Abstract:

【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.

Key words: microRNA, Liver cancer, Bmi1, miR-200b

摘要：

【摘要】 目的 研究miR-200b在肝癌中对Bmi1的调控作用,并分析其结合位点。方法 首先利用生物信息学软件预测人Bmi1 3′-UTR上miR-200b可能的结合位点;然后构建Bmi1 3′-UTR野生型和突变型报告载体,应用双荧光素酶报告基因分析检测Bmi1 3′-UTR上miR-200b的结合位点;最后利用real time PCR和Western blot在人肝癌细胞HepG2中验证miR-200b对Bmi1的调控作用。结果〓人Bmi1 3′-UTR上存在3个miR-200b的潜在结合位点;双荧光素酶报告基因分析提示,转染miR-200b-3p mimic后野生型组荧光素酶活性明显低于突变型组;real time PCR和Western blot结果提示在HepG2细胞中过表达miR-200b可明显下调Bmi1的表达。结论 miR-200b在肝癌中可通过靶向结合Bmi1基因3′-UTR特异性调控Bmi1基因的表达。

关键词: microRNA, 肝癌, Bmi1, miR-200b