HBV polymerase overexpression due to large core gene deletion enhances hepatoma cell growth by binding inhibition of microRNA-100

// Ya-Hui Huang 1, 2 , Ying-Hsin Tseng 1, 2 , Wey-Ran Lin 1, 3 , George Hung 4 , Tse-Ching Chen 5 , Tong-Hong Wang 5 , Wei-Chen Lee 6 , Chau-Ting Yeh 1, 2 1 Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan 2 Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan 3 Department of Hepato-Gastroenterology, Chang Gung Memorial Hospital, Taoyuan, Taiwan 4 Department of Molecular Biology, Princeton University, NJ, USA 5 Department of Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan 6 Division of Liver and Transplantation Surgery, Department of General Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan Correspondence to: Chau-Ting Yeh, e-mail: chautingy@gmail.com Keywords: hepatitis B, hepatocellular carcinoma, molecular oncology, mutation screening, microRNA-100 Received: August 03, 2015 Accepted: January 17, 2016 Published: January 25, 2016 ABSTRACT Different types of hepatitis B virus (HBV) core gene deletion mutants were identified in chronic hepatitis B patients. However, their clinical roles in different stages of natural chronic HBV infection remained unclear. To address this issue, HBV core genes were sequenced in three gender- and age-matched patient groups diagnosed as chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC), respectively. Functional analysis of the identified mutants was performed. A novel type of large-fragment core gene deletion (LFCD) was identified exclusively in HCC patients and significantly associated with unfavorable postoperative survival. The presence of LFCDs resulted in generation of precore-polymerase fusion protein or brought the polymerase reading frame under direct control of HBV precore/core promoter, leading to its over-expression. Enhanced cell proliferation and increased tumorigenicity in nude mice were found in hepatoma cells expressing LFCDs. Because of the epsilon-binding ability of HBV polymerase, we hypothesized that the over-expressed polymerase carrying aberrant amino-terminal sequence could bind to cellular microRNAs. Screening of a panel of microRNAs revealed physical association of a precore-polymerase fusion protein with microRNA-100. A binding inhibition effect on microRNA-100 by the precore-polymerase fusion protein with up-regulation of its target, polo-like kinase 1 (PLK1), was discovered. The binding inhibition and growth promoting effects could be reversed by overexpressing microRNA-100. Together, HCC patients carrying hepatitis B large-fragment core gene deletion mutants had an unfavorable postoperative prognosis. The growth promoting effect was partly due to polymerase overexpression, leading to binding inhibition of microRNA-100 and up-regulation of PLK1.