Methylation-associated silencing of miR-200b facilitates human hepatocellular carcinoma progression by directly targeting BMI1.
Methylation-associated silencing of miR-200b facilitates human hepatocellular carcinoma progression by directly targeting BMI1.
复制标题
miR-200b 甲基化相关沉默通过直接靶向 BMI1 促进人肝细胞癌进展
DOI：
10.18632/oncotarget.7629
复制
发表时间：
2016-04-05
期刊：
影响因子：
--
通讯作者：
Liu C
中科院分区：
文献类型：
--
作者：
Wu WR;Sun H;Zhang R;Yu XH;Shi XD;Zhu MS;Zeng H;Yan LX;Xu LB;Liu C
本研究旨在探讨microRNA-200b及其预测靶点BMI1在人肝细胞癌中的生物学功能。采用定量聚合酶链式反应检测肝细胞癌组织中microRNA-200b和BMI1的表达。用荧光素酶报告实验验证BMI1作为microRNA-200b的直接靶点。在体内外研究了microRNA-200b对肝癌进展的影响。用甲基化特异性聚合酶链式反应(MSP)和亚硫酸氢盐测序聚合酶链式反应(BSP)检测microRNA-200b启动子的甲基化状态。在83.3%的肝细胞癌组织中，microRNA-200b的表达显著下调。相比之下，BMI1在66.7%的肝细胞癌组织中显著过表达。荧光素酶检测结果证实BMI1是microRNA-200b的直接靶基因。在肝癌细胞中强制表达microRNA-200b显著抑制了细胞的增殖、克隆形成、细胞周期进展和侵袭。此外，microRNA-200b与5-氟尿嘧啶在体外协同诱导细胞凋亡，并在体内抑制致瘤性。此外，MSP分析和BSP分析显示，在肝癌组织中，microRNA-200b启动子区域的CpG位点广泛甲基化，并伴随着microRNA-200b表达的下调。此外，经5-氮胞苷处理后，肝癌细胞中的microRNA-200b被激活，而BMI1的表达明显下调。我们的结果表明，microRNA-200b被DNA超甲基化部分沉默，它可以通过直接靶向肝癌中的BMI1来抑制肿瘤的进展。
This study aims to investigate the biological function of microRNA-200b and BMI1, predicted target of microRNA-200b in human hepatocellular carcinoma (HCC). MicroRNA-200b and BMI1 expression in HCC tissues were evaluated by qPCR. A luciferase reporter assay was used to validate BMI1 as a direct target of microRNA-200b. The effect of microRNA-200b on HCC progression was studied in vitro and in vivo. Methylation specific PCR (MSP) and bisulfite sequencing PCR (BSP) were used to detect the methylation status of the microRNA-200b promoter. Significant downregulation of microRNA-200b was observed in 83.3% of HCC tissues. By contrast, BMI1 was significantly overexpressed in 66.7% of HCC tissues. The results of the luciferase assay confirmed BMI1 as a direct target gene of microRNA-200b. Forced expression of microRNA-200b in HCC cells dramatically repressed proliferation, colony formation, cell cycle progression, and invasion. Moreover, microRNA-200b synergized with 5-fluorouracil to induce apoptosis in vitro and suppressed tumorigenicity in vivo. In addition, MSP analysis and BSP revealed that CpG sites in the promoter region of microRNA-200b were extensively methylated in HCC, with concomitant downregulation of microRNA-200b expression. Furthermore, microRNA-200b was activated in HCC cells after treatment with 5-azacytidine, whereas BMI1 expression was clearly downregulated. Our results indicate that microRNA-200b is partially silenced by DNA hypermethylation and that it can repress tumor progression by directly targeting BMI1 in HCC.