The Role And Mechanism Of IL-17A In Cell Invasion,Epithelial Mesenchymal Transition And PD-L1 Expression In Non-small Cell Lung Cancer

Background:Lung cancer is currently the second most prevalent cancer worldwide and is also the leading cause of cancer deaths.Current immunotherapies have significantly improved the survival of lung cancer patients and have become first-line therapeutic agents,but innate or acquired drug resistance still exists.The search for relevant molecules in the tumour microenvironment involved in influencing the efficacy of immune checkpoint inhibitors may improve drug resistance.The pro-inflammatory factor interleukin 17A(IL-17A)is involved in creating a suppressive immune microenvironment in lung cancer tissues,promoting tumour development and influencing the response to immunotherapy.This study will investigate the impact of IL-17A in the development of NSCLC and anti-PD-L1 therapy,and investigate the mechanism of its occurrence.Methods:1.Thirty-nine cases of tumor and adjacent tissues of NSCLC patients were collected,and the expression of IL-17A in the tissues was detected by immunohistochemistry.The correlation between baseline data and pathological data of patients was analyzed.2.NSCLC cell lines A549 and SPC-A-1 were selected as the research objects in vitro.The level of PD-L1 and epithelial-mesenchymal transition(EMT)related proteins was detected after IL-17A protein stimulation.3.IL-17A knockdown and overexpression A549 and SPC-A-1 cell models were constructed,and cell migration,invasion and plate colony formation abilities were detected.Western blot and immunofluorescence were used to detect the expression of PD-L1 and EMT-related proteins.4.To detect the effect of IL-17A on autophagy in NSCLC cells.Immunofluorescence assay,T cell killing assay and western blot were used to detect the level of autophagy and PD-L1 in A549 and SPC-A-1 cells with IL-17A knockdown or overexpression.The level of autophagy in clinical samples was detected by immunohistochemistry and tissue immunofluorescence.5.To investigate the effect of IL-17A on oxidative stress in NSCLC cells.The content of superoxide anion and the level of H2O2 were measured.Western blot and immunofluorescence were used to detect p62 and Nrf2.6.In vivo experiment,Subcutaneous tumor mouse models overexpressed with IL-17A LLC were constructed.Mice were divided into control group,OE-IL-17A group,anti-PD-L1 group and OE-IL-17 A+anti-PD-L 1 treatment group.Subcutaneous tumor volume,weight and mouse body weight were monitored,and autophagy,Nrf2 and PD-L1 expression levels in tumor tissues were detected by immunohistochemistry.The number of T cells in tumor tissue was detected by immunofluorescence.7.The LLC subcutaneous tumor mouse model was established in vivo and divided into control group,anti-IL-17A group,anti-PD-L1 group and combined treatment group.The subcutaneous tumor volume,weight,and body weight of mice were monitored.The autophagy level,Nrf2 and PD-L1 expression levels in tumor tissues were detected by immunohistochemistry.Results:1.IL-17A was commonly expressed in NSCLC tumor tissues.87.2%of the 39 lung cancer pathological tissues were positive for IL-17A and 12.8%were negative.There was a positive correlation between IL-17A and PD-L1(r=0.6121,P<0.0001).2.Exogenous IL-17A increased the PD-L1 level in NSCLC cells,and promoted the expression of mesenchymal markers N-cadherin,Vimentin and Snail.3.IL-17A knockdown and overexpression cell models were constructed.The migration,invasion and colony forming ability of IL-17A overexpression cells were significantly enhanced.The expression of mesenchymal cell markers such as N-cadherin,Snail and Twist increased,while the expression of epithelial cell marker E-cadherin decreased.Knockdown of IL-17A reversed these abilities.4.Overexpression of IL-17A increased the expression of LC3Ⅱ/Ⅰ and p62 in NSCLC cells and blocked autophagic flow,accompanied by an increase in PD-L1 expression.Knockdown of IL-17A increased the expression of LC3Ⅱ/Ⅰ and decreased P62 in NSCLC cells.Autophagic flow was activated and PD-L1 level was decreased.5.Autophagy regulates the expression of PD-L1 in NSCLC cells.Treatment of A549 and SPC-A-1 cells with autophagy agonist decreased the expression of PD-L1.The expression of PD-L1 was increased in A549 and SPC-A-1 cells after treatment with autophagy inhibitor.6.In vivo,PD-L1 inhibitors were used to reduce tumor volume more significantly in mice whose tumor tissues overexpressed IL-17A.Immunohistochemical results suggested that higher expression of IL-17A in tumor tissues was accompanied by higher expression level of PD-L1.7.In the LLC subcutaneous tumor mouse model,anti-PD-L1 can significantly reduce the tumor volume and weight.Anti-IL-17A activated the autophagy of tumor cells,decreased the expression of PD-L1 and reduced the tumor volume.When anti-IL-17A was combined with anti-PD-L1,PD-L1 expression decreased significantly compared with the control group and tumour volume and weight increased compared with anti-PD-L1 alone.It is suggested that the effect of anti-PD-L1 drugs is weakened when combined with anti-IL-17A.Conclusion:We found that IL-17A promoted the progression of NSCLC and inhibited autophagy through the ROS/Nrf2/p62 pathway,resulting in increased expression of PD-L1 in cancer cells,IL-17A may affect the efficacy of immunotherapy by regulating autophagy.