Hepatitis C Virus Core Protein Down-Regulates p21^Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cells

<div><p>Background</p><p>Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma, but HCV core-modulated cellular microRNAs are unknown. The HCV core protein regulates <i>p21^Waf1/Cip1</i> expression. However, the mechanism of HCV core-associated <i>p21^Waf1/Cip1</i> regulation remains to be further clarified. Therefore, we attempted to determine whether HCV core-modulated cellular microRNAs play an important role in regulating <i>p21^Waf1/Cip1</i> expression in human hepatoma cells.</p> <p>Methods</p><p>Cellular microRNA profiling was investigated in core-overexpressing hepatoma cells using TaqMan low density array. Array data were further confirmed by TaqMan real-time qPCR for single microRNA in core-overexpressing and full-length HCV replicon-expressing cells. The target gene of microRNA was examined by reporter assay. The gene expression was determined by real-time qPCR and Western blotting. Apoptosis was examined by annexin V-FITC apoptosis assay. Cell cycle analysis was performed by propidium iodide staining. Cell proliferation was analyzed by MTT assay.</p> <p>Results</p><p>HCV core protein up- or down-regulated some cellular microRNAs in Huh7 cells. HCV core-induced microRNA-345 suppressed <i>p21^Waf1/Cip1</i> gene expression through targeting its 3′ untranslated region in human hepatoma cells. Moreover, the core protein inhibited curcumin-induced apoptosis through p21^Waf1/Cip1-targeting microRNA-345 in Huh7 cells.</p> <p>Conclusion and Significance</p><p>HCV core protein enhances the expression of microRNA-345 which then down-regulates <i>p21^Waf1/Cip1</i> expression. It is the first time that HCV core protein has ever been shown to suppress <i>p21^Waf1/Cip1</i> gene expression through miR-345 targeting.</p> </div

HCV core-induced microRNA-345 inhibits <i>p21^Waf1/Cip1</i> gene expression in HepG2 cells and curcumin-stimulated Huh7 cells.

<p>(<b>A</b>) HCV core (amino acid 1–191 or 1–173)-expressing HepG2 cells were transfected with miR-345 inhibitor (5 nM or 10 nM). At 24 hours after transfection, <i>p21^Waf1/Cip1</i> gene expression at protein level was analyzed by Western blotting. β-actin served as an internal control. (<b>B</b>) HCV core (amino acid 1–191 or 1–173)-expressing Huh7 cells in response to curcumin stimulation were transfected with miR-345 inhibitor (5 nM or 10 nM). At 24 hours after transfection, <i>p21^Waf1/Cip1</i> gene expression at protein level was analyzed by Western blotting. β-actin served as an internal control (upper panel). Apoptosis was analyzed by fluorescence microscopy (middle panel) and FACS Calibur (lower panel) using Annexin V-FITC apoptosis assay. Original magnifications ×200. Cells from early apoptotic stage were stained with annexin V-FITC, and appeared green. Cells from late apoptotic stage were stained with both annexin V-FITC and PI, and merged to be yellow. Data was shown as the means ± S.D. from triplicate experiments. *<i>P</i><0.05, **<i>P</i><0.001.</p

MicroRNA-345 down-regulates <i>p21^Waf1/Cip1</i> gene expression through targeting its 3′UTR but not microRNA-93 in human hepatoma cells.

<p>(<b>A</b>) Two luciferase reporter vectors, pGL3-Control-p21 3′UTR Sense (S) and pGL3-Control-p21 3′UTR Antisense (AS) which bear the sense and antisense 3′UTRs from human <i>p21^Waf1/Cip1</i> gene, respectively, were constructed (upper panel). Huh7 cells and HepG2 cells were transfected with p21 3′UTR sense or antisense luciferase reporter vector in combination with miR-345 mimic, miR-93 mimic or a mixture of miR-345 and miR-93 mimics. At 24 hours after transfection, Huh7 cells (left lower panel) and HepG2 cells (right lower panel) were collected for luciferase reporter assay. (<b>B</b>) Base-pairing between mature hsa-miR-345 and target site in human <i>p21^Waf1/Cip1</i> 3′UTR is shown (left panel). One luciferase reporter vector, pGL3-Control-p21 3′UTR Mutant (mutated in the seed sequence of miR-345), was constructed. Mutant miR-345 mimic was synthesized. The mutated site is underlined (left panel). Huh7 cells were transfected with wild-type p21 3′UTR sense or mutant p21 3′UTR luciferase reporter vector in combination with wild-type or mutant miR-345 mimic. At 24 hours after transfection, Huh7 cells were collected for luciferase reporter assay. The experiment with mutant miR-345 mimic and mutant p21 3′UTR with restoring complementarity was performed (right panel). (<b>C</b>) HepG2 cells were transiently transfected with the increased amount of miR-345 mimic for 24 hours. The <i>p21^Waf1/Cip1</i> gene expression was analyzed by real-time qPCR (left panel) and Western blotting (right panel). β-actin served as an internal control. Data was shown as the means ± S.D. from triplicate experiments. *<i>P</i><0.05.</p

Correction: Hepatitis C Virus Core Protein Down-Regulates p21^Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cells

Correction: Hepatitis C Virus Core Protein Down-Regulates p21^Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cell

HCV core protein up-regulates miR-345 and miR-93 expression in human hepatoma cells.

