Identification of lncRNA dual targeting PD-L1 and PD-L2 as a novel prognostic predictor for gastric cancer

Although breakthroughs have been achieved in gastric cancer (GC) therapy with immune checkpoint inhibitors (ICIs) targeting programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1), the acquisition of high response rate remains a huge challenge for clinicians. It is imperative to identify novel biomarkers for predicting response to immunotherapy and explore alternative therapeutic strategy for GC.

The transcriptomic profiles and clinical information of GC patients from The Cancer Genome Atlas (TCGA)-stomach adenocarcinoma (STAD) database was used to screen differentially expressed lncRNAs between the tumor specimens and the paracancerous tissues. The TargetScan, miRDB and miRcode database were then utilized to construct competing endogenous RNA (ceRNA) networks and identify pivotal lncRNAs. An independent dataset from GEO (GSE70880) and 23 pairs of GC specimens of our cohort were subsequently performed for external validity. The relationship between clinical variables and gene expression were evaluated by Kruskal-wallis test and Wilcoxon signed-rank. The prognostic value of the candidate genes was assessed using Kaplan-Meier analysis and Cox regression models. CIBERSORT and Gene set enrichment analysis (GSEA) were used to determine immune cell infiltration. Gastric adenocarcinoma AGS cells and human embryonic kidney 293T (HEK293T) cells with knockdown of LINC01094 were generated by siRNA transfection, followed by detecting the alteration of the target miRNA and PD-L1/PD-L2 by RT-qPCR. Besides, the interaction between lncRNA and the miRNA-PD-L1/PD-L2 axis were verified by dual luciferase reporter assay.

Twenty-two intersecting lncRNAs were identified to be PD-L1/PD-L2-related lncRNAs and LINC01094-miR-17-5p-PD-L1/PD-L2 was constructed as a potential ceRNA network. LINC01094 was increased in tumor specimens than adjacent normal samples and was positively associated with advanced tumor stages and EBV and MSI status. Furthermore, LINC01094 expression was an independent risk factor for poor overall survival (OS) in GC patients. CD8⁺ T cell exhaustion-related genes were enriched in high-LINC01094 tissues and high-PD-L2 group. A strong positive association of LINC01094 expression was established with M2 macrophages, IL-10⁺ TAM, as well as PD-L1 and PD-L2 levels, therefore a LINC01094-miR-17-5p-IL-10 network was proposed in macrophages. Using the exoRBase database, LINC01094 was assumed in blood exosomes of GC patients The results of knockdown experiments and luciferase reporter assays revealed that LINC01094 interacted with miR-17-5p and served as a miRNA sponge to regulate the expression of PD-L1 and PD-L2.

LINC01094 dually regulates the expression of PD-L1 and PD-L2 and shapes the immunosuppressive tumor microenvironment via sponging miR-17-5p. LINC01094 may serve as a potential prognostic predictor and therapeutic target in GC.