MiRNA-mediated gene silencing of epithelial cell adhesion molecule inhibits growth of human gallbladder cancer cells in vitro

Abstract

Objective To observe the effect of epithelial cell adhesion molecule (EP-CAM) gene silencing by RNA interfering (RNAi) technology on the proliferation, apoptosis of GBC-SD cells. Methods Recombinant plasmid pcDNA? 6. 2-GW/EraGFPmiR-EPCAM-l, -2, -3 and -4 targeting human EP-CAM gene were respectively transfected into GBC-SD cells by lipofectamine? 2000 after DNA sequencing. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of EP-CAM and adopted to select the recombinant plasmid which showed the most optimal inhibition effct. After tranfection by pcDNA?6. 2-GW/EmGFPmiR-EPCAM-l with the highest inhibition rate, cell proliferation was analyzed by methyl thiazol tetrazolium (MTT) assay, and apoptosis was assayed by flow cytometry. Results Four EP-CAM-targeted microRNA (miRNA)-l, -2, -3 and -4 were successfully inserted into the plasmid vector pcDNA?6. 2-GW/EmGFPmiR, and the coding sequences of the obtained miRNA were consistent with the designed fragments. RT-PCR results showed that the mRNA expression of EP-CAM gene in GBC-SD cells transfected with recombinant plasmid-1 ,-2,-3 and -4 were 0. 26 ± 0. 02, 0. 79 ± 0. 03, 0. 37 ± 0. 02, 0.45 ± 0.04 respectively, which was significantly lower than that in blank and negative control groups (0. 94 ± 0. 03, 0. 92 ± 0. 03, respectively, P ＜ 0. 01). Western blotting revealed that the relative expression of EP-CAM protein in GBC-SD cells transfected with recombinant plasmid-1, -2, -3 and 4 was 0. 22 ±0. 03, 0.71 ±0.03, 0.32 ±0.02, and 0.41 ±0.04 respectively, which was significantly lower than that in blank and negative control groups (0. 85 ±0.03, 0. 84 ±0.04, respectively,P＜0. 01). After transfection with pcDNA?6. 2-GW/EmGFPmiR-EPCAM-l, the proliferation ability of the cells was significantly inhibited and the inhibition rate was 39% after 48 h. As compared with the control groups, the difference was significant (P ＜ 0. 05). However cell apoptosis had no significant change ( P ＞ 0. 05 ). Conclusion The recombinant plasmid pcDNA? 6. 2-GW/EmGFPmiR miRNA targeting human EP-CAM gene could effectively down-regulate EP-CAM expression and inhibit proliferation of GBC-SD cells in vitro. Key words: Gallbladder carcinoma; Epithelial cell; Adhesion molecule; RNA interference; Proliferation