The Role Of IL-23 In The Development Of Hepatocellular Carcinoma

Part one Construction of murine single chain IL-23 expressing plasmid and its hepa1-6 IL-23-expressing cell line.Objective: To construct murine single chain IL-23 expressing plasmid and hepa1-6 IL-23-expressing cell line.Methods: The fragment of p19 and p40 were amplified by RT-PCR from the total RNA extracted from hepa1-6 cells. p19 and p40 fragment were linked via a linker(Gly4Ser)3 by overlap extension PCR to obtain IL-23 fusion gene. And the gene was inserted into the Venus lentiviral vector to form the plasmid Venus-IL-23. Lentivirus was generated by transfecting Venus-IL-23 or Venus into the 293 T packaging cell line. hepatocellular carcinoma(HCC) cells were infected for 24 h with 10 ml of IL-23 or vector lentiviral supernatant. Then single cells were sorted into 96-well plates by flow cytometry. The sorted cells were cultured in DMEM containing 10% FBS, and then stably infected cell lines expressing similar levels of YFP were harvested.Results: Sequence analysis showed that IL-23 plasmid was correctly constructed.Conclusion: Murine single chain IL-23 expressing plasmid and hepa1-6 IL-23-expressing cell line were successfully established.Part two Impacts of IL-23 expression on HCC cell proliferation, cell cycle, apoptosis and migrationObjective: To analyze the impact of IL-23 expression on HCC cell proliferation, cell cycle, apoptosis and migration.Methods: In vitro experiments were performed to analyze the impact of IL-23 on HCC cell proliferation, cell cycle, apoptosis and migration. MTT assay and Ki-67 staining were used detect the impact of IL-23 expression on the proliferation of HCC cells. Flow cytometry was used to analyze the apoptosis and cell cycle of HCC cells. Wound healing and transwell assay were performed to detect the role of IL-23 in the migration of HCC cells.Results: MTT assay and Ki-67 staining showed that IL-23 promoted the proliferation of HCC cells; flow cytometry results demonstrated that IL-23 had no effect on the cell apoptosis of HCC cells; cell cycle results showed that IL-23 expression increased the HCC cells in S and G2 phases; wound healing and transwell assay suggested that IL-23 promoted the migration of HCC cells.Conclusion: IL-23 could directly promote HCC cell proliferation and increase the percent of cells in G2/S phases in vitro, but did not affect the apoptosis. The direct effect of IL-23 also involved enhancing the mobility of HCC cells.Part three The role of IL-23 in the development of HCCObjective: To explore the role of IL-23 in the development of HCC.Methods:(1) Generation of murine HCC model:(a) hepa1-6 cells stably expressing IL-23 or vector control(1 × 106/2 ml) were injected into mice using hydrodynamic gene transfer(HGT)method, 3 weeks later, mice were sacrificed and the number of tumor nodes were counted(b) Mice were given drinking water with antibiotic(80000 U/250 ml) before the surgery. hepa1-6 cells stably expressing IL-23 or control vector(1×106/25 μl) were in situ transplanted into the mice by surgery. 2 weekslater, mice were sacrificed and the tumor volumes were measured;(c.) 14 days-old mice were injected with DEN(25 mg/kg) intra-peritoneally. IL-23 or vector micro-plasmid were injected hydropdymically every other month. 8 months later, mice were sacrificed and tumor volumes and the number of tumor nodes were measured.(2) The percentages of effect CD4+ T cells, CD8+ T cells, myeloid derived suppressor cells(MDSC) and Treg cells were analyzed by flow cytometer. Cytokine productions by the effector cells, including IFN-γ, TNF-α and IL-17 A, were measured by intracellular staining. IL-17 A knockout mice were used to confirm whether the effect of IL-23 in HCC development was mediated by IL-17 A. The cytokine levels in serum were measured by CBA.Results: The promoting role of IL-23 in the development of HCC was confirmed by three HCC models. The flow cytometry results demonstrated that the percentages of MDSCs and Tregs were significantly increased during the late phase of HCC development. The percentage of IFN-γ+TNF-α- CD8+ T cells were also slightly increased, while the percentages of IFN-γ+TNF-α+ CD8+ T cells and IFN-γ-TNF-α+ CD8+ T cells were decreased significantly. During the early phase of HCC development, IL-23 significantly promoted the production of IL-17 A, especially the IL-17 A produciton by group 3 innate lymph cells(ILC3) cells.Conclusion: IL-23 promoted the development of HCC through its enhancement of IL-17 A production mainly by ILC3 cells. The increased IL-17 A production further suppressed the anti-tumor immune response.