Figure 4
MiR-98, miR-101 and miR-214 posttranscriptionally down-regulates EZH2 expression in ESCC cell line. (A-C) 1 × 106 Eca109 cells were cotransfected with 50 pmol of miRNAs (or control miRNA) and 1 μg of Luc-EZH2 (or matched Luc-EZH2-mut) plasmid, respectively. Luciferase reporter assay were performed at 48 hr posttransfection. Results showed that cells transfected with miR-98 + Luc-EZH2 (A), miR-101 Luc-EZH2 (B), miR-214 Luc-EZH2 (C) exhibited a significant decrease of reporter activity in comparison with those cotransfected with control microRNA + Luc-EZH2 plasmid. However, the reporter activity of cells cotransfected with miRNAs and Luc-EZH2-mut plasmid showed no significant difference with that of cells cotransfected with control miRNA and Luc-EZH2-mut plasmid. (D, E) 1 × 106 Eca109 cells were transfected with 50 pmol of miRNAs (or control microRNA), respectively. The expression level of EZH2 mRNA and protein was detected by qPT-PCR an Western Blot at 48 hr posttransfection and normalized to that of GAPDH and β-actin, respectively. Results showed that the level of EZH2 protein (D) was significantly decreased in cells transfected with miRNAs (including miR-98, miR-101 and miR-214) as compared to the cells transfected with control microRNA, while the expression level of EZH2 mRNA (E) exhibited no significantly difference between cells transfected with miRNAs and those transfected with control microRNA. Data represent mean ± SEM from 4 independent experiments; *, P < 0.05, **, P < 0.01 by t test.