HCV core protein-induced down-regulation of microRNA-152 promoted aberrant proliferation by regulating Wnt1 in HepG2 cells - PubMed (original) (raw)
HCV core protein-induced down-regulation of microRNA-152 promoted aberrant proliferation by regulating Wnt1 in HepG2 cells
Shifeng Huang et al. PLoS One. 2014.
Abstract
Background: Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs (miRNAs). The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC), but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152) by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells.
Methods: MiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR). Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting.
Results: HCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT-PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001), miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels.
Conclusion: These findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1, which might, at least partially lead to cell proliferation of liver cancer cells. MiR-152 may have a therapeutic potential to suppress liver cancer proliferation.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Over-expression of the HCV core protein down-regulated miR-152 expression and up-regulated Wnt1 expression.
HepG2 cells were mock infected, or infected with Ad-HCV core or Ad-EGFP for 48 h, and were harvested for preparation of total RNA and protein. (A) Infection efficiency of the adenovirus observed under the fluorescent microscope (×10). (B) Western blot analysis for HCV core protein expression. (C) SLqRT-PCR analysis for miR-152 expression. After normalization to endogenous RNU6B expression, the expression of miR-152 in each group of HepG2 cells was expressed as fold changes in comparison to the levels observed in mock-infected HepG2 cells. (D) Real time RT-PCR analysis for Wnt1 mRNA expression. (E) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (HepG2: mock-infected HepG2 cells; Hcv: HepG2 cells infected with the Ad-HCV core; Co: HepG2 cells infected with the Ad-EGFP control; Advirus-Hcv: Adenovirus expressing the HCV core protein, Ad-HCV core; Advirus-Ctrl: Adenovirus expressing the EGFP control protein, Ad-EGFP).
Figure 2. HCV core protein promoted cell growth, G1/S transition and colony formation efficacy of HepG2 cells via its down-regulation on miR-152.
HepG2 cells were transfected with double-stranded miR-152 mimics, single-stranded miR-152 inhibitor, or their relative negative control RNA at a final concentration of 5 nM with a HiPerFect Transfection Reagent kit as indicated for 48 h, and were collected for preparation of MTT, flow cytometry and colony formation assay. (A) Results of MTT assay. (B) Results for cell cycle profiles. (C) Results for colony formation assay. Data are shown as means and standard deviations from triplicate experiments. * P<0.05, ** P<0.01. (Advirus-Hcv: Adenovirus expressing the HCV core protein; Advirus-Ctrl: Adenovirus expressing the EGFP control protein; inh NC: scrambled oligonucleotides as the negative control for miR-152 inhibitor; inh-152: miR-152 inhibitor; miR NC: scrambled oligonucleotides as the negative control for miR-152 mimics; miR-152: miR-152 mimics; HCV+ miR-152: Adenovirus expressing the HCV core protein+miR-152 mimics).
Figure 3. Sequence-specific suppression of Wnt1 gene expression by miR-152.
The two WT plasmids, pGL3-WNT1-1-wt-3′UTR and pGL3-WNT1-2-wt-3′UTR, including 90 nt spanning each seed match at the 3′UTR, as well as the two mutant controls, pGL3-WNT1-1-mut-3′UTR and pGL3-WNT1-2-mut-3′UTR, were generated based on the firefly luciferase expressing vector pGL3-promoter and confirmed by sequencing. HepG2 Cells were co-transfected with 250 ng of pGL3-WNT1-WT or pGL3-WNT1-Mut constructs with 23.5 nM of miR-152 mimics, miR-152 inhibitor, or their respective negative control. Each sample was co-transfected with 25 ng of pRL-TK plasmid expressing renilla luciferase to monitor the transfection efficiency. A luciferase activity assay was performed 48 hours after transfection with the dual luciferase reporter assay system (Promega). The relative luciferase activity was normalized with renilla luciferase activity. (A) WT and Mut 3′-UTRs of WNT1, indicating the interaction sites between miR-152 and 3′-UTR of WNT1. (B) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-1-WT or WNT1-1-Mut 3′-UTR and miR-152 mimics or its negative control (NC). (C) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-2-WT or WNT1-2-Mut 3′-UTR and miR-152 mimics or miR-152 mimics NC. (D) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-2-WT or WNT1-2-Mut 3′-UTR and miR-152 inhibitor or miR-152 inhibitor NC. Data are shown as means and standard deviations from at least three independent experiments. ** P<0.01.
Figure 4. MiR-152 regulates Wnt1 expression at both mRNA and protein level.
HepG2 cells were transfected with double-stranded miR-152 mimics, single-stranded miR-152 inhibitor, or their relative negative control RNA at a final concentration of 5 nM with a HiPerFect Transfection Reagent kit as indicated for 48 h, and were collected for preparation of total RNA and protein. (A) Real time RT-PCR analysis for Wnt1 mRNA expression. (B) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (inh NC: scrambled oligonucleotides as the negative control for miR-152 inhibitor; inh-152: miR-152 inhibitor; miR NC: scrambled oligonucleotides as the negative control for miR-152 mimics; miR-152: miR-152 mimics; Hcv+miR-152: Adenovirus expressing the HCV core protein+miR-152 mimics).
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