| Background:The data about the main death of cancer in 2015 from?CA:a cancer journal for clinicians?showed that liver cancer is ranked second in males and six in females.The incidence of liver cancer in China went account for 50%of the total number of patients with liver cancer,and more than 90%liver cancer is hepatocellular carcinoma.Cancer immunoediting holds that the immune system could recognize and eliminate tumors,while tumor could escape from the surveillance of immune system and that is regarded as one of the tumor special signs.Researchers showed that glycosylation plays an important role in tumor immune escape.Protein glycosylation is completed by glycosyltransferase which catalyze glycan in the form of a covalent bond connecting with ploypeptide.Sialylation is one of the important ways of glycosylation,mainly by the action of sialyltransferases(STs)which catalyzed the transfer of sialic acids through an?2,3,?2,6 or?2,8-bonds to the end of glycoproteins and glycolipids.In addition,aberrant glycosylation is one of the most prominent feature of carcinogenesis that influences tumor immunity,angiogenesis and multiple steps of tumor progression.Hypersialylation is one of the most common glycosylation changes in cancer and its capacity to promote tumor growth has been recognized in the late 1960s.Meanwhile distinct glycosylation patterns evolving from this‘microevolutionary'process could confer advantages in terms of tumor growth,tumor dissemination,and immune escape.Studies have shown that,the up-regulated expression of?2,6 sialylation catalyzed by?-galactoside?2,6 sialyltranferase-1(ST6Gal-?)has been observed in many types of the cancers,including breast,ovarian and cervix.However,the mechanisms of immune evasion induced by ST6Gal-? mediated?2,6-sialylation in HCC still remains poorly understood.Objective:1.To detect the role of exogenous expression ST6Gal-? in co-culture system of hepatocellular carcinoma cells and T cells(Jurkat cells).2.To explore the effects of ST6Gal-? upregulation on the secretion of immunosuppressive and immunoactivative cytokines in co-culture system.3.To detect ST6Gal-? upregulation on the expression of migration and apoptosis related proteins in HepG2 cells and the changes of apoptosis related proteins in Jurkat cells,meanwhile to investigate the molecular mechanisms involved in ST6Gal-? induced tumor immune escape.4.To observe the effect on tumor cells growth in C57BL/6J mice induced by the change of ST6Gal-? expression,and the mouse T cell infiltration in tumor tissue,meanwhile we have preliminary explored its function on tumor immune escape in vivo.Method:1.Establishment of stable cell lines with exogenous expression of ST6Gal-? in HepG2 cells,the mRNA and protein levels of ST6Gal-? were measured using real-time PCR and Western-blot assays and SNA lectin blot was used to determine the?2,6-linked sialic acid levels.2.Cell Counting Kit-8 and Transwell migration assays were used to determine the proliferation ability and migration ability of HepG2 and HepG2/ST cells,meanwhile Annexin V-FITC/PI double staining and TUNEL assay were used to determine the apoptosis of Jurkat cells and tumor cells after co-cultured with each other.3.ELISA assay was performed to examine the secretion of relative cytokines after Jurkat cells co-cultured with tumor cells,meantime Western blot assay and Agarose SNA immunoblotted to determine the evels of?2-6 sialylation for CD147,Fas and integrin-?3 after ST6Gal-? overexpression.4.Western blot assay was performed to analysis the expression of CD147/MMPs and Fas-FasL signaling pathway and Bcl-2family in HepG2 cells after ST6Gal-? overexpression,also to analysis the changes of PI3K/Akt/NF-?B signaling pathway and mitochondrial apoptosis pathway and Bcl-2family in Jurkat cells.5.Hepa1-6 and Hepa1-6/ST cells were inoculated subcutaneously into the inguina of C57BL/6J mice,respectively,inflammatory cells infiltration was observed with the method of paraffin tissue sections for H&E and IHC stainings for CD4~+and CD8~+T cells,even qPCR and ELISA assays were performed to detect the secretion of TNF-?and IFN-?in tumor and serum.6.Sorting T cells of high purity from C57BL/6J mouse spleen,tumor tissue and human blood by FCM,then make them co-cultured with different ST6Gal-? expressed mouse or human hepatocellular carcinoma cells,CCK8 was used to detect the proliferation of CD8~+T cells and ELISA was used to detect the secretion of IL-2 from CD4~+T cells.Result:1.The mRNA,protein expression and?2,6-linked sialic acid levels ofST6Gal-? in HepG2 monoclonal cell line were significantly increased.2.ST6Gal-? overexpression promoted the proliferation and migration of hepatocellular carcinoma HepG2 cells,inhibited the survival of Jurkat cell and reduced apoptosis in HepG2 cells after co-cultured in vitro.3.ST6Gal-? overexpression in tumor cells significantly increased the levels of immunosuppressive cytokines TGF-?1 and IL-10,while diminished the levels of immunoactivative cytokines IL-6,TNF-?,IFN-?and IL-2 in HCC microenvironment,respectively,increased the amount of?2-6-sialylation on CD147 and Fas,which increased more obviously after co-cultured with Jurkat cells.4.CD147,MMP2/7/9,Bcl-2 and FasL levels were significantly increased in HepG2/ST1,2cells,while Bax,Bad expression were decreased and Fas expression were no difference.The levels of Fas,p-GSK-3?,Bad,Bax,cytochrome c and the cleaved caspase-9,3were significantly increased in the Jurkat cells,while PI3K,p-Akt,p65-NF-?B,Bcl-2expression were decreased and FasL,Akt,GSK-3?expression were no difference.5.The tumor size of Hepa1-6/ST graft and Hca-P/ST graft were significantly increased compared to the control group,meanwhile ST6Gal-? overexpression significantly decreased cell intratumoral penetration by CD8~+T lymphocytes in both models and decreased the expression levels TNF-?and IFN-?.6.The proliferation of splenic,human blood and intratumoral CD8~+T lymphocytes were significantly inhibited after co-cultured with ST6Gal-? up-regulated tumor cells compared to the control groups and the content of IL-2 from CD4~+T cells was significantly decreased.Conclusion:1.ST6Gal-? overexpression could promote the proliferation and migration of hepatocellular carcinoma HepG2 cells in vitro,then reduce its apoptosis mediated by immune cells.2.ST6Gal-? overexpression in tumor cells could inhibit the proliferation of Jurkat cells and promote the apoptosis.3.ST6Gal-? overexpression in tumor cells significantly diminished the levels of immunoactivative cytokines and increased the levels of immunosuppressive cytokines,then effected the tumor microenvironment.4.ST6Gal-? upregulation could promoted the CD147/MMPs signaling pathway in HepG2 cells and inhibit the Fas mediated apoptosis,which induced tumor immune escape.5.ST6Gal-? overexpression in tumor cells down-regulates the PI3K/Akt/NF-?B signaling pathway in Jurkat cells,which induced that cell anergy.6.ST6Gal-?-upregulation could enhance the tumorigenicity of tumor cells and suppress intratumoral penetration of CD8~+T Lymphocytes,then reduce the expression levels of TNF-?,IFN-?and IL-2 to suppress the anti-tumor activity of immune cells. |
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