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. 2015 Jul 8;10:19.
doi: 10.1186/s13027-015-0014-0. eCollection 2015.

Viral Non-Coding RNA Inhibits HNF4α Expression in HCV Associated Hepatocellular Carcinoma

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Free PMC article

Viral Non-Coding RNA Inhibits HNF4α Expression in HCV Associated Hepatocellular Carcinoma

Zhao Wang et al. Infect Agent Cancer. .
Free PMC article

Abstract

Background: Hepatitis C virus (HCV) infection is an established cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC); however, it is unclear if the virus plays a direct role in the development of HCC. Hepatocyte nuclear factor 4α (HNF4α) is critical determinant of epithelial architecture and hepatic development; depletion of HNF4α is correlated with oncogenic transformation. We explored the viral role in the inhibition of HNF4α expression, and consequent induction of tumor-promoting genes in HCV infection-associated HCC.

Methods: Western blot analysis was used to monitor the changes in expression levels of oncogenic proteins in liver tissues from HCV-infected humanized mice. The mechanism of HNF4α depletion was studied in HCV-infected human hepatocyte cultures in vitro. Targeting of HNF4α expression by viral non-coding RNA was examined by inhibition of Luciferase HNF4α 3'-UTR reporter. Modulation of invasive properties of HCV-infected cells was examined by Matrigel cell migration assay.

Results: Results show inhibition of HNF4α expression by targeting of HNF4α 3'-UTR by HCV-derived small non-coding RNA, vmr11. Vmr11 enhances the invasive properties of HCV-infected cells. Loss of HNF4α in HCV-infected liver tumors of humanized mice correlates with the induction of epithelial to mesenchymal transition (EMT) genes.

Conclusions: We show depletion of HNF4α in liver tumors of HCV-infected humanized mice by HCV derived small non-coding RNA (vmr11) and resultant induction of EMT genes, which are critical determinants of tumor progression. These results suggest a direct viral role in the development of hepatocellular carcinoma.

Figures

Fig. 1
Fig. 1
HNF4α protein in HCV-infection associated liver tumor: (a). Representative Western blots of three controls (c), and three liver tumors (T) are shown. Change in HNF4α α protein level was normalized to β-Actin loading control; relative values are indicated underneath each lane. b Quantitative change in HNF4α protein levels was estimated by similar Western blot analyses of 7 controls and 8 liver tumors from HCV infected chimeric mice. The relative values of HNF4α shown were based on Western blots run in triplicates (mean +/- SE) (*p < 0.01)
Fig. 2
Fig. 2
Altered expression of Vimentin and EMT markers in liver tumors of HCV-infected MUP-uPA/SCID/Bg mice: ( 2a ) Induction of Vimentin in liver tumor was compared to the liver tissue from human hepatocyte engrafted but uninfected chimeric mice. Representative Western blots of four tumors and four control liver is shown in the upper panel with β-Actin loading control. Lower panel shows relative increase in Vimentin in liver tumors was compared to the uninfected controls. ( 2b) Expression levels of EMT regulatory genes in liver tumors. Liver protein from seven uninfected controls and eight HCV infected humanized mice were compared by Western blotting (run in triplicate); Relative values (Mean +/- SE, normalized to β-Actin loading control) of each protein is given underneath
Fig. 3
Fig. 3
Inhibition of HNF4α in human hepatocytes transfected with HCV genomic RNA. Upper part (a) shows Western blots of HNF4α (from left to right), from mock transfected control cells, cells transfected with 1 μg HCV (H77) genomic RNA and (far right lane), cells transfected with 1 μg HCV RNA plus 50nM LNA-vmR-11antagomir. Cells were harvested 48 h post transfection. Lower part (b) shows relative change in HNF4α protein levels as compared to the mock-transfected cells (analyzed in triplicate Mean =/- SE, *p < 0.03)
Fig. 4
Fig. 4
Inhibition of HNF4α by vmr11: Upper part (a), shows Western blot of HNF4α from control (mock transfected) cells, cells transfected with 50nM vmir-11 “mimic” oligonucleotides, and (far right lane), cells transfected with 50nM vmir-11 mimic plus 50nM vmir-11 antagomir. Transfections with vmr11 oligonucleotides were repeated at 24-h intervals. Cells were harvested at 48 h. Lower part (b), shows relative changes in HNF4α protein levels normalized to β-Actin loading control (*p < 0.03)
Fig. 5
Fig. 5
HNF4α mRNA levels in HCV RNA or vmr11 transfected cells. Upper panel (a): RT-PCR analysis of HNF4α mRNA from mock transfected, HCV (H77) genomic RNA transfected or HCV (H77) genomic RNA plus vmiR-11 antagomir co-transfected cells; Lower panel (b); RT-PCR analysis of HNF4α mRNA from mock transfected cells, or cells transfected with wild-type vmr11 (“mimic”) oligonucleotides or vmiR-11 mimic oligonucleotides plus LNA-vmr11 antagomir. The transfection conditions were as described in Fig. 4. Relative values (SEM) of HNF4α mRNA were estimated from three independent RT-PCR runs (*p < 0.01)
Fig. 6
Fig. 6
Luc-HNF4a 3’-UTR assay: Indicated concentrations of 22 nucleotide vmr11 “mimic” oligonucleotides or 2’-Fluoro modified vmr11 mimic oligonucleotides were co-transfected with Luciferase HNF4α 3’UTR reporter plasmid [13] and luciferase activity was quantitated three days post transfection as described
Fig. 7
Fig. 7
Matrigel cell invasion assay: (From left to right): Hepatocytes transfected with 50nM scrambled (Scr.) nonspecific control oligonucleotides, Scrambled plus H77 (HCV1a) genomic RNA, 2’-Fluoro modified vmr11 mimic (F-vMir-11-m), HCV 1a genomic RNA (H77) plus anti-vmr11 (anti-vMir-11), H77 plus 2’-Fluoro anti-vmr11 (F-vmr11-R), and Fluoro-vmr11-mimic plus Fluoro-vmr11-R. Three days post-transfection, the cells were processed for Matrigel assay. Data are mean + S.E.M. from 6 independent experiments for each bar graph. *Significantly different from Scr. Control by ANOVA with post-hoc Dunnett’s test (P < 0.05)

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