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Page 4 of 9 Sukowati et al. Hepatoma Res 2019;5:2 I http://dx.doi.org/10.20517/2394-5079.2018.106

Nanog, Klf4, as well as CSC markers EpCAM and β-catenin. The presence of HBx proteins stimulated cell
[25]
growth and migration . This in vitro data were then confirmed by using HBx transgenic mice where a high
[23]
number of EpCAM cells with characteristics of human progenitor cells was observed . Transformation of
rat oval cells with HBx and the subsequent injection in nude mice treated with aflatoxin B1 in vivo, gave rise
to tumor that expressed markers of adult hepatocytes as albumin and CK18, undifferentiated marker alpha-
[26]
fetoprotein (AFP), and oncoprotein c-Myc .
The truncation of HBx protein in the C-terminal region (HBx-ΔC) is a common event because of HBV X
sequence integration in the genome. A recent study had shown that HBx-ΔC promoted the appearance
[27]
of a CD133 hepatic CSC subset and confer cancer and stem cell-like features in HCC . It is associated
with cancer cell invasiveness and reduction of apoptotic response, tumorigenicity, chemoresistance, and
migration [27,28] .

Regardless of the data provided, the exact mechanism by which HBV proteins altered the early fate of the
cells was still unclear. Several studies had shown that DNA demethylation could be a major mechanism in
[29]
the increase of the expression of CSC markers in normal hepatocytes , also correlated with HBV DNA
[31]
[30]
integration and the axis of HBx-DLL3 (Delta-like 3) of Notch receptor . In the last study, the treatment
[31]
of HBV-transformed cells with a histone deacetylase inhibitor induced DLL3 expression . In a recent
2018 study, it was shown that in the very early stage of HCC, the global DNA methylation 5hmC and 5fC
contents were decreased significantly. It was found to be correlated with HBV infection, decreased ten-eleven
[32]
translocation enzyme activity and uncoordinated expression of DNA methylation-related enzymes .
HCV
HCV, a member of flaviviridae family, is a single stranded RNA virus with 9.6 kb genome size. HCV genome
is processed into structural proteins core, E1, and E2, and non-structural (NS) proteins p7, NS2, NS3, NS4A,
[33]
NS4B, NS5A, and NS5B . Since HCV being an RNA virus cannot integrate into human genome, at the
beginning, the mechanism in HCV-related HCC pathogenesis is supposed exclusively to indirect via chronic
inflammation and oxidative stress. Subsequently, it leads to fibrosis and eventually cirrhosis as observed
[34]
in the other HCC etiologies . However, current literature in experimental models also showed direct
oncogenic effect of the HCV proteins, including on the involvement of the CSC.

A previous study showed that HCV-infected hepatocytes transformed into sphere formation with a number
of epithelial-mesenchymal transition (EMT) and CSC markers, including high level of the stem cell factor
receptor c-Kit. These spheres were potent in promoting tumor growth in immunodeficient mice. However,
these spheres were highly sensitive to cell death from the treatment of sorafenib, a multikinase inhibitor,
[35]
and stattic, an inhibitor of the Stat3 molecule . Furthermore, by inserting HCV sub-genomic replicon in
cultured cells, the acquisition of CSC traits, including an enhanced expression of doublecortin and CaM
kinase-like-1, Lgr5, CD133, AFP, CK19, Lin28, and c-Myc, was demonstrated. Conversely, curing of the
replicon from these cells diminished the expression of these factors. In vivo analysis of liver tissues from
[36]
HCV-positive patients and liver tissue microarrays supported these observations .

It had been shown that HCV core and NS proteins, can induce cell transformation in vitro and in vivo mice
[37]
transgenic model . By using intercross breeding of transgenic mouse models, HCV NS5A protein induced
the toll-like receptor 4 (TLR4). This induction mediated liver damage and tumor formation in synergy with
alcohol-induced endotoxemia. Consequently, the expression of stem cell marker Nanog and the presence of
[38]
CD133/Nanog-positive cells were observed .

This study was then continued by in vivo animal study of NS5A mice fed with high in cholesterol and
saturated fat diet (HCFD). Liver tissues of these HCFD mice had increased levels of TLR4, Nanog,
phosphorylated signal transducer and activator of transcription (pStat3), and Twist1. Further analysis of