Epstein-Barr virus-encoded miR-BART5-5p upregulates PD-L1 through PIAS3/pSTAT3 modulation, worsening clinical outcomes of PD-L1-positive gastric carcinomas.

Abstract

Epstein-Barr virus (EBV) is etiologically associated with ~ 10% of all gastric carcinomas. However, the molecular mechanisms and roles of EBV miRNAs in gastric carcinoma oncogenesis are yet to be elucidated. MicroRNA microarray and TaqMan quantitative real-time RT-PCR were conducted. RT-PCR and luciferase reporter assay for PIAS3, western blotting for 20 proteins, immunofluorescence for STAT3, transfection with miRBART5-5p-plasmid, STAT3-plasmid, miRBART5-5p mimic, or PIAS3-siRNA, and in vitro assays for biological effects of PD-L1 were implemented. In situ hybridization for EBV-encoded small RNAs and immunohistochemistry were performed on gastric carcinoma tissues. Transfecting miR-BART5-5p into EBV(-) gastric carcinoma cell lines caused a decrease in PIAS3 3'-UTR reporter activity, PIAS3 downregulation, and subsequent STAT3 activation followed by PIAS3/pSTAT3-dependent PD-L1 upregulation. Interestingly, due to PD-L1 knockdown, apoptosis was increased, while the rate of cell proliferation, invasion capacity, and migration were decreased in miR-BART5-5p-transfected cells. In EBV(+) gastric carcinoma cells, anti-miR-BART5-5p reduced PD-L1 levels through PIAS3/pSTAT3 control. Among 103 patients with EBV-associated gastric carcinomas, overall survival was significantly shortened for those with PD-L1(+) tumors compared to those with PD-L1(-) tumors (P = 0.049). Our findings imply that miR-BART5-5p directly targets PIAS3 and augments PD-L1 through miR-BART5/PIAS3/pSTAT3/PD-L1 axis control. This contributes to antiapoptosis, tumor cell proliferation, invasion and migration, as well as immune escape, furthering gastric carcinoma progression and worsening the clinical outcome, especially in the PD-L1(+) group of patients with EBV-associated gastric carcinomas. miR-BART5-5p may, therefore, be amenable to PD-1/PD-L1 immune checkpoint inhibitor therapy.