ABSTRACT
Objective To explore the effects of zinc finger E-box binding protein (ZEB)2 3′UTR gene transfection on proliferation, invasion and migration in human gastric epithelial cell line GES-1. Methods The synthetic ZEB2 3′UTR and miR-200b micmics were transfected into GES-1 cell line by lipofectamine 2000. We set up control grop, the mutation group and ZEB2 3′UTR group. Real-time quantitative PCR was performed to evaluate the expression levels of miR-200a/b/c and ZEB1/ZEB2 mRNAs after transfection.And then we set up control group, ZEB2 3′UTR group, ZEB2 3′UTR+negative control group and ZEB2 3′UTR+miR-200b micmics group. The protein expression levels of ZEB1, ZEB2, matrix metallopro-teinases (MMP) 2/9 and proliferating cell nuclear antigen (PCNA) were detected by Western blot assay. The invasion and mi-gration capability were analyzed by transwell assay and wound healing test. MTT assay was used to detect the proliferation ability. Results Compared with control group and mutation group, the expressions of miR-200a/b/c were significantly de-creased, especially for miR-200b. And the expressions of ZEB1/ZEB2 were significantly increased at both mRNA and pro-tein levels after transfected with the ZEB2 3′UTR, enhancing the capability of migration,invasion,and proliferation (P <0.05). Compared with ZEB2 3′UTR group, the capabilities of proliferation,invasion and migration were significantly lower in combined group. Conclusion ZEB2 3′UTR can increase the ability of cell proliferation, invasion and metastasis through regulating the levels of miR-200a/b/c, and then influence the regulation of transcription of the target gene, which could lead to malignant transformation of GES-1 cells.
ABSTRACT
Objective:To explore the effect and mechanism of miRNA sponge on the epithelial-mesenchymal transition (EMT) of gastric carcinoma cell lines SGC7901. Methods:Synthetic ZEB2 3'UTR plasmid and siRNA targeting ZEB2 were transfected into the SGC7901 cell line by Lipofectamine 2000. Real-time quantitative polymerase chain reaction was performed to evaluate the expres-sion levels of miR-200a/b/c. Finally, the migratory, invasive, and proliferative activities of the gastric carcinoma cells in vitro were ana-lyzed by the scratch test, the Transwell cell invasion, and the cell cloning assay. The expression of the target protein was detected by Western blot. Results:Compared with the control group, the expressions of miR-200a/b/c significantly decreased, and their migration, invasion, and proliferation capabilities were considerably higher after they were transfected with ZEB2 3'UTR. Although the expres-sions of miR-200a/b/c significantly increased, the migratory, invasive, and proliferative activities of SGC7901 cells also degraded after they were transfected with siRNA targeting ZEB2. The expression of ZEB2 increased, and that of E-cadherin decreased at the protein level after they were transfected with ZEB2 3'UTR. The protein expression of Vimentin in SGC7901 cells significantly increased. The indicators show the opposite trend when cells were transfected with siZEB2, and the differences between the control and mutation groups were insignificant. Conclusion:ZEB2 3'UTR can regulate EMT course by regulating the miR-200a/b/c expression in gastric car-cinoma, consequently regulating the invasion and migration of carcinoma cells.