Association between MicroRNA-373 and Long Noncoding RNA NORAD in Hepatitis C Virus-Infected Hepatocytes Impairs Wee1 Expression for Growth Promotion

Abstract

Chronic hepatitis C virus (HCV) infection may lead to end-stage liver disease, including hepatocellular carcinoma (HCC). We have shown previously that microRNA-373 (miR-373) is upregulated in HCV-infected human liver biopsy specimens. To gain insight into the role of miR-373 in HCV-mediated pathogenesis, we investigated its interacting partner for hepatocyte growth regulation. Transcriptome sequencing (RNA-seq) data revealed that Wee1 is associated with miR-373 and is a direct target. Interestingly, higher expression of Wee1 was noted in HCV-infected hepatocytes than in uninfected hepatocytes, suggesting that other factors may block miR-373-mediated Wee1 inhibition. We subsequently found an association between the long noncoding RNA NORAD (LINC00657) and miR-373, and we demonstrated that NORAD binds to miR-373 and Wee1 independently. However, the high level of Wee1 expression in HCV-infected hepatocytes suggested that miR-373 forms a complex with NORAD. Depletion of miR-373 or the inhibitor Wee1 reduces the growth of Huh7.5 cells harboring the HCV genome as well as reducing Wee1 expression. Taken together, our data demonstrate a novel mechanism of hepatocyte growth promotion during HCV infection involving a miR-373-NORAD-Wee1 axis, which may be a target for future therapy against HCV-associated HCC.IMPORTANCE The mechanism of HCV-mediated liver pathogenesis is poorly understood. In this study, we observed that HCV infection upregulates miR-373 and Wee1, a pivotal player in the G₂ checkpoint in the cell cycle, although Wee1 is a direct target for miR-373. Subsequent investigation demonstrated that miR-373 forms a complex with the long noncoding RNA NORAD, resulting in the release of their common target, Wee1, in HCV-infected cells, which, in turn, favors uncontrolled cell growth. Our study suggested a previously unknown mechanism for hepatocyte growth promotion following HCV infection, and this pathway can be targeted for future therapy against HCV-mediated liver pathogenesis.

Keywords: NORAD; Wee1; hepatitis C virus; miRNA-373.

FIG 1

miR-373 targets Wee1. (A) miR-373-transfected cell lysates were immunoprecipitated with an Ago2-specific monoclonal antibody or an unrelated IgG2a isotype control. RNA was isolated from immunoprecipitates by using an RNeasy kit, and Wee1 expression was analyzed by quantitative RT-PCR. (B) IHH were cotransfected with the 3′ UTR of Wee1 cloned into the pMIR-Report luciferase vector (500 ng) and either a control miR (mock), a miR-373 mimic, a miR-19a mimic (positive control), or a miR-10b mimic (negative control), each at 25 nM. Relative luciferase activity was measured after 48 h of transfection. In silico analysis also suggested a potential binding site of miR-373 in the Wee1 3′ UTR (shown above the graph). (C) RNA was isolated from Huh7.5 cells transfected with either a control miR, a miR-373 mimic, or anti-miR-373 (25 nM each), and relative mRNA expression was analyzed by qRT-PCR using specific primers. The 18S rRNA gene was used as an internal control. Data are presented as the means and standard deviations from three independent experiments. (D) Huh7.5 cell lysates transfected with a control miR or a miR-373 mimic were prepared after 48 h of transfection. (Left) Wee1 expression was analyzed by Western blotting using a specific antibody. The blot was reprobed with an antibody to actin for normalization. (Right) Densitometric analysis was performed using ImageJ software. (E) (Left) Cell lysates were also analyzed for p-Cdc2 and total-Cdc2 expression by Western blotting using specific antibodies, and blots were reprobed with an antibody to GAPDH for normalization of the protein load. (Right) Densitometric analysis was performed using ImageJ software. (F) miR-373 expression was analyzed by qRT-PCR of RNA from mimic-transfected cells. Results after normalization with U6 as an internal control are presented. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

Increased Wee1 expression in HCV-infected hepatocytes. (A) Huh7.5 cells were either mock treated or infected with HCV (multiplicity of infection, 1), and RNA was isolated after 48 h. The relative expression of Wee1 mRNA was analyzed by qRT-PCR. 18S rRNA was used as an endogenous control and for target gene normalization. (B) (Left) Mock-treated or HCV-infected Huh7.5 cell lysates were subjected to Western blot analysis for Wee1 expression using a specific antibody. The blot was reprobed with an antibody to actin for normalization. (Right) Densitometry analysis was performed using ImageJ software. (C) (Left) Lysates from mock-treated or HCV-infected cells were subjected to Western blot analysis for p-Cdc2, total Cdc2, or an actin antibody. (Right) Densitometry analysis was performed using ImageJ software. Bands in panels B and C were spliced from the same gel for labeling purposes. Data are presented as the means and standard deviations from three independent experiments. *, P < 0.05; **, P < 0.01.

