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

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

Xiaomin Liu et al. Med Sci Monit. 2016.

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.

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Figures

Figure 1

Figure 1

Expression of miR-18a was downregulated in HBV-positive tissue samples compared with HBV-negative tissue samples, while other miRNAs, including miR-122, miR-143, miR-122, miR-210, miR-345 and miR-148a, showed no apparent differences.

Figure 2

Figure 2

(A) CTGF was identified as the target of miR-18a, with its possible binding region CTGF 3′UTR and its mutant. (B) Relative luciferase activity of cells treated with constructs containing wild-type CTGF 3′UTR segments was significantly lower compared with the negative controls, while that of cells treated with constructs containing mutant CTGF 3′UTR segments was merely different compared with the negative controls.

Figure 3

Figure 3

(A) Expression level of CTGF mRNA in HBV-positive HCC tissue samples was upregulated compared with the HBV-negative tissue samples. (B) Expression level of CTGF protein in HBV-positive HCC tissue samples was upregulated compared with the HBV-negative tissue samples.

Figure 4

Figure 4

(A) Western blot analysis of HBX in HBX-expressing cells compared with the controls. (B) Expression level of miR-18a was inhibited by the presence of HBX compared with the controls. (C) Protein expression level of CTGF was higher among HBX-expressing cells compared with the controls. (D) The mRNA expression of CTGF was upregulated in the presence of HBX compared with the controls.

Figure 5

Figure 5

(A) Viability of HBX-expressing cells increased when compared with the controls. (B) Cell cycle status of HBX-expressing cells in phase G0, S, and G2/M compared with the controls.

Figure 6

Figure 6

(A) CTGF protein expression level of cells treated with miR-18a mimics and CTGF siRNA were equally low when compared with the scramble controls. (B) CTGF mRNA expression level of cells treated with miR-18a mimics and CTGF siRNA were equally low when compared with the scramble controls.

Figure 7

Figure 7

(A) Viability of cells treated with miR-18a mimics and CTGF siRNA showed similar low levels compared with the scramble controls. (B) Cell cycle status of cells treated with miR-18a mimics and CTGF siRNA in phase G0, S, and G2/M compared with the scramble controls.

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