Fig 1.
Determination of the effect of HCV on COX-2 expression.
(A, B) Regulation of COX-2 expression in hepatoma cells is mediated by HCV. pCOX-2-Luc was transfected into Huh7, Ava5, and JFH-1-infected Huh7 cells to measure the effect of COX-2 on HCV protein expression. The cell lysates of Huh7, Ava5 (harboring HCV subgenomic replicon), and JFH-1-infected Huh7 cells were subjected to western blotting. (C) Identification of the viral proteins of HCV that mediate COX-2 regulation. Huh7 cells were co-transfected with pCOX-2-Luc and different viral protein expression vectors (0.5 μg). (D) HCV NS5A induces COX-2 transcriptional activity. Huh7 cells were co-transfected with pCOX-2-Luc and different amounts of pCMV-NS5A-Myc (0–2 μg). After incubation for 3 days, the cell lysates were subjected to the luciferase activity assay to measure the induction of the COX-2 promoter. The activity of Ava5 cells was the positive control. “Mock” indicates the co-transfection of empty vector, pcDNA4/myc, and pCOX-2-Luc. Data shown are mean ± SE; n = 3. *p < 0.05. **p < 0.01.
Fig 2.
Elucidation of the regulation of IL-8 expression in hepatoma cells with HCV infection and HCV NS5A.
(A) Induction of IL-8 transcription by HCV infection and HCV NS5A expression. Huh7 cells were infected with JFH-1 and transfected with pCMV-NS5A-Myc. After 3 days, the total cellular RNA was extracted to quantify the relative RNA levels of IL-8 normalized to gapdh expression using qRT-PCR. (B) Induction of IL-8 production by HCV NS5A expression. Huh7 cells were transfected with pCMV-NS5A-Myc and the relative RNA and protein levels of IL-8 were analyzed by using qRT-PCR and western blot. (C) After 3 days, the cell supernatant was analyzed to quantify the amount of IL-8 using an ELISA kit, as described in the Materials and Methods. Mock control indicates Huh7 cells transfected with the vehicle vector. Data shown are mean ± SE; n = 3. *p < 0.05. **p < 0.01.
Fig 3.
Analysis of the role of IL-8 in the regulation of COX-2 expression mediated by HCV.
Induction of COX-2 (A) promoter activity, (B) RNA levels, (C) protein expression in a dose-dependent manner. The Huh7 cells were co-transfected with pCOX-2-Luc and different amounts of pCMV-IL-8-Myc (0–2 μg). After incubation for 3 days, the cell lysates were subjected to luciferase activity assay to measure the induction of COX-2 promoter by Steady-Glo luciferase assay system. The Huh7 cells were transfected with different amounts of pCMV-IL-8-Myc (0–2 μg). After 3 days incubation, the total RNA and cell lysates were extracted and analyzed by qRT-PCR and western blot, respectively. GAPDH shows the loading control. (C, D) Determination of the effect of IL-8 shRNA on COX-2 expression by NS5A. Huh7 cells were co-transfected with pCOX-2-Luc, pCMV-NS5A-Myc (1 μg), and different amounts of IL-8 shRNA (0–1 μg). After 3 days of incubation, the total cell lysates were extracted and analyzed using the luciferase assay and western blotting. (F) Determination of the effect of HCV and IL-8 on PGE2 expression. The Huh7 cell were transfection with pCMV-NS5A-Myc combined with or without sh-IL-8 or treated with recombinant IL-8. The Huh7 cells transfected with pCMV-COX-2-Myc served as a positive control of PGE2 production. After 3 days, the intercellular PGE2 levels were assayed with the Biotrak PGE2 enzyme immunoassay system. The relative IL-8 and COX-2 RNA levels were normalized by gapdh expression. GAPDH was used as an equal loading control. Data shown are mean ± SE; n = 3. *p < 0.05. **p < 0.01.
Fig 4.
Analysis of the effects of ERK and JNK signaling pathways on COX-2 expression mediated by HCV.