<p>(<b>A</b>) Huh7 cells were transiently transfected with empty vector (labeled with Mock) and three HCV core gene-expressing vectors, pT-REx-HA-Core191 (labeled with HA-Core191), pT-REx-HA-Core173 (labeled with HA-Core173), and pT-REx-HA-Core153 (labeled with HA-Core153), for core protein with amino acids 1–191, 1–173 and 1–153, respectively. At 48 hours after transfection, the expression of HCV core protein was analyzed by immunoprecipitation. (<b>B</b>) Huh7 cells were transiently transfected with empty vector and two HCV core gene-expressing vectors, pT-REx-HA-Core191, and pT-REx-HA-Core173, for core protein with amino acids 1–191 and 1–173, respectively. At 48 hours after transfection, cellular microRNA profiling was analyzed by TaqMan low density array. Three microRNAs, miR-21, miR-345 and miR93, of thirty-one microRNAs were indicated. (<b>C</b>) Huh7 and HepG2 cells were transiently transfected with empty vector (labeled with Mock) and three HCV core gene-expressing vectors, pT-REx-HA-Core191 (labeled with HA-Core191), pT-REx-HA-Core173 (labeled with HA-Core173), and pT-REx-HA-Core153 (labeled with HA-Core153), for core protein with amino acids 1–191, 1–173 and 1–153, respectively. At 48 hours after transfection, relative expression of miR-345 or miR-93 was determined by TaqMan real-time qPCR in Huh7 cells (left upper panel) and HepG2 cells (right upper panel). The expression of HCV core protein was analyzed by Western blotting (left lower and right lower panels). (<b>D</b>) The genotype 1b strain of full-length HCV replicon (HCV-N) and control replicon HCV-A357T which expresses only the initial five amino acids of the core protein due to introduction of a termination codon, was created by the change of one nucleotide at HCV nt 357 in pHCV-N (upper panel). HCV core and NS5B gene expression in full-length HCV replicon-expressing cells was analyzed by immunoprecipitation followed by Western blotting and Western blotting only respectively (left lower panel). The relative expressions of miR-345 and miR-93 were determined by TaqMan real-time qPCR in full-length HCV replicon-expressing Huh7 cells (right lower panel). Data was shown as the means ± S.D. from triplicate experiments. *<i>P</i><0.05, **<i>P</i><0.001.</p

MicroRNA-345 inhibits curcumin-mediated apoptosis through down-regulation of <i>p21^Waf1/Cip1</i> gene expression in Huh7 cells.

<p>(<b>A</b>) Huh7 cells were treated with different doses (6.25, 12.5, 25 and 50 µM) of curcumin for 24 hours. DMSO served as control (labeled with 0 µM). The <i>p21^Waf1/Cip1</i> gene expression at protein level was determined by Western blotting (upper panel). β-actin served as an internal control. Apoptosis was determined by annexin V-FITC apoptosis assay (middle panel). Cell proliferation was analyzed by MTT assay (left lower panel). Cell cycle distribution was examined by cell cycle analysis (right lower panel). (<b>B</b>) Huh7 cells were transfected with the increased amount of <i>p21^Waf1/Cip1</i> siRNA in response to curcumin stimulation (50 µM) for 24 hours. Apoptosis was analyzed by DNA fragmentation analysis. The <i>p21^Waf1/Cip1</i> gene expression at protein level was examined by Western blotting. (<b>C</b>) Huh7 cells were transfected with the increased amount of miR-345 mimic in response to curcumin stimulation (50 µM) for 24 hours. The <i>p21^Waf1/Cip1</i> gene expression at protein level was examined by Western blotting (upper panel). β-actin served as an internal control. Apoptosis was analyzed by fluorescence microscopy (left middle panel) and FACS Calibur (left lower and right lower panels) using Annexin V-FITC apoptosis assay. Original magnifications ×200. Cells from early apoptotic stage were stained with annexin V-FITC, and appeared green. Cells from late apoptotic stage were stained with both annexin V-FITC and PI, and merged to be yellow. Cell proliferation was analyzed by MTT assay (right middle panel). Data was shown as the means ± S.D. from triplicate experiments. *<i>P</i><0.05, **<i>P</i><0.001.</p

Additional file 1: Table S1. of Quantitative methylation analysis reveals distinct association between PAX6 methylation and clinical characteristics with different viral infections in hepatocellular carcinoma

Top 50 abnormally methylated genes in HCC with different etiologic factors. (PDF 27.4 kb

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