MiR-128-3p Affects Differentiation Of CD4~+Treg Cells By Targeting IL-16 In Gastric Cancer

Objective:This study investigated whether miR-128-3p is involved in the process of gastric cancer(GC)cells releasing immune-related cytokines or affecting the infiltration of immune cells in the tumor microenvironment(TME)of GC,thereby promoting tumor immune escape.Methods:Bioinformatics analysis was performed to predict putative downstream target genes regulated by miR-128-3p in GC.Gene Set Enrichment Analysis(GSEA)was used to analyze the correlation between different immune cells and the expression of IL-16 and miR-128-3p.Seven pairs of fresh GC tissue samples and peripheral blood of 7 GC patients were collected,and quantitative real-time reverse(qRT-PCR)and western blot(WB)were used to analyze the expression of miR-128-3p mRNA and IL-16 protein in GC tissue.Luciferase assay were conducted to explore the mechanisms by which miR-128-3p regulates IL-16.The concentrations of IL-16 in the supernatants of GC cells were detected by ELISA.Then lymphocytes were separated from peripheral blood of GC patients and cocultured with GC cells,and the proportion of Treg subgroups was evaluated by flow cytometry.Results:In the present study,the differentially expressed analysis of relevant cytokines between the high miR-128-3p group and the low miR-128-3p group were identified in The Cancer Genome Atlas(TCGA)GC samples.It was found that the expression of cytokines such as IL-16,SEMA4C and STC1 were related to low miR-128-3p group,and may be the direct target genes of miR-128-3p.The expression of IL-16 in low miR-128-3p group was significantly increased in fresh GC tissues.In addition,we found that two potential binding sites of miR-128-3p exist in the 3’UTR region of IL-16.Luciferase reporter gene assay showed that miR-128-3p could negatively regulate the expression of IL-16.GSEA was used to analyze the correlation between different immune cells and the expression of IL-16 and miR-128-3p.among different immune cells,Tregs were most significant positively correlated with the expression of IL-16 and had a negative correlation with miR-128-3p expression.At the cellular level,Increasing IL-16 secretion level or recombinant human IL-16 significantly stimulated the proportion of CD4~+CD25~+FOXP3~+Tregs in cultured lymphocytes in vitro.Conclusion:The abnormal activation of miR-128-3p/IL-16 axis in the TME of GC can promote the differentiation of CD4~+CD25~+FOXP3~+Tregs,thus promoting the immune escape of gastric cancer.These results suggest that the targeting strategy of blocking the interaction between miR-128-3p,IL-16 and Tregs may have the potential to resist the immune escape of GC.