FIG 3

miR-373 interacts with the lncRNA NORAD. (A) miR-373-transfected cell lysates were immunoprecipitated with an Ago2-specific monoclonal antibody or an unrelated IgG2a isotype control. RNA was isolated from immunoprecipitates by using an RNeasy kit, and the expression of NORAD mRNA was analyzed by quantitative RT-PCR. (B) Predicted binding site for miR-373 in NORAD. IHH were cotransfected with a NORAD luciferase reporter plasmid contacting the miR-373 binding site (200 ng) and control miR or miR-373 mimic (25 or 50 nM [+ or ++, respectively]). The relative luciferase activity of NORAD was measured as described for Wee1 in the legend to Fig. 1B. (C) Luciferase activity was measured from IHH cotransfected with a NORAD luciferase reporter plasmid contacting the miR-373 binding site (200 ng) and a control miR or a miR-130a mimic (50 nM). (D) Huh 7.5 cells were either mock treated or infected with HCV, and RNA was isolated after 48 h of infection. The relative expression of NORAD mRNA was analyzed by qRT-PCR. GAPDH was used as an internal control. Data are presented as the means and standard deviations from three independent experiments. *, P < 0.05; **, P < 0.01.

FIG 4

NORAD regulates Wee1 expression. (A and B) IHH were transfected with a control plasmid or a plasmid containing full-length NORAD. After 48 h of transfection, RNA was isolated, and cell lysates were prepared. (A) The relative expression of Wee1 mRNA was analyzed by qRT-PCR. The 18S rRNA gene was used as an endogenous control. (B) (Left) Wee1 protein expression was analyzed by Western blotting using a specific antibody, and the membrane was reprobed with an antibody to actin for normalization. The bands were spliced from the same gel for labeling purposes. (Right) Densitometry analysis was performed using ImageJ software. (C) IHH were cotransfected with NORAD plasmid DNA (250 ng [+] or 500 ng [+]) and the 3′ UTR of a Wee1 reporter construct (250 ng), and the relative luciferase activity of the Wee1 3′ UTR was measured as described above. (D) Wee1 3′ UTR luciferase reporter plasmid DNA (250 ng) and either a miR-373 mimic (25 nM) or full-length NORAD plasmid DNA (250 ng), or both, were transfected into hepatocytes, and luciferase activity was measured. (E) NORAD mRNA expression in IHH transfected with control or NORAD plasmid DNA was determined by qRT-PCR. Results after normalization with GAPDH as an internal control are presented. Data are presented as the means and standard deviations from three independent experiments. *, P < 0.05; **, P < 0.01.

FIG 5

miR-373 and NORAD regulate PUM1 expression. (A and B) Huh7.5 cells were transfected with either NORAD plasmid DNA (250 ng), or 25 nM miR-373 mimic, or control plasmid DNA. Western blot analysis for PUM1 expression was performed. The membranes were reprobed with an antibody to actin for normalization. The control lane is same for panels A and B. (C) miR-373-transfected cell lysates were immunoprecipitated with an Ago2-specific monoclonal antibody or an unrelated IgG2a isotype control. RNA was isolated from immunoprecipitates, and PUM1 expression was analyzed by qRT-PCR. (D) PUM1 expression in mock-infected or HCV-infected Huh7.5 cells was analyzed using a specific antibody. Densitometry analysis was performed using ImageJ software. Data are presented as the means and standard deviations from three independent experiments. **, P < 0.01.

FIG 6

Silencing miR-373 reduces the growth of hepatocytes harboring HCV. (A) (Left) Huh7.5 cells harboring genome-length HCV were transfected with 25 nM control or anti-miR-373. Viable cells were counted using trypan blue at the indicated time points. (Right) HCV-harboring Huh7.5 cells were treated with a vehicle (control) or 300 nM MK1775, and viable cells were counted at the indicated time points. Data are presented as means and standard deviations from at least three independent experiments. (B) HCV-infected Huh7.5 cells were transfected with 25 nM control or anti-miR-373, and RNA was isolated after 48 h. The relative expression of Wee1 mRNA was analyzed by qRT-PCR. 18S rRNA was used as an endogenous control and for target gene normalization. (C) (Left) HCV-infected cells were transfected with 25 nM control or anti-miR-373, and cell lysates were subjected to Western blot analysis using the indicated antibodies. The membrane was reprobed with an antibody to actin for normalization. (Center and right) Densitometry analysis was performed using ImageJ software. Data are presented as the means and standard deviations from three independent experiments. *, P < 0.05; **, P < 0.01.

FIG 7

Schematic diagram of miR-373 and NORAD in the regulation of Wee1 in HCV-infected cells.