The Role And Mechanism Of CircRAPGEF5 In Non-small Cell Lung Cancer Treated With PD-1 Inhibitors

Background: Programmed cell death protein 1(PD-1)inhibitors are currently approved as first-line drugs for the treatment of non-small cell lung cancer(NSCLC),but only some patients benefit from them.The current problem is the lack of effective biomarkers to predict their efficacy.Circular RNAs(circ RNAs)are a class of abundant and highly conserved non-coding RNAs that may serve as biomarkers for NSCLC diagnosis and potential therapeutic targets.Studies have demonstrated that some circ RNAs can affect the efficacy of PD-1inhibitors in the treatment of NSCLC.However,there are currently no studies on screening efficacy-associated circ RNAs from NSCLC patients treated with PD-1 inhibitors.Objective: To screen efficacy-associated circRNAs from NSCLC patients treated with PD-1 inhibitors,and to explore the effect and mechanism of candidate circular RNA circ RAPGEF5 on the efficacy of PD-1 inhibitors in the treatment of NSCLC.Methods: The circRNAs expression profile of NSCLC patients treated with anti-PD-1 was identified using find＿circ,CIRI2 and CIRIexplorer2.According to the response of patients to PD-1 antibody,the circ RNA expression profile was divided into the response group and the non-response group.DEseq2 was used to screen the differentially expressed circ RNA between the two groups.RT-q PCR and fluorescence in situ hybridization(FISH)were used to verify the expression and localization of circ RAPGEF5 in NSCLC cells.CCK8 was used to detect the effect of circ RAPGEF5 on the proliferation of H1299 cells and the effect of circ RAPGEF5 on cell migration was detected in the Transwell chamber.In addition,the effect of circ RAPGEF5 on apoptosis was detected by flow cytometry.In vitro T cell-mediated tumor cell killing assay and in vivo anti-PD1 experiments were used to study the role of circ RAPGEF5 in anti-tumor immunity of NSCLC.Secreted IFN-γ and TNF-α from cocultured PBMCs were detected by ELISA.RNA sequencing analysis was performed on H1299 cells before and after the circ RAPGEF5 knockout.The differentially expressed genes were used to search for downstream genes regulated by circ RAPGEF5.The regulation of circ RAPGEF5 on PD-L1 and IRE1α protein levels was detected by Western blot,and the effect of circ RAPGEF5 on PD-L1 expression levels was detected by RT-q PCR.Results: A total of 690 differentially expressed circRNAs were identified in the response group and non-response group,and circ RAPGEF5 was selected as a candidate marker for further analysis.Patients with low circ RAPGEF5 expression presented with a significantly better prognosis than those with high circ RAPGEF5 expression.RTq PCR verified the expression of circ RAPGEF5 in NSCLC cells and its resistance to RNase R treatment.FISH showed that circ RAPGEF5 was mainly located in the cytoplasm.Functional experiments showed that silencing circ RAPGEF5 inhibited the growth,migration and invasion of NSCLC cells,whereas circ RAPGEF5 overexpression exhibited the opposite effect.In vitro T cell-mediated tumor cell killing test results and in vivo anti-PD1 experiments showed that circ RAPGEF5 knockdown increases the sensitivity of NSCLC to PD-1 inhibitors therapy.Circ RAPGEF5 knockdown suppressed the protein expression level of IRE1α and suppressed the transcription and protein expression levels of PD-L1.Conclusion: CircRAPGEF5 promotes the proliferation and metastasis of NSCLC cells,and promotes the resistance of NSCLC to PD-1 inhibitor treatment.circ RAPGEF5 can regulate PD-L1 expression,and circ RAPGEF5 has potential as a biomarker for predicting anti-PD-1treatment response in NSCLC.