Studies On The Expression And Underlying Mechanisms Of DEPDC1 In Lung Adenocarcinomas

Objective: DEP domain protein 1(DEPDC1)is ectopically expressed in various cancer tissues and is closely related to the survival of patients,but its expression and mechanism in lung adenocarcinoma are not completely clear.This paper was aimed to investigate the expression of DEPDC1 in normal lung tissues and LUAD,its prognostic value in LUAD patients,and the underlying mechanisms.Methods:(1)DEPDC1 expression in lung adenocarcinoma and its relationship with the survival of patients: DEPDC1 expression in LUAD from The Cancer Genome Atlas(TCGA)LUAD dataset,and Gene Expression Omnibus(GEO)datasets GSE75037 GSE31210 was analyzed.The correlation between the expression level of DEPDC1 in LUAD and patient survival was analyzed with Kaplan-Meier Plotter.(2)Proliferation,invasion and migration studies: A549 cell line was used.Small interfering RNA(si RNA)was used to silence the expression of DEPDC1 for 48 h,and then colony formation assay,Transwell experiments,and wound-healing experiments were performed to detect effect of DEPDC1 knockdown on proliferation,invasion and migration.(3)Autophagy study: After 48 h of silencing DEPDC1 expression with si RNA,A549 cells were treated with the autophagy inhibitor Bafilomycin A1(100 ng/ml)for 1 hour.Finally,Western blot analyisis was used to detect the molecular markers of autophagy-P62 and LC3-B;(4)Study on the mechanism of DEPDC1: a)using Western blot analyisis determine the effects of silencing DEPDC1 on basal levels and epidermal growth factor(EGF 100 ng/ml)-induced ERK1/2 and AKT phosphorylation and total protein;b)Western blot analysis was used to detect the effects of silencing DEPDC1 on RAS expression,and S6K1 phosphorylation;c)A549 cells were treated with ERK1/2 inhibitor GDC-0994(25 μM/L)and RAS inhibitor FTS(100 μM/L)for 24 hours,and then treated with autophagy inhibitor Bafilomycin A1(100 ng/ml).After 1 hour,Western lot analysis was used to detect the changes of autophagy marker protein LC3-B at the protein level and S6K1 phosphorylation.Results:(1)DEPDC1 expression was up-regulated in LUAD,which was significantly different from normal tissues(P<0.05).(2)The expression level of DEPDC1 in LUAD was negatively correlated with the survival of patients with LUAD,that is,the higher the DEPDC1 expression level,the worse the patient survival.(3)Knocking down DEPDC1 inhibited the proliferation,migration and invasion of A549 cells,and induced autophagy in A549 cells.(4)The bioinformatical analysis and in vitro experiments revealed that DEPDC1 activates ERK1/2 phosphorylation by up-regulating RAS expression,and inhibition of RAS-ERK1/2 signaling can activate autophagy in A549 cells.Conclusion:(1)Oncoprotein DEPDC1 is highly expressed in LUAD,and the high expression of DEPDC1 is negatively correlated with the survival of patients with LUAD;(2)DEPDC1 can promote the proliferation,migration and invasion of A549 cells,and inhibit autophagy of A549 cells;(3)DEPDC1 regulates the proliferation,invasion,migration,and autophagy of A549 cells through the RAS-ERK-m TOR pathway.