Effect Of ARID1A On Tumor Immunity Of Gastric Cancer And Its Significance In Immunotherapy

Part1 Correlation between expression level of ARID1 A in gastric carcinoma and tumor immune microenvironment and its clinicopathological significance Objective: The expression of ARID1 A in gastric cancer was investigated in our study.The correlation between the expression of ARID1 A and tumor immune microenvironment and its clinicopathological clinicopathology was analyzed.Methods: 90 gastric carcinoma specimens were harvested from Union Hospital of Tongji Medical College of Huazhong University of Science and Technology.Expression of ARID1 A and CD8 in gastric cancer tissues was evaluated by immunohistochemical test,then the relationship between the expression level of ARID1 A and tumor immune microenvironment and its clinicopathology was analyzed.Results: The expression of ARID1 A was high in 69 cases and low in 21 cases(23.3%).Chi-square test confirmed that low ARID1 A expression correlated with a female sex(0.025),deep invasion(0.049),advanced TNM stage(0.005)and low CD8+ T lymphocytes expression score(< 0.001),but had no obvious correlation with the age(0.145),tumor location(0.732),differentiation(0.732),Lymph node positive(0.110),N stage(0.732)and blood vessel invasion(0.148).Pearson test showed that the content of CD8+T lymphocytes in gastric cancer tissues was positively correlated with the expression level of ARID1A(P<0.0001).Conclusions: Low expression of ARID1 A is correlated with deep depth invasion and advanced TNM stage,suggesting a poor oncology prognosis.The content of CD8+T lymphocytes in gastric cancer tissues was positively correlated with the expression level of ARID1 A,and the low expression of ARID1 A was correlated with the decreased content of CD8+T lymphocytes in gastric cancer tissues.Part2 Effect of ARID1 A on immune microenvironment of gastric cancer Objective: To study the effect of ARID1 A on tumor immune microenvironment of gastric cancer and its mechanism.Methods: Lentivirus transfection was used to construct stable knockdown of ARID1 A in AGS,SNU-1,SNU719 and MFC cell lines.Western blot and q RT-PCR were used to verify the knockdown efficiency of ARID1 A.Using q RT-PCR and ELISA to test the m RNA and protein expression of Th1-type chemokines(CXCL9 CXCL10)respectively.Using ARID1 A knockdown MFC cells to build gastric xenograft model.Tumor growth was observed in each group.Flow cytometry was used to test the content lymphocytes in transplanted tumor tissures.The CD8+ T lymphocyte infiltration was detected by using the immunohistochemical.Results: The decreased expression level of ARID1 A in gastric cancer cells down-regulated the expression level of Th1-type chemokine in human gastric cancer cell lines.Tumor volume were increased more rapidly in the ARID1 A knockdown group,and poor oncology prognosis was observed.Low expression of ARID1 A reduced the CD8+T lymphocytes infiltration in tumor microenvironment and weaken the autoimmune anti-tumor immunity.Conclusions: The decreased expression level of ARID1 A gene in gastric cancer cells caused the decrease of CD8+T lymphocytes in the tumor immune microenvironment by reducing the expression of Th1 chemokine in gastric cancer,thus downregulating the autoimmune anti-tumor immunity.Part3 The therapeutic effect of epigenetic regulation on ARID1A low expression gastric cancer and its mechanism Objective: To investigate the therapeutic effect of an epigenetic regulator,HDAC inhibitor(LBH589),on low expression of ARID1 A in gastric cancer and its molecular mechanism Methods: The histone acetylation level of gastric cancer cells after ARID1 A knockdown was analyzed by Western blot.Then,the ARID1 A knockdown gastric cancer cells were treated with histone deacetylase inhibitor LBH589,and their acetylation level was analyzed by Western blot.The expression of Th1 chemokines in ARID1 A knockdown gastric cancer after treatment with LBH589 was tested by q RT-PCR and ELISA.Primers were designed for CXCL9 and CXCL10 promoters,and chromatin immunoprecipitation test was conducted to verify that LBH589 treatment could promote the binding of STAT1 to CXCL9 and CXCL10 promoters.Immunofluorescence staining was used to detect the nucleus protein expression in gastric cancer cell nucleus protein to analyze its character change of chromatin.Using ARID1 A knockdown MFC cells to build gastric xenograft model.Flow Cytometry was used to detect the contents of T lymphocytes,TNF ? and IFN-? in tumor tissues.Results: The level of histone acetylation was decreased in ARID1 A knockdown gastric cancer.LBH589 could significantly up-regulate the histone acetylation level of ARID1 A knockdown gastric cancer and increase the expression of CXCL9 and CXCL10.Chromatin immunoprecipitation assay confirmed that LBH589 treatment can promote the binding of STAT1 to CXCL9 and CXCL10 promotors,thereby promoting the transcription of CXCL9 and CXCL10.Immunofluorescence staining showed that after knockdown of ARID1 A in gastric cancer,HP-1? protein expression in the nucleus was up-regulated,LBH589 could down-regulate HP-1? protein expression.Mouse experiments showed that LBH589 could significantly reduce the volume of xenograft tumor of the ARID1 A knockdown group and improve the oncology prognosis of the tumor.Flow cytometry showed that LBH589 could significantly up-regulate the content of T lymphocytes,TNF-? and IFN-? in the tissues of ARID1 A knockdown group.Conclusion: The knockdown of ARID1 A down-regulated the level of histone acetylation,resulting the change of chromatin character,by increasing the closure of nucleosome,thus down-regulating the "accessibility" of related genes encoding Th1-type chemokine,which leads to the decrease of the expression level of Th1-type chemokine.By reversing this epigenetic change,the histone deacetylase inhibitor LBH589 up-regulated the histone acetylation level of ARID1 A knockdown gastric cancer cells,opened the nuclear chromatin of ARID1 A knockdown gastric cancer cells,restored the "accessibility" of key genes,and thus restored the expressions of CXCL9 and CXCL10.In vivo experiments,we confirmed that LBH589 could effectively up-regulate the CD8+T lymphocyte content in the tumor microenvironment and improve the immunosuppressive state of the ARID1 A knockdown gastric cancer,thus effectively treating the gastric cancer with low expression of ARID1 A.Part4 Epigenetic regulation enhances the therapeutic effect of ARID1 A low expression gastric cancer against PD-L1 antibodyObjective: To study the therapeutic effect of Histone deacetylase inhibitor combined with PD-L1 antibody on ARID1 A low expression gastric cancer,and to explore the sensitization effect of epigenetic therapy on immune checkpoint inhibitorMethods: A model of implanted tumor model with complete immune function were constructed by using ARID1 A knockdown mice gastric cancer cells.The therapeutic effect of PD-L1 combined with LBH589 on ARID1 A knockdown gastric cancer cells was evaluated in vivo,and the therapeutic effect of monotherapy and combined treatment was analyzed and compared.Results: The efficacy of PD-L1 antibody alone in the treatment of ARID1 A knockdown group was undesirable.The combination of histone deacetylase inhibitor LBH589 with PD-L1 could effectively treat the knockdown group.Conclusions: HDAC inhibitor--LBH589 enhances the therapeutic response of tumor cells to PD-L1 antibodies,immuno-checkpoint inhibitor,by regulating epigenetic changes in gastric cancer with low expression of ARID1 A.