Targeted inhibition of microRNA-200c on expression of AP-2α to enhance the proliferation of colon cancer cells in vitro

Abstract

Objective To investigate the impact of miR-200c overexpression on colon cancer cell proliferation ability and the related mechanism. Methods MicroRNAs which may combined with the transcription factor AP-2α were screened and forecasted by the bioinformatics database, while its eukaryotic expression plasmids and specific inhibitor were synthesized. Plasmids PEZX-miR-200c, PEZX-NC, pmir-GLO-AP-2α3'UTR, pmir-GLO and the specific inhibitors miR-67-inhibtor, miR-200c-inhibitor were transfected in vitro into colon cancer HCT-116 and SW480 cells and the HEK293T cell by Lipofectamine2000. The expression of AP-2α mRNA and protein in colon cancer cells was analyzed by qRT-PCR,Western blot and immunocytochemical staining. CCK-8 assay and flow cytometry were adopted to observe the effect of miR-200c on colon cancer cells proliferation and apoptosis. Dual-Luciferase assay experiments were performed to observe the relative luciferase activity induced by miR-200c. Results The proliferation activity was significantly decreased in anti-miR-200c /SW480 group, while in PEZX-miR-200c/HCT-116 group, it was higher than that in PEZX-NC/HCT-116 group. The apoptosis ability was significantly increased in anti-miR-200c/SW480 group [(78±0.7)% vs (66±1.1)%, P<0.05]. The expression of AP-2α both in mRNA and protein levels was decreased in PEZX-miR-200c/HCT-116 group, while the protein level was increased in Anti-miR-200c/SW480 group. The relative luciferase activity inhibited by miR-200c was decreased in HEK-293T cells transfected with PEZX-miR-200c and pmir-GLO-AP-2α3'UTR (0.51±0.09 vs 0.98±0.04, P<0.01). Conclusion MicroRNA-200c could promote cell proliferation ability by targeting transcriptional factor AP-2α in human colorectal cancer cells. Key words: Transcription factor AP-2α; microRNA-200c; Colonic neoplasms