The Expression And Function Of ATP6V0d2 In Tumor Microenvironment And Immune Cells

Purpose:To investigate the cell-type expression and localization of ATP6V0d2 in gastric carcinoma,and the expression and function of ATP6V0d2 in immune cells including T cells and macrophages.Methods:1.RNA-Seq data of 29 pairs of gastric cancer tissues and adjacent tissues were obtained from TCGA public database.The R language was used to analyze the expression of ATP6V0d2.In addition,freshly isolated gastric cancer tissues were collected from patients and the expression of ATP6V0d2 protein was detected by Western Blot.2.mRNA and protein were extracted from three gastric cancer cell lines.Real-time quantitative PCR?RT-PCR?and Western Blot were used to detect the expression of ATP6V0d2 at mRNA and protein levels.3.Using anti-ATP6V0d2antibody,cell type expressing ATP6V0d2 in freshly isolated gastric tumor tissues was determined by immunohistochemistry.4.Flow cytometry sorted na?ve T cells were differentiated under conditions of different CD4⁺subsets differentiation for 3 days.The expression of ATP6V0d2 in CD4⁺subsets was detected by RT-PCR.5.ATP6V0d2 retrovirus plasmid was constructed and transfected into packaging cells PlatE to generate ATP6V0d2 retrovirus.6.Using overexpressing ATP6V0d2 CD4⁺T cell or ATP6V0d2^-/-T cells,the function of ATP6V0d2 in the regulation of CD4⁺T cell differentiation was determined by Flow Cytometry.7.The expression of ATP6V0d2 in different CD4⁺T subset cells was detected by Western Blot.8.The co-localization of ATP6V0d2 with CD3 or IL-17 in primary gastric cancer tissues was determined by immunofluorescence assay.9.mRNA was extracted from different tissues and immune cells.The expression of ATP6V0d2 was detected by RT-PCR.Proteins were isolated from bone marrow-derived macrophages?BMDMs?and the expression of ATP6V0d2 protein in BMDMs was detected by Western Blot.10.The BMDMs cells were stimulated with different concentrations of IL-4,LPS alone or in the presence of BAY11-7802 for the indicated time.mRNA and protein were isolated followed by RT-PCR and Western Blot to detect the expression of ATP6V0d2 in BMDMs.Results:1.ATP6V0d2 was highly expressed in human gastric cancer tissues.Instead of tumor cells,ATP6V0d2 was expressed by infiltrating immune cells in tumor microenvironment.The expression of ATP6V0d2 in immune cells is positively correlated with the differentiation status of gastric cancer.2.Different CD4⁺T cell subsets express different levels of ATP6V0d2 mRNA.Th17subset expressed high amount of ATP6V0d2 mRNA,and the expression was inhibited by IL-2.Although IL-6 could induce ATP6V0d2 expression via STAT3 activation,ATP6V0d2 did not affect CD4⁺T cell differentiation.Moreover,the expression of ATP6V0d2 protein in all T cell subsets and tumor infiltrating T cells was minor.3.ATP6V0d2 was expressed in BMDMs,as well as macrophage-enriched tissues.In BMDMs,IL-4 induced expression of ATP6V0d2 in a time and dose-dependent manner,whereas LPS suppressed ATP6V0d2 expression via NF-?B pathway.Conclusion:ATP6V0d2 is not expressed in human gastric cancer cells and has no effect on the differentiation of CD4⁺T cells.ATP6V0d2 is expressed in BMDMs and regulated by IL-4 and LPS antagonistically.ATP6V0d2 may play a role in tumorigenesis by regulating the functions of tumor associated macrophages in tumor microenvironment.