Aberrant Expression Of P-STAT3 In Peripheral Blood CD4~+ And CD8~+ T Cells Related To Hepatocellular Carcinoma Development

Aim:To detect expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in peripheral blood CD4+ and CD8+T cells from hepatocellular carcinoma (HCC) patients to investigate whether aberrant expression of p-STAT3 in peripheral CD4+ and CD8+ T cells relate to HCC pathogenesis.Methods:In Vitro Group:Establish co-culture system of peripheral blood mononuclear cells (PBMCs) and hepatoma cell line Huh-7 in vitro. Huh-7 cells were seeded in twelve-well plates at densities of 1.25×105/0.5ml,2.5×105/0.5ml, 5×105/0.5ml cells per well. Samples were incubated for 4 h, and 0.5 ml of PBMCs (5×105/ml) was added to each well. After 24 h,36 h,48 h and 60 h, PBMCs were harvested. p-STAT3 levels in CD4+ and CD8+ T cells of PBMCs were detected by flow cytometry. In addition, the supernatants were collected, the production of IL-4, IFN-y, IL-10, and IL-6 in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Spearman’s correlation analysis was used to determine the correlation between the ratio of IFN-y/IL-4 and levels of IFN-y, IL-4, IL-6, IL-10, and p-STAT3 in CD4+T and CD8+T cells in the co-culture system of 48h. In Clinical Trials Group:Anticoagulated and non-anticoagulated peripheral blood was collected from patients with HCC and healthy controls. p-STAT3 levels in peripheral blood CD4+and CD8+T cells were detected by flow cytometry. Serums were extracted, the production of IFN-y, IL-4, IL-6, and IL-10 in serums were measured by ELISA. Spearman’s correlation analysis was used to determine the correlation between the ratio of IFN-y/IL-4 and levels of IFN-γ, IL-4, IL-6, IL-10, and p-STAT3 in CD4+ and CD8+ cells in patient samples.Results:In vitro Group:p-STAT3 levels in CD4+T cells from PBMCs co-cultured with Huh7 cells were increased than in PBMCs cultured with medium alone(p<0.05), and p-STAT3 levels in CD8+ T cells from PBMCs co-cultured with Huh7 cells were also increased than in PBMCs cultured with medium alone(p<0.05). The expressin of IFN-y were down-regulated whereas IL-4, IL-6, and IL-10 were up-regulated in supernatants of Huh7 cells co-cultured with PBMCs than in controls (p<0.01). However, with time of co-culture and with the increasing ratio of PBMCs to Huh7 cells, the expression of p-STAT3 in CD4+cells was increased (p<0.05), the expression of p-STAT3 in CD8+cells was also up-regulated (p<0.05). The expressin of IFN-y were decreased while IL-4, IL-6 and IL-10 were increased in supernatants of Huh7 cells co-cultured with PBMCs than in controls (p<0.01). The ratio of IFN-y/IL-4 and the levels of IFN-γ correlated negatively with p-STAT3 in CD4+T cells from PBMCs co-cultured with Huh-7 cells, and the ratio of IFN-y/IL-4 and the levels of IFN-y also correlated negatively with p-STAT3 in CD8+T cells. IL-4, IL-6, and IL-10 levels correlated positively with p-STAT3 levels in CD4+T cells, as well as IL-4, IL-6, and IL-10 levels correlated positively with p-STAT3 levels in CD8+T cells. In Clinical Trials Group:The expression of p-STAT3 in CD4+T cells T cells were all higher in HCC patients than in healthy controls (p<0.001), and the expression of p-STAT3 in CD8+T cells T cells were also all higher in HCC patients than in healthy controls(p<0.001). IFN-γ levels were lower whereas IL-4, IL-6, and IL-10 levels were higher in serum of HCC patients than in controls (p<0.001). The ratio of IFN-γ/IL-4 and the levels of IFN-γ correlated negatively with p-STAT3 in peripheral CD4+ cells in patient samples, as well as, the ratio of IFN-y/IL-4 and the levels of IFN-γ correlated negatively with p-STAT3 in peripheral CD8+ cells..however, IL-4, IL-6, and IL-10 levels correlated positively with p-STAT3 levels in CD4+T cells, as well as, IL-4, IL-6, and IL-10 levels correlated positively with p-STAT3 levels in CD8+T cells.Conclusions:The expression of p-STAT3 was up-regulated in peripheral CD4+and CD8+T cells of HCC patients resulted in abnormal immune surveillance and contributed to HCC pathogenesis.