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16 July 2016 : Laboratory Research  

HBX Protein-Induced Downregulation of microRNA-18a is Responsible for Upregulation of Connective Tissue Growth Factor in HBV Infection-Associated Hepatocarcinoma

Xiaomin LiuBCDE, Yingjian ZhangABCDFG, Ping WangCD, Hongyun WangCD, Huanhuan SuCD, Xin ZhouCD, Lamei ZhangCD

DOI: 10.12659/MSM.895943

Med Sci Monit 2016; 22:2492-2500

Abstract

BACKGROUND: This study was designed to improve our understanding of the role of miR-18a and its target (connective tissue growth factor (CTGF), which are mediators in HBX-induced hepatocellular carcinoma (HCC).

MATERIAL AND METHODS: We first investigated the expression of several candidate microRNAs (miRNAs) reported to have been aberrantly expressed between HepG2 and HepG2.2.15, which is characterized by stable HBV infection, while the CTGF is identified as a target of miR-18a. Furthermore, the expression of CTGF evaluated in HepG2 was transfected with HBX, while the HepG2.2.15 was transfected with miR-18a and CTGF siRNA. We examined the cell cycle at the same time.

RESULTS: We found that the expression of miR-18a was abnormally reduced in the HBV-positive HCC tissue samples compared with HBV-negative HCC samples. Through the use of a luciferase reporter system, we also identified CTGF 3’UTR (1046–1052 bp) as the exact binding site for miR-18a. We also observed a clear increase in CTGF mRNA and protein expression levels in HBV-positive HCC human tissue samples in comparison with the HBV-negative controls, indicating a possible negatively associated relationship between miR-18a and CTGF. Furthermore, we investigated the effect of HBX overexpression on miR-18a and CTGF, as well as the viability and cell cycle status of HepG2 cells. In addition, we found that HBX introduction downregulated miR-18a, upregulated CTGF, elevated the viability, and promoted cell cycle progression. We transfected HepG2.2.15 with miR-18a mimics and CTGF siRNA, finding that upregulated miR-18a and downregulated CTGF suppress the viability and cause cell cycle arrest.

CONCLUSIONS: Our study shows the role of the CTGF gene as a target of miR-18a, and identifies the function of HBV/HBX/miR-18a/CTGF as a key signaling pathway mediating HBV infection-induced HCC.

Keywords: Case-Control Studies, Carcinoma, Hepatocellular - virology, Cell Cycle Checkpoints - genetics, Connective Tissue Growth Factor - metabolism, Hepatitis B virus - metabolism, Hepatitis B, Chronic - virology, Liver Neoplasms - virology, RNA, Messenger - metabolism, Signal Transduction, Trans-Activators - metabolism

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750