The Mechanism Of TRIP13 In Regulating Lung Cancer Proliferation And Invasion Through Wnt Signaling Pathway As Well As EMT Process

Lung cancer is one of the most common malignancies,and its incidence is increasing every year.Approximately 85–90% of lung cancer cases are diagnosed as non-small-cell lung cancer(NSCLC).NSCLC is classified into lung adenocarcinomas,squamous cell carcinomas,large cell carcinomas,and so on,with adenocarcinomas being the most prevalent.The 5-year survival rate of NSCLC remains very low;it varies from 4% to 17% depending on the tumor region and stage.Therefore,exploring new biomarkers(oncogenes / suppressor genes)plays an important role in the prevention,diagnosis and treatment of non-small cell lung cancer.The Wnt signaling pathway has been confirmed to be related to the proliferation and invasion of cancer cells.In the Wnt signaling pathway,Wnt proteins bind to Frizzled(Fz)family receptors,including the lipoprotein receptor-related protein LRP5 / 6.Thereby,it inhibits the activity of the GSK3 complex and it promotes the accumulation of ?-catenin in the cytoplasm,so that it can enter the nucleus.Then,through binding to TCF / LEF transcription factor,the activation of target genes of the Wnt pathway was induced.TRIP13 is a member of the ATPase and it is a mitotic checkpoint-related protein firstly found in mice.A large number of studies have confirmed that TRIP13 can regulate mitotic checkpoint-related proteins and it is related to the occurrence and development of tumors such as prostate cancer,colorectal cancer,and epithelial ovarian cancer.Our study found that TRIP13 is highly expressed in both lung squamous cell carcinoma and lung adenocarcinoma,and the expression level of TRIP13 is correlated with the prognosis of lung cancer patients.In vitro experiments,it shows that TRIP13 can affect the ability of proliferation and invasion in lung cancer cells.TRIP13 can interact with the Wnt pathway receptor LRP6 and activate the Wnt signaling pathway as well as epithelial-to-mesenchymal transition(EMT)process.Therefore,TRIP13 may be used as a biochemical indicator for early diagnosis of lung cancer.Objective: To examine the expression levels of TRIP13 in lung cancer tissues,and analyze the relationship between the expression of TRIP13 and the prognoses,clinical pathological factors of lung cancer patients.To investigate the role and molecular mechanism of TRIP13 in lung cancer cells.Methods: We analyzed the expression of TRIP13 in lung squamous cell carcinoma and lung adenocarcinoma tissues through the UALCAN based on the TCGA and other tumor databases,and analyzed the relationship between TRIP13 and patient prognosis and clinicopathological factors using Kaplan Meier curve.We collected 16 pairs of fresh lung cancer tissues and the corresponding normal tissues surgically removed in the First Hospital of China Medical University.All the samples were stored at-80°C after surgery.We detected the expression of TRIP13 in lung cancer tissues and lung cancer cells using Western blot.We also examined the location of TRIP13 in lung cancer cells by immunofluorescence.TRIP13 plasmid was transfected in A549 cells and H1299 cells to increase its expression,and small interfering RNA(SiRNA)of TRIP13 was used to interference its expression.We explored the effects of TRIP13 on proliferation and invasion in non-small cell lung cancer cells using cell proliferation assay,clone formation assay,and matrigel invasion assay.We detected the protein levels of Wnt signaling pathway and EMT-related proteins using western blot after increasing or decreasing the expression of TRIP13 in lung cancer cells.The relationship between TRIP13 and LRP6 was explored using co-immunoprecipitation,and its location detection was performed using immunofluorescence.Results:1.The expression level of TRIP13 in lung squamous cell carcinoma and lung adenocarcinoma tissues was higher than those in normal lung tissues,and it was asscciated with the poor prognosis of lung cancer patients.The expression of TRIP13 in lung cancer cells was higher than that in normal bronchial epithelial cells(HBE)and it is localized in the cytoplasm.2.Overexpression of TRIP13 in H1299 and A549 cells can significantly promote the ability of proliferation,colony formation and invasion in lung cancer cells.Knockdown of TRIP13 can inhibit the ability of proliferation,colony formation and invasion in lung cancer cells.3.Overexpression of TRIP13 in H1299 cells and A549 cells can significantly reduce the expression level of GSK3?,increase the expression levels of LRP6,active ?-catenin,TCF4,LEF1 and target proteins of Wnt signaling pathway,including c-Myc,MMP7 and Cyclin D1.Knockdown of TRIP13 in H1299 cells and A549 cells can increase the expression level of GSK3? and decrease the expression levels of LRP6,active ?-catenin,TCF4,LEF1 and target proteins of Wnt signaling pathway,including c-Myc,MMP7 and Cyclin D1.4.Overexpression of TRIP13 in H1299 cells and A549 cells can decrease the expression level of E-cadherin,increase the expression levels of N-cadherin,Snail and Vimentin.Knockdown of TRIP13 in H1299 cells and A549 cells can increase the expression level of E-cadherin,decrease the expression levels of N-cadherin,Snail and Vimentin.5.We revealed that TRIP13 can bind to LRP6 in H1299 cells by bidirectional endogenous immunoprecipitation.The laser confocal immunofluorescence detection also displayed the co-localization of LRP6 and TRIP13 in A549 cells and H1299 cells.Conclusion:1.TRIP13 is highly expressed in lung cancer tissues and it is associated with poor prognosis in lung cancer patients.2.TRIP13 promotes the proliferation and invasion of lung cancer cells by activating the Wnt signaling pathway and EMT.3.TRIP13 might promote the activity of Wnt signaling pathway by binding with LRP6.