【Abstract】〓Objective〓To investigate the regulating effect of miR-200b on Bmi1 gene in liver cancer, and analysis the binding sites. Methods〓First, bioinformatics software was used to predict the putative miR-200b binding sites on the human Bmi1 3'-UTR. Next, Bmi1-3'-UTR-wild type and Bmi1-3'-UTR-mutant type reporter vectors were generated and luciferase reporter assays were performed to determine the binding sites. Last, real time PCR and Western blot analysis were used to validate the regulating effect of miR-200b on Bmi1 gene in human liver cancer cell line HepG2. Results〓The results obtained by bioinformatics software showed that human Bmi1 3′-UTR had three putative miR-200b binding sites. Dual luciferase reporter gene analysis showed that the luciferase activity of Bmi1-3'-UTR-wild type group was significantly reduced whereas the Bmi1-3′-UTR-mutant type group was unchanged after miR-200b-3p mimic transfection. Real time PCR and Western blot analysis confirmed that over-expression of miR-200b can significantly repress the expression of Bmi1. Conclusion〓miR-200b can regulate the expression of Bmi1 by base pairing to the 3'-UTR of Bmi1 mRNA in human liver cancer.
