The Role And Mechanism Of DEK Regulating The Proliferation And Invasion Of Non-small Cell Lung Cancer Via Wnt Signaling Pathway And EMT

The incidence rate and mortality rate of lung cancer are high in the world,and non-small cell lung cancer(NSCLC)is the majority of all lung cancer types.With the individualized treatment schemes such as immunotherapy and molecular targeted therapy are more and more refined,we have further improved our understanding of the molecular mechanism and potential biomarkers of lung cancer.But we still need to find more new markers for the early diagnosis and prognosis of lung cancer,and clarify their role in the development of lung cancer in order to promote the further development of lung cancer therapy.Wnt/?-catenin signaling pathway is one of the regulators for maintaining normal embryonic development and tissue stability,and is correlated with the formation of tumor.The induction of epithelial mesenchymal transition in epithelial tumor cells enables them to destroy the adhesion with adjacent cells thus enhance the ability of migration and invasion to the adjacent tissues.Wnt / ?-catenin signaling pathway and EMT have been confirmed to be of great significance in the development of breast cancer,lung cancer,pancreatic cancer and other tumors.DEK is a highly conserved chromatin related non-histone phosphorylated protein.DEK is related to epigenetic and transcriptional regulation and affects the biological processes of many cells.Studies have reported that DEK was involved in the formation of a variety of tumors,and could regulate Wnt / ?-catenin signaling pathway and EMT,so it is considered to be a potential therapeutic target.However,the research on mechanism of DEK in tumor is very limited,especially the expressions of DEK in NSCLC and the mechanism of DEK in lung cancer development are still unclear,and the relevant reports are very few.Objective: In this study,we used the online database data,as well as immunohistochemistry,Western blot,cell function experiments and other molecular experimental methods to explore the expressions of DEK in lung cancer and analyzed its relationship with clinicopathological factors included tumor differentiation,lymph node metastasis,TNM stage and the prognosis of lung cancer patients,and clarified the role and molecular mechanism of DEK in the invasion and metastasis of lung cancer.Methods: 1.By using UALCAN and GEPIA online databases to retrieve clinical samples after operation,the expression of DEK in lung cancer and normal lung tissues were compared,the relationship between DEK and clinicopathological factors was analyzed,and the prognostic value of DEK gene in lung cancer was evaluated.2.130 NSCLC samples and 60 normal lung tissues(5 cm away from cancer tissue)adjacent to cancers were collected in the First Affiliated Hospital of China Medical University from January 2013 to December 2018.79 cases were adenocarcinoma and 51 cases were squamous cell carcinoma.The expression of DEK was detected by immunohistochemistry,and the relationships between DEK and clinicopathological factors were analyzed.In addition,the expressions of DEK protein in 24 fresh NSCLC tissues resected in 2018(including 16 adenocarcinoma and 8 squamous cell carcinoma)were detected by Western blot.3.Some lung cancer cell lines were cultured in order to confirm the expressions of DEK,and performed the in vitro experiments.The total proteins were extracted from those cells and detected by Western blot.4.Two lung cancer cells A549 and H1299 were selected to transfect with the DEK or DEK-sh RNA plasmid.The transfection efficiency was observed and verified by Western blot.The effects of DEK in the proliferation,migration and invasion of lung adenocarcinoma cells were studied by cell proliferation assay,colony forming assay,migration and invasion experiments.5.We used Western blot to detect the changes of key proteins of Wnt signaling pathway,and the EMT related proteins.At the same time,we detected the expressions of EGFR,ALK,and KRAS.6.Gsk3? inhibitor CHIR-99021 was applied to observe the effect of DEK on the activity of ?-catenin,the key protein of Wnt signaling pathway.Results: 1.UALCAN database showed that DEK expression was higher in lung adenocarcinoma and lung squamous cell carcinoma than in normal lung tissues(P < 0.05).DEK expression was positively correlated to the poor prognosis of lung adenocarcinoma patients(P < 0.05).GEPIA database showed that in lung adenocarcinoma,the higher expression of DEK was positively correlated with EGFR and KRAS,respectively(P < 0.05),but not with ALK and ROS1.In lung squamous cell carcinoma,the expressions of DEK were not correlated with the expressions of EGFR,KRAS,ALK and ROS1.2.In 130 cases of lung cancers,the expression of DEK was detected by immunohistochemistry.The higher expression of DEK was observed in 77 cases(59.23%)and the lower expression of DEK was in 53 cases(40.77%).In normal lung tissues,17 cases(28.33%)showed high expression of DEK.The expression of DEK in lung cancers was significantly higher than that in normal lung tissues(P < 0.05).Western blot showed that the expression of DEK in lung cancer tissues was significantly higher than that in normal lung tissues(P < 0.05).In addition,by analyzing the relationship between the expressions of DEK and the clinicopathological factors of lung cancers,we found that the high expression of DEK were correlated with the differentiation,TNM stage and lymph node metastasis of lung cancers(P < 0.05).But it was not related to the histological type,tumor size,age and gender.In lung adenocarcinomas,the expression of DEK was positively correlated with EGFR(r = 0.256,P = 0.013)and KRAS(r = 0.232,P = 0.022),respectively,but not with ALK.In lung squamous cell carcinoma,the expression of DEK was not correlated with the expressions of EGFR,KRAS and ALK.3.Overexpression of DEK by DEK transfection enhanced the proliferation rate and the ability of colony formation(P < 0.05,respectively)of H1299-DEK and A549-DEK cells compared to the control cells.In contrast,knock-down of DEK expression by Sh RNA inhibited proliferation rate and the ability of colony formation(P < 0.05,respectively).Furthermore,enhanced DEK expression promoted,but knock-down of DEK expression inhibited,the migrative and invasive abilities of A549-DEK and H1299-DEK cells compared to the control cells(P < 0.05,respectively).4.Enhanced expression of DEK up-regulated the levels of active-?-catenin and LEF1,cyclin D1,c-myc,MMP7.Enhanced expression of DEK in A549 and H1299 cells also increased the levels of EGFR,KRAS,vimentin,snail,and N-cadherin,and decreased the level of GSK3?,Axin,E-cadherin(P < 0.05).The opposite results were obtained on knock-down of DEK expression(P < 0.05).Treatment with GSK3? inhibitor CHIR-99021,the downregulation of active-?-catenin induced by koncking-down of DEK was reversed.Conclusion: 1.DEK expression was higher in lung cancer tissues than in normal lung tissues and was associated with poor histological differentiation,lymph node metastasis,advanced TNM stage of lung cancers.2.High expression of DEK indicated poor prognosis of lung adenocarcinoma patients.3.DEK can activate Wnt signaling pathway and EMT process,and promote the proliferation,migration and invasion of lung cancer cells.4.In lung adenocarcinoma,DEK expression was positively correlated with the expressions of EGFR and KRAS,respectively.