5.1 cells with HCV JFH-1 for different times. Western references blot analyses revealed that the HCV NS3 protein was detected at 3 days postinfection, indicating that HCV replicated well in infected cells (Fig. 1E). The levels of the p-STAT3, MMP-2, and Bcl-2 proteins in infected cells were increased in a time-dependent manner, the levels of STAT3 proteins were slightly increased by 1.17-fold at 6 days postinfection, and the level of ��-actin proteins remained relatively constant during HCV infection (Fig. 1E). It has been reported that phosphorylation of STAT3 is regulated by the JNK and ERK signaling cascade (4, 9, 61). We also revealed that the levels of the p-ERK and p-JNK proteins were increased over time after HCV infection, while the levels of the ERK, JNK, and ��-actin proteins were relatively unchanged during HCV infection (Fig.

1E). In addition, Huh7.5.1 cells were infected with JFH-1 at different concentrations. The results showed that the levels of the NS3, p-STAT3, MMP-2, Bcl-2, p-ERK, and p-JNK proteins were enhanced in cells infected with HCV in a dose-dependent fashion and the level of the STAT3 protein was slightly increased by 1.22-fold, while the levels of the ERK, JNK, and ��-actin proteins were relatively unchanged during HCV infection (Fig. 1F). Thus, we demonstrate that HCV stimulates STAT3 activity through the JNK and ERK signaling cascades, resulting in the activation of MMP-2 and Bcl-2 in hepatocytes. These in vitro results (in Huh7.5.1 cells transiently infected with HCV) are consistent with our in vivo data (in PBMCs chronically infected with HCV).

The NS4B protein of HCV activates MMP-2 and Bcl-2 expression by repressing SOCS3 production and stimulating STAT3 activity. We then determined which of the 10 proteins of HCV is responsible for the regulation of STAT3. Huh7 cells were cotransfected with the reporter plasmid pGL3-APRE-Luc, along with plasmids expressing each of the 10 HCV genes constructed previously (42). A luciferase activity assay showed that STAT3 promoter activity was activated by NS4B or NS5A, while other proteins had a slight or no effect on the STAT3 promoter (Fig. 2A). These results suggest that the HCV NS4B and NS5A proteins are involved in the regulation of STAT3 expression. Fig 2 Effects of HCV proteins in the regulation of STAT3, MMP-2, and Bcl-2 expression.

(A) Huh7 cells were cotransfected with the reporter pGL3-APRE-Luc containing the luciferase gene under the Anacetrapib control of the STAT3 promoter and pCMV-Flag2A, pCMV-core, pCMV-E1, … HCV NS5A is known to activate STAT3, interact with Bax, and inhibit apoptosis in hepatocellular carcinoma (10, 21, 55). Moreover, it has been reported that the nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without HA-Ras cotransfection (14). Thus, we explored only the role of NS4B in STAT3 activation in this study.