The Effect And The Potential Mechanism Of CUL4B Knockdown On The Proliferation,Invasion,Migration And EMT In Non-small Cell Lung Cancer Cells

Lung cancer is one the leading cause of cancer death in the world.More than 80%cases are classified as non-small cell lung cancer(NSCLC).In recent years,despite great progress in clinical diagnosis and treatment,patients with NSCLC have a 5-year survival rate of less than 15%.Thus,understanding the molecular mechanisms underlying NSCLC development and progression is of significance to the development of therapeutic strategies for NSCLC patients.Cullin-RING ligases(CRLs)are multi-subunit complexes composed of a cullin,RING protein and substrate-recognition subunit,play important roles in regulating diverse cellular processes,such as cell cycle progression,transcription and signal transduction.Cullin 4B(CUL4B),a scaffold protein in the Cullin 4B-RING E3 ligase complex(CRL4B),was found to be highly expressed in most human cancers.High CUL4B expression was significantly as sociated with the cancer invasion,lymph node metastasis,distant metastasis,histological differentiation,vascular invasion,and tumor stage.Moreover,knockdown of CUL4B.reduced the proliferation,colony formation,and invasiveness of carcinoma cells in vitro and inhibited tumor growth in vivo.However,the expression pattern and role of CUL4B in NSCLC remains largely unknown.Therefore,in the present study,we investigated the role of CUL4B in NSCLC,and the underlying mechanism was also explored.This research was divided into three parts:Part One:The expression of CUL4B in human non-small cell lung cancer tissue and cell lines;Part Two:The effect of CUL4B knockdown on the proliferation,invasion,migration and EMT in non-small cell lung cancer cells;Part Three:The potential mechanism by which CUL4B knockdown suppresses invasion,migration and EMT in non-small cell lung cancer cells.Part One:The expression of CUL4B in human non-small cell lung cancer tissue and cell linesMethods1.The mRNA and protein expression of CUL4B in human non-small cell lung cancer tissue was detected by using real-time PCR and western blot analysis.2.Real-time PCR was performed to measure the mRNA expression of CUL4B in the human lung bronchus epithelial cell line BEAS-2B and non-small cell lung cancer cell lines A549,H157,H1299,and H1650.3.Western blot analysis was conducted to examine the protein level of CUL4B in the human lung bronchus epithelial cell line BEAS-2B and non-small cell lung cancer cell lines A549,H157,H1299,and H1650.Results1.The mRNA and protein expression of CUL4B was up-regulated in human non-small cell lung cancer tissue.2.The mRNA expression of CUL4B was significantly increased in non-small cell lung cancer cell lines A549,H157,H1299,and H1650,compared with that in the human lung bronchus epithelial cell line BEAS-2B.3.The protein level of CUL4B was significantly increased in non-small cell lung cancer cell lines A549,H157,H1299,and H1650,compared with that in the human lung bronchus epithelial cell line BEAS-2B.Part Two:The effect of CUL4B knockdown on theproliferation,invasion,migration and EMT in non-smallcell lung cancer cellsMethods1.Western blot analysis was used to detect the expression of CUL4B in non-small cell lung cancer cell lines A549 and H1299 cells transfected with siRNA-CUL4B.2.CCK-8 proliferation assay and colony formation assay were performed to determine the effect of CUL4B knockdown on the proliferation of non-small cell lung cancer cell lines A549 and H1299 cells.3.The effect of CUL4B knockdown on the migration and invasion of non-small cell lung cancer cell lines A549 and H1299 cells was determined by the Transwell migration assay.4.The protein levels of the epithelial-mesenchymal transition markers E-cadherin,N-cadherin and Vimentin was detected by western blot analysis.Results1.Western blot analysis confirmed the low expression of CUL4B in non-small cell lung cancer cell lines A549 and H1299 cells,as compared to controls.2.CCK-8 proliferation assay and colony formation assay showed that knockdown of CUL4B obviously decreased the proliferation of non-small cell lung cancer cell lines A549 and H1299 cells,compared with respective controls.3.The Boyden chamber transwell assay demonstrated that non-small cell lung cancer cell lines A549 and H1299 cells derived from siRNA-CUL4B-transfectants displayed a lower ability of migration and invasion,compared with that derived from scramble-CUL4B-transfected cells.4.The results of western blot analysis indicated that CUL4B knockdown inhibited the expression of the epithelial-mesenchymal transition,and the markers E-cadherin was significantly increased,and N-cadherin and Vimentin were decreased.Part Three:The potential mechanism by which CUL4B knockdown suppresses proliferation,invasion,migrationand EMT in non-small cell lung cancer cellsMethods1.Western blot analysis was used to detect the expression of Wnt/?-catenin signaling pathways related proteins P-catenin,cyclinD1 and c-Myc in non-small cell lung cancer cell line A549 cells transfected with siRNA-CUL4B.2.CCK-8 proliferation assay and colony formation assay were performed to detect the effect of Wnt/?-catenin signaling pathways inhibitor,XAV939 on the proliferation of non-small cell lung cancer cell line A549 cells.3.The effect of Wnt/?-catenin signaling pathways inhibitor,XAV939 on the migration and invasion of non-small cell lung cancer cell line A549 cells was determined by the Transwell migration assay.4.Western blot analysis was performed to comfirm the effect of XAV939 on the expression of the epithelial-mesenchymal transition markers E-cadherin,N-cadherin and Vimentin.Results1.Western blot analysis confirmed CUL4B knockdown inhibited the expression of Wnt/?-catenin signaling pathways related proteins ?-catenin,cyclinD1 and c-Myc in non-small cell lung cancer cell line A549 cells.2.CCK-8 proliferation assay and colony formation assay showed that the Wnt/?-catenin signaling pathways inhibitor,XAV939 obviously decreased the proliferation of non-small cell lung cancer cell line A549 cells,compared with respective controls.3.The Boyden chamber transwell assay demonstrated that non-small cell lung cancer cell line A549 cells treated with the Wnt/?-catenin signaling pathways inhibitor,XAV939 displayed a lower ability of migration and invasion,compared with that treated with control media.4.Western blot analysis indicated that the Wnt/?-catenin signaling pathways in hibitor,XAV939 inhibited the expression of the epithelial-mesenchymal transition,and the markers E-cadherin was significantly increased,and N-cadherin and Vimentin were decreased.Conclusions1.CUL4B is overexpressed in in human non-small cell lung cancer tissue and cell lines.2.CUL4B knockdown suppressed the proliferation,invasion,migration and EMT in non-small cell lung cancer cells.3.The Wnt/?-catenin signaling pathways inhibitor,XAV939 could suppressed the proliferation,invasion,migration and EMT in non-small cell lung cancer cells.4.CUL4B knockdown inhibited the proliferation,invasion,migration and EMT in non-small cell lung cancer cells through suppressing Wnt/?-catenin signaling pathways.