Molecular mechanism of exosomal delivery of miR-15a/16 targeting PD-L1 to inhibit immune escape in gastric cancer

QU Yajing; NING Tao; LI Zhihong; SHI Xiaoxiong; HUO Lili; CHEN Hui; REN Guibing

doi:10.3969/j.issn.1009-0460.2025.02.001

Abstract：Objective To determine whether artificially modified exosomes transporting miR-15a/16 target programmed death receptor-ligand 1(PD-L1)and inhibit immune escape in gastric cancer.Methods The protein expression levels of PD-L1 in gastric cancer and normal tissues were analyzed by western blotting.The levels of miR-15a and miR-16 in plasma and tissue of gastric cancer patients were detected by quantitative real-time polymerase chain reaction(qRT-PCR).Bioinformatics database was used to predict PD-L1 mRNA of downstream regulatory target gene of miR-15a/16,and the luciferase reporter gene experiment verified the targeted binding relationship.In vitro,NC,miR-15a,miR-16,miR-15a/16 mimics and inhibitors were transfected into gastric cancer cell line,and PD-L1 protein and mRNA levels were detected in each group.The modified exosomes exo-miR-15a,exo-miR-16,and exo-miR-15a/16 loaded with miR-15a,miR-16,and miR-15a/16 were constructed,and PD-L1 protein and mRNA levels in each group were detected.In vivo,mouse tumor models were used to evaluate the tumor inhibitory effect of exo-miR-15a/16.Flow cytometry was used to analyze the effects of exo-miR-15a,exo-miR-16 and exo-miR-15a/16 on CD8+INF-γ+T cells;and the survival rate of mice was analyzed.Results PD-L1 was highly expressed in gastric cancer tissues.The levels of miR-15a and miR-16 in plasma and tissue of gastric cancer patients were decreased,and the later the tumor stage,the more significantly their expression levels decreased(P＜0.05).MiR-15a,miR-16 and PD-L1 mRNA had a targeted binding relationship.In vitro,when miR-15a and miR-16 levels were up-regulated,PD-L1 expression was decreased,and the lowest expression was reached when miR-15a/16 mimics was co-transfected.PD-L1 expression was increased when miR-15a and miR-16 levels were down-regulated,and the highest expression was reached when miR-15a/16 inhibitors was co-transfected(P＜0.05).Exo-miR-15a,exo-miR-16 and exo-miR-15a/16 inhibited PD-L1 expression in gastric cancer cells and tumor growth in vivo,and exo-miR-15a/16 had the most significant effect(P＜0.05).Exo-miR-15a/16 relatively increased the proportion of CD8+INF-γ+T cells,thereby promoting immune enhancement and inhibiting the growth of gastric cancer,and improved survival rate of mice.Conclusion As a novel nano liposome,modified exosomes loaded with miR-15a/16 negatively regulated PD-L1 expression in vivo and in vitro,inhibited immune escape and tumor growth of gastric cancer.