Tristetraprolin Overexpression In Gastric Cancer Cells Suppresses PD-L1 Expression And Inhibits Tumor Progression By Enhancing Antitumor Immunity

Objective:The RNA-binding protein tristetraprolin(TTP)binds to AU-rich elements in the 3’-untranslated region of messenger RNAs and facilitates rapid degradation of target mRNAs.Therefore,it regulates the expression of multiple cancer and immunity-associated transcripts by decreasing their mRNA stability.Furthermore,a lack of TTP in cancer cells influences cancer progression and predicts poor survival.Although the functions of TTP on cancer cells have previously been researched,the mechanism of TTP on the interaction between cancer cells with their microenvironment remains undiscovered.In this study,we attended to find out the role of cancer cell TTP during the interaction between tumor and immune cells,specifically regulatory T cells(Tregs)to understand underlying mechanism of TTP on controlling antitumor immunity.Methods:Plasmid transfection was used to increase the expression of TTP in GC cells and trypan blue dye exclusion assay was performed to measure viability rate of GC cells.Western blotting was performed to assess Bcl-2 and caspase 3 expression to evaluate the effect of TTP on apoptosis.LDH release assay was performed to measure peripheral blood mononuclear lymphocytes(PBMLs)-mediated cytotoxicity against GC cells after co-culture.Flow cytometry and qRT-PCR were performed to measure the percentage changement of Tregs in PBMLs and Tregs infiltration into tumor cells.qRT-PCR and western blotting were used to study the relationship between TTP and PD-L1.Actinomycin D(Act D)was used to analyze whether TTP reduces PD-L1 mRNA stability.Luciferase assay was performed to detect whether TTP binds to AREs in the PD-L1 3’-UTR directly.Treatment of recombinant human PD-L1 or neutralizing antibody was used to study the effect of PD-L1 on the function of TTP in GC cells.Moreover,we explored correlations between TTP and PD-L1 or Tregs by IHC and conducted clinicopathological and survival analysis.Results:TTP overexpression in GC cells markedly promoted apoptosis and reduced cell survival.TTP overexpression enhanced PBMLs-mediated cytotoxicity against GC cells.Foxp3+Tregs in PBMLs or infiltration into GC cells is decreased after cocultivation in when TTP is overexpressed.TTP was found to bind to AREs in the PD-L1 3’-UTR directly and decreased its mRNA stability to suppress the expression of PD-L1 in GC cells.PD-L1 is involved in the TTP-mediated strengthening of antitumor immunity.TTP expression was remarkably reduced in GC and inversely correlated with PD-L1 expression and infiltration of Foxp3+Tregs in GC tissue,and they were also closely associated with invasion depth,lymph node metastasis,advanced TNM stage,as well as survival rate.Conclusions:Our results suggest TTP in GC cells not only affects cell survival and apoptosis but also enhances PBMLs-mediated cytotoxicity against GC cells to decelerate tumor progression.Moreover,we identified PD-L1 as a critical TTP-regulated factor that contributes to inhibiting antitumor immunity.