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Background Limited response to programmed death ligand-1 (PD-L1)/programmed death 1 (PD-1) immunotherapy is a major hindrance of checkpoint immunotherapy in non-small cell lung cancer (NSCLC). The abundance of PD-L1 on the tumor cell surface is crucial for the responsiveness of PD-1/PD-L1 immunotherapy. However, the negative control of PD-L1 expression and the physiological significance of the PD-L1 inhibition in NSCLC immunotherapy remain obscure.Methods Bioinformatics analysis was performed to profile and investigate the long non-coding RNAs that negatively correlated with PD-L1 expression and positively correlated with CD8+T cell infiltration in NSCLC. Immunofluorescence, in vitro PD-1 binding assay, T cell-induced apoptosis assays and in vivo syngeneic mouse models were used to investigate the functional roles of LINC02418 and mmu-4930573I07Rik in regulating anti-PD-L1 therapeutic efficacy in NSCLC. The molecular mechanism of LINC02418-enhanced PD-L1 downregulation was explored by immunoprecipitation, RNA immunoprecipitation (RIP), and ubiquitination assays. RIP, luciferase reporter, and messenger RNA degradation assays were used to investigate the m6A modification of LINC02418 or mmu-4930573I07Rik expression. Bioinformatics analysis and immunohistochemistry (IHC) verification were performed to determine the significance of LINC02418, PD-L1 expression and CD8+T cell infiltration.Results LINC02418 is a negative regulator of PD-L1 expression that positively correlated with CD8+T cell infiltration, predicting favorable clinical outcomes for patients with NSCLC. LINC02418 downregulates PD-L1 expression by enhancing PD-L1 ubiquitination mediated by E3 ligase Trim21. Both hsa-LINC02418 and mmu-4930573I07Rik (its homologous RNA in mice) regulate PD-L1 therapeutic efficacy in NSCLC via Trim21, inducing T cell-induced apoptosis in vitro and in vivo. Furthermore, METTL3 inhibition via N6-methyladenosine (m6A) modification mediated by YTHDF2 reader upregulates hsa-LINC02418 and mmu-4930573I07Rik. In patients with NSCLC, LINC02418 expression is inversely correlated with PD-L1 expression and positively correlated with CD8+T infiltration.Conclusion LINC02418 functions as a negative regulator of PD-L1 expression in NSCLC cells by promoting the degradation of PD-L1 through the ubiquitin-proteasome pathway. The expression of LINC02418 is regulated by METTL3/YTHDF2-mediated m6A modification. This study illuminates the underlying mechanisms of PD-L1 negative regulation and presents a promising target for improving the effectiveness of anti-PD-L1 therapy in NSCLC.

Abstract Background OTUB1 (ovarian tumor domain protease domain-containing ubiquitin aldehyde-binding proteins)-mediated deubiquitination of FOXM1 (Forkhead box M1) participates in carcinogenesis of various tumors. We aim to investigate the effect and mechanism of OTUB1/FOXM1 on RCC (renal cell carcinoma) progression. Expression levels of OTUB1 in RCC tissues and cell lines were examined by qRT-PCR (quantitative real-time polymerase chain reaction) and immunohistochemistry. Cell proliferation was measured with CCK8 (Cell Counting Kit-8) and colony formation assays. Wound healing and transwell assays were used to determine cell migration and invasion, respectively. The effect of OTUB1 on FOXM1 ubiquitination was examined by Immunoprecipitation. Western blot was used to uncover the underlying mechanism. In vivo subcutaneous xenotransplanted tumor model combined with immunohistochemistry and western blot were used to examine the tumorigenic function of OTUB1. Results OTUB1 was up-regulated in RCC tissues and cell lines, and was associated with poor prognosis of RCC patients. Knockdown of OTUB1 inhibited cell viability and proliferation, as well as migration and invasion of RCC cells. Mechanistically, knockdown of OTUB1 down-regulated FOXM1 expression by promoting its ubiquitination. Down-regulation of FOXM1 inhibited ECT2 (epithelial cell transforming 2)-mediated Rho signaling. Moreover, the inhibition of RCC progression caused by OTUB1 knockdown was reversed by FOXM1 over-expression. In vivo subcutaneous xenotransplanted tumor model also revealed that knockdown of OTUB1 could suppress in vivo RCC growth via down-regulation of FOXM1-mediated ECT2 expression. Conclusions OTUB1-mediated deubiquitination of FOXM1 up-regulates ECT-2 to promote tumor progression in RCC, providing a new potential therapeutic target for RCC treatment.