(A, B) Huh7 cells were infected with HCV JFH-1 or transfected with pCMV-NS5A-Myc. Following infection or transfection, the cells were grown in complete medium. The cell lysates were collected and subjected to western blotting with specific antibodies for MAPK (ERK, p38, and JNK) and phospho-MAPK. (C-F) To identify the COX-2 activation through ERK and JNK activation upon HCV NS5A expression or HCV infection, Huh7 cells were co-transfected with pCOX-2-Luc with pCMV-NS5A-Myc or infected with HCV JFH-1. Following transfection or infection, the cells were grown in complete medium with or without the specific ERK inhibitor U0126 and JNK inhibitor SP600125. The cell lysates were collected and subjected to the luciferase activity assay to measure COX-2 promoter activity and western blotting with antibodies specific for COX-2 and GAPDH. (G) Huh7 cells were co-transfected with pCMV-NS5A-Myc (1 μg), and different amounts of IL-8 shRNA (0–1 μg). After 3 days, the cell lysates were collected and subjected to western blotting with antibodies specific for MAPK (ERK, p38, and JNK) and phospho-MAPK. GAPDH was used an equal loading. Data shown are mean ± SE; n = 3. *p < 0.05. **p < 0.01.
Fig 5.
Determination of the effects of C/EBP on COX-2 expression regulated by HCV.
(A) Diagrams of COX-2 promoter with series deletion containing binding element of transcription factor, which includes the wild-type COX-2 promoter (WT, −891/+9) and series deletion (ΔGRE, ΔGRE/NF-κB, ΔGRE/ NF-κB /C/EBP). (B, C) Huh7, JFH-1 infected Huh7, and Ava5 cells were co-transfected with each of the COX-2 reporter clones for analyzing the induction of COX-2 transcription factors. (D) The COX-2 reporter vector, pCOX-2(ΔGRE/NF-κB)-Luc or pCOX-2(ΔGRE/NF-κB/C/EBP)-Luc (1 μg) was transfected into Huh7 cells with or without pCMV-NS5A-Myc. After 3 days of incubation, the cell lysates were subjected to the luciferase assay to measure the induction of COX-2 promoter activity. (E) The each transcription factor influenced induction fold change of COX-2 promoter in Ava5 cells or HCV NS5A-expression Huh7 cells. (F) Huh7 cells were transfected with pCOX-2(ΔGRE/NF-κB-C/EBPmut)-Luc with NS5A overexpressing and JFH-1-infected cells. The promoter activity was assayed by the luciferase system. (G, H) Huh7 cells were transfected with pCMV-NS5A-Myc or infected with HCV JFH-1 and the total expression (upper panel) and nucleus translocation level (lower panel) of C/EBP was analyzed by western blotting. (I, J) Huh7 cells were co-transfected with pCOX-2-Luc with pCMV-NS5A-Myc or infected with HCV JFH-1 combined with C/EBP knock-down. The cell lysates were collected and subjected to western blotting and luciferase assay analysis, respectively. Lamin B1 and GAPDH were used as an equal loading controls. Data shown are mean ± SE; n = 3. *p < 0.05. **p < 0.01.
Fig 6.
Determination of the effects of IL-8 knockdown on inflammatory cytokine production mediated by HCV NS5A expression and HCV infection.
(A-D) Huh7 cells were transfected with IL-8 shRNA expression vector (0.5 μg) and then infected with HCV JFH-1 for 6 h. After 3 days of incubation, the total cellular RNA was extracted and analyzed using qRT-PCR. (E-H) Huh7 cells were co-transfected with pCMV-NS5A-Myc (1 μg) and IL-8 shRNA expression vector (0.5 μg). After 3 days of incubation, the total cellular RNA was extracted and analyzed using qRT-PCR. The relative TNF-α, iNOS, IL-1, and COX-2 RNA levels were normalized by gapdh expression. Data shown are mean ± SE; n = 3. *p < 0.05. **p < 0.01.
Fig 7.
Identification of the roles of IL-8 and COX-2 in HCV replication.
(A) Ava5 cells were transfected with IL8-shRNA in a concentration-dependent manner (0–2 μg). After 3 days of incubation, the total cell lysates were extracted and HCV protein synthesis was analyzed by western blotting. GAPDH was used as the equal loading control. (B) Huh7 cells were transfected and treated with pCMV-NS5A-Myc, pCMV-COX-2-Myc, recombinant IL-8 protein, and PGE2. The transfected and treated cells were incubated with or without NS398 for 3 days. The relative HCV RNA levels were analyzed using qRT-PCR. The HCV RNA level of JFH-1-infected Huh7 cells was defined as 1. Data shown are mean ± SE; n = 3. *p < 0.05. **p < 0.01.
Fig 8.
A proposed model of COX-2 up-regulation by HCV infection.