Role Of TGF?3-Smads-Sp1 Axis In DcR3-mediated Immune Escape Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most malignant tumors in the world,and is also one of the highest incidence of morbidity and mortality in China.At present,surgery,chemotherapy and radiotherapy methods are still the primary treatment for HCC,but their effects are not very satisfactory.Therefore,it is of great significance to explore molecular mechanism of HCC and to seek new strategies for the treatment of HCC based on its molecular mechanism.DcR3(decoy receptor 3,also known as TR6),a new tumor necrosis factor receptor superfamily member,is composed of 271 amino acid residues,and it is a soluble secretory protein lacking a transmembrane sequence.Currently,studies have shown that in tumor tissues,high expression of DcR3 can promote tumorigenesis,metastasis and immune escape though inducing tumor cells to escape the recognition and killing of immune cells in a variety of ways.However,molecular mechanism of high expression and immune escape of DcR3 in HCC have not been elucidated.In this study,the expression of cytokines TGF?3 and DcR3 in HCC tissues were found significantly higher than those in adjacent normal liver tissues by cytokines chip,immunofluorescence and immunohistochemistry(IHC).Real-time PCR,western blotting and IHC were used to detect the expression of DcR3 in fresh and paraffin specimens of HCC.It was found that the expression of DcR3 in HCC was up-regulated,which was related to HbsAg infection and cirrhosis of patients with HCC,as well as related to the degree of differentiation of HCC and the patient's poor prognosis.And then using co-immunoprecipitation(CoIP),chromatin immunoprecipitation assay(ChIP)and double luciferase reporter experiments,we confirmed that the up-regulated DcR3 in HCC was regulated by Smads/Sp1,which was activated by TGF?3 cytokine.Following that,we constructed stable HCC cell lines with high expression and knockdown of DcR3,and proved that high expression of DcR3 in HCC cells promoted the proliferation and migration of HCC cells and inhibited the differentiation and secretion of CD4+T lymphocytes in vivo and in vitro.Finally,we established a co-culture system of HCC cells and CD4+T lymphocytes,and proved that high expression of DcR3 in HCC cells suppressed the immune regulation of CD4+T cells by binding to ligand LIGHT in CD4+T lymphocytes by flow cytometry,immunofluorescence confocal microscopy and enzyme-linked immunosorbent assay(Elisa),and combined with cytokine chip results.Based on the above,in this study,we investigated the molecular mechanism of DcR3 expression,and confirmed that TGF?3-Smads/Spl signal pathway regulated the expression of DcR3 in HCC cells.The high expression of DcR3 in HCC cells promoted the proliferation and migration of HCC cells.At the same time,high expression of DcR3 in HCC cells inhibited the immunoregulatory function of CD4+T cells by competitively binding to LIGHT in CD4+T lymphocytes.Therefore,this study explores the preliminary mechanism of TGF?3-Smads/Spl-DcR3 signaling pathway axis-mediated immunosuppression of hepatocellular carcinoma,which provides a theoretical basis for seeking new strategies for immunotherapy of HCC.