Figure 3

miR-214 post-transcriptionally regulates PTEN expression by targeting the 3’-UTR of PTEN. (A, B) Luciferase reporter assay were performed at 48 hr post-transfection. BGC-823 and SGC-7901 gastric cancer cell lines were transfected respectively with the Renilla luciferase expression construct pRL-TK and pGL3-PTEN-3’-UTR firefly luciferase expression construct, along with either anti-miR-214 or control anti-miRNA. Results showed that cells co-transfected with miR-214 and pGL3-PTEN plasmid exhibited a significant increase of reporter activity in comparison with those co-transfected with the control anti-miRNA and pGL3-PTEN plasmid. However, the reporter activity of cells co-transfected with anti-miR-214 and pGL3-PTEN-mut plasmid showed no significant difference with that of cells cotransfected with control microRNA and pGL3-PTEN-mut plasmid. (C, D) The expression level of PTEN protein was detected by Western Blot at 48 hr post-transfection and normalized to that of β-actin. Results showed that the level of PTEN protein was significantly increased in cells transfected with anti-miR-214 as compared to the cells transfected with control anti-miRNA. (E) The expression level of PTEN mRNA was detected by qRT-PCR at 48 hr posttransfection and normalized to that of GAPDH. Results showed that the expression level of PTEN mRNA exhibited no significantly difference between cells transfected with anti-miR-214 and those transfected with control anti-miRNA. Data represent mean ± SEM from three independent experiments; *P < 0.05 by t test, **P < 0.01 by t test.