Methylation-associated silencing of miR-200b facilitates human hepatocellular carcinoma progression by directly targeting BMI1

// Wen-rui Wu 1, 2, * , Hong Sun 3, * , Rui Zhang 1, 2, * , Xian-huan Yu 1, 2 , Xiang-de Shi 1, 2 , Man-sheng Zhu 1, 2, 4 , Hong Zeng 5 , Li-xu Yan 6 , Lei-bo Xu 1, 2 , Chao Liu 1, 2 1 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, China 2 Department of Biliary-Pancreatic Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, China 3 Department of Respiratory, The First Affiliated Hospital/School of Clinical Medical, Guangdong Pharmaceutical University, Guangzhou, 510080, China 4 Department of Thoracic Surgery, Cancer Center of Guangzhou Medical University, Guangzhou, 510095, China 5 Department of Pathology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510120, China 6 Department of Pathology, Guangdong General Hospital, Guangdong Academy of Medical Science, Guangdong, 510080, China * These authors have contributed equally to this work Correspondence to: Lei-bo Xu, email: mdxuleibo@hotmail.com Chao Liu, email: mdliuchao@hotmail.com Keywords: hepatocellular carcinoma, progression, microRNA-200b, methylation, BMI1 Received: November 09, 2015 Accepted: February 09, 2016 Published: February 23, 2016 ABSTRACT 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.