| Background and Objective Lung cancer is the highest incidence and mortality cancer.On the global scale,there are 1.8 million new cases and 1.6 million deaths in 2012.Lung cancer consists of small cell lung cancer,large cell carcinoma,squamous cell carcinoma and adenocarcinoma,and the latter three belong to the non-small cell lung cancer(NSCLC)and accounts for 80–85% of all lung cancer cases.Though enormous progression has been made in treatment of NSCLC,the 5-year survival rate is less than 15%,especially,is approximately 1% for the stage IV patients.Patients with non-small cell lung cