer MKN-28 cells, and up-regulation of RASAL1 inhibited the proliferation, invasion and migration of BGC-823 gastric cancer cells by regulating the RAS/ERK signaling pathway. Thus, our results suggest that RASAL1 may play an important role as a tumor suppressor gene in gastric cancer.
本研究旨在分别利用RNA干扰和基因转染技术，研究RASAL1基因在高分化胃癌细胞系MKN-28和低分化胃癌细胞系BGC-823细胞中的生物学特性。用RASAL1的shRNA转染MKN-28细胞，并用含RASAL1的pcDNA 3.1质粒载体转染BGC-823细胞。然后用RT-PCR和蛋白质印迹法检测RASAL1 mRNA和蛋白质的表达。通过下拉法和蛋白质印迹法分析RAS和细胞外信号调节激酶1/2的活性。还分别通过细胞计数试剂盒-8细胞增殖测定、碘化丙啶/膜联蛋白V染色结合流式细胞术以及transwell小室测定法，检测MKN-28或BGC-823细胞的增殖能力、凋亡率、侵袭和迁移潜能。对两种细胞中RASAL1 mRNA和蛋白质表达的检测表明，RASAL1的shRNA和RASAL1-pcDNA3.1质粒已成功转染至这两种细胞中。此外，MKN-28细胞中RASAL1表达的降低导致RAS-GTP和p-ERK1/2表达增加。有趣的是，RASAL1表达的降低抑制了细胞凋亡并促进了细胞增殖、侵袭和迁移。BGC-823细胞中RASAL1表达的增加导致RAS-GTP和p-ERK1/2表达下降，并促进了细胞凋亡，抑制了细胞增殖、侵袭和迁移。RASAL1的下调促进了胃癌MKN-28细胞的增殖、侵袭和迁移，而RASAL1的上调通过调节RAS/ERK信号通路抑制了BGC-823胃癌细胞的增殖、侵袭和迁移。因此，我们的结果表明，RASAL1可能作为胃癌中的肿瘤抑制基因发挥重要作用。
