Circular RNA CircHMGB2 Drives Immunosuppression And Anti-PD-1 Resistance In Non-small-cell Lung Cancer Via The MiR-181a-5p/CARM1 Axis

Background and Objective:Lung cancer is one of the most frequent malignancies,and is the leading cause of cancer-related death in the world.Non-small-cell lung cancer(NSCLC)accounts for approximately 85%of all lung cancer cases.Although curative resection for early-stage NSCLC has achieved promising results,most patients are diagnosed at an advanced stage.They lose the opportunity for surgery.Their treatment mainly relies on chemotherapy,targeted therapy and immunotherapy,and the benefits are limited.The latest clinical studies confirmed that the overall 5-year survival rate of NSCLC is only16%.Therefore,further studies on the occurrence and progression of NSCLC have potential clinical value and may contribute to developing new therapeutic strategies for NSCLC.Circular RNAs(circRNAs),derived from the back-splicing of exons in pre-m RNA,are a class of noncoding RNAs characterized by covalently closed loops.Circular RNAs have a variety of biological functions.While some studies have reported the role of circ RNAs in the oncogenesis and progression of NSCLC,the potential effects of most circ RNAs on NSCLC remain unclear and further research is needed.High-mobility group box 2(HMGB2)is a ubiquitous human nucleic protein,responsible for the active status of chromatin domains.Previous studies have confirmed the oncogenic role of HMGB2,while the biological functions of HMGB2-derived circ RNAs remain unknown.Thus,we intended to study the potential role of HMGB2-derived circ RNAs in NSCLC.Methods:The expression of 3 circ RNAs derived from HMGB2 in 8 pairs of NSCLC and paired normal lung tissues was assessed by q RT-PCR,and hsa＿circ＿0071452(circHMGB2)was filtered out.The expression of circHMGB2 was assessed via q RT-PCR in 120 NSCLC tissues and paired normal tissues.The correlation between the level of circHMGB2 and the clinicopathological characteristics and prognosis of patients was analyzed.The role of circHMGB2 in NSCLC was detected by Matrigel Transwell,wound healing assay,CCK-8,clone formation assay and nude mice subcutaneous tumor model.Subcutaneous tumor models were established with LLC cell line in immunocompetent mice C57BL/6 to observe the growth of subcutaneous tumors.Flow cytometry and immunohistochemistry were used to detect infiltration of immune cell subsets in subcutaneous tumors.Then,the efficacy of anti-PD-1 therapy was observed.In addition,in vivo circ RNA precipitation,RNA immunoprecipitation,luciferase reporter assays and RNA pulldown assays were applied to explore the underlying mechanism by which circHMGB2 promoted NSCLC progression.Results:Three circular RNAs(hsa＿circ＿0071452,hsa＿circ＿0071453,hsa＿circ＿0071454)derived from HMGB2 can be obtained from circ Base,and the expression of circHMGB2(hsa＿circ＿0071452)was significantly upregulated(P<0.001.In 120NSCLC and matched normal tissues,circHMGB2 was highly expressed in 104 NSCLC tissues,and the expression of circHMGB2 in 57 NSCLC tissues was more than twofold higher than that in normal tissues.The study found that higher expression of circHMGB2 was related to larger tumor size and greater lymph node metastasis in NSCLC patients(p<0.05)and circHMGB2 expression was increased as clinical stage increased.Survival analysis confirmed that the expression of circHMGB2 was closely related to the overall survival and recurrence of NSCLC patients(p<0.05).Multivariate Cox regression analysis showed that circHMGB2 expression was an independent prognostic factor in NSCLC patients.Matrigel Transwell and wound healing assays results demonstrated that circHMGB2 had no significant effect on the invasion and migration of NSCLC cells.However,the CCK-8 and plate cloning assay results revealed that circHMGB2 had significant effect on the proliferation of NSCLC cells.The LLC-control and LLC-circHMGB2 cells were used to construct subcutaneous tumor models in immunocompetent mice C57BL/6.The results showed that overexpression of circHMGB2 significantly promoted the growth of NSCLC(p<0.01).Flow cytometry analysis showed that in tumors subcutaneously inoculated with LLC-circHMGB2 cells,CD8~+T cells(p<0.05),DCs(p<0.05)and NK cells(p<0.05)were significantly lower than those in the control group,whereas CD4~+T cells(p<=0.8392),TAM(p=0.8811),Treg(p=0.7638)cells were no difference.Furthermore,the study found that upregulation of circHMGB2 reduced the efficacy of anti-PD-1 therapy(P<0.0001).Mechanistically,circHMGB2 relieved the inhibition of downstreamCARM1 by sponging mi R-181a-5p,which resulted in immune evasion and resistance to anti-PD-1 therapy in NSCLC.Conclusion:Circ HMGB2 overexpression reshapes the tumor immune microenvironment of NSCLC and promotes resistance to anti-PD-1 therapy,participates in the progression of NSCLC,and may be a new biomarker and a potential therapeutic target for the prognosis of patients with NSCLC.