Investigation Of The Effect Of IFN-γ On Epithelial-mesenchymal Transition In Lung Adenocarcinoma Cells And Its Underlying Molecular Mechanism

Background: Active IFN-γ signaling is a common feature of tumor response to PD-1checkpoint blockade.IFN-γ,the most important anti-tumor cytokine,can not only directly act on tumor cells but also indirectly inhibit tumor progression through lymphocytes.It has been reported that gene mutation of its related pathway is the key factor of immune checkpoint inhibitor resistance in tumor patients.However,recent studies have shown that IFN-γ can also promote cancer.After treatment with immunosuppressive agents,the expression level of IFN-γ in tumor tissue is significantly increased.Therefore,identifying the tumor-promoting effect of IFN-γ and its underlying molecular mechanism may be a crucial step to maximize the efficacy of anti-tumor immunotherapy.Methods: This study used real-time quantitative polymerase chain reaction(RT-q PCR)and immunohistochemistry(IHC)methods to explore the correlation between IFN-γand ZEB1.The expression of Epithelial-mesenchymal transition(EMT)related genes was analyzed by bioinformatics.Western bolt,Cell Counting Kit-8(CCK-8),plate clone formation assay,Transwell assay,and immunofluorescence assay were used to evaluate the cell proliferation ability,the induction of EMT,and the signal transduction status of different treatment groups.Immunoprecipitation assay was used to verify the binding of H3K27me3 in the ZEB1 promoter region.Enzyme-linked immunosorbent assay(ELISA)was used to determine the secretion of chemokine CXCL10 in different treatment groups.Meanwhile,in vivo experiments,the research group constructed the tail vein injection of tumor lung metastasis mouse model and short-term drug treatment of clinical lung adenocarcinoma in vitro samples to verify the corresponding in vitro experimental results.Results: Here,the research group found a significant positive correlation between IFNG and ZEB1 m RNA expression in 42 patients with stage ⅢA lung adenocarcinoma.Next,using lung adenocarcinoma cells,the research group showed that IFN-γupregulates the expression levels of ZEB1 at the m RNA and protein levels.Moreover,treatment of A549 and HCC827 cells with IFN-γ led to decreased expression of Ecadherin and increased expression of Vimentin.Knockdown of ZEB1 by si RNA reversed the regulation of E-cadherin and Vimentin by IFN-γ.The expression patterns of EMT-related genes were significantly altered upon the IFN-γ treatment.Using Transwell and mouse assay,the research group found that the migration and metastasis mediated by IFN-γ are mainly through the expression of transcription factor ZEB1.In mechanism,IFN-γ can upregulate the m RNA and protein expression of JMJD3 by activating the JAK1/2-STAT1 pathway.IFN-γ induced JMJD3 significantly reduced H3K27 trimethylation in the promoter of the ZEB1 gene,thereby activating ZEB1 transcription.GSK-J4,the JMJD3 inhibitor,inhibited IFN-γ induced ZEB1 expression.ZEB1 and mi R200 c have negative feedback regulation and play an important role in inducing EMT.The research group found that IFN-γ upregulated ZEB1 first and then downregulated mi R200 c expression.And IFN-γ-induced mi R200 c and PD-L1 protein were reversed by knockdown of ZEB1.This study previously demonstrated that IFN-γmediated antitumor responses,including inhibition of cell proliferation and induction of chemokine CXCL9 and CXCL10 expression,are STAT1-IRF1 dependent.The research group further found that ZEB1 knockout could reduce IFN-γ-mediated cell migration and metastasis,but did not affect the expression of STAT1 and IRF1,cell proliferation,and the induced expression of chemokine CXCL9 and CXCL10.Conclusion:IFN-γ significantly reduces H3K27 trimethylation in the promoter of the ZEB1 gene through the JAK1/2-STAT1-JMJD3 pathway,thus activating ZEB1 transcription.ZEB1 knockout decreased IFN-γ-mediated cell migration and metastasis but did not affect the expression of STAT1,IRF1,and PD-L1,the inhibition of cell proliferation,and the expression of chemokines CXCL9 and CXCL10.These results reveal the mechanism of IFN-γ promoting tumor EMT and provide a potential target to minimize the tumor-promoting effect of IFN-γ while preserving its anti-tumor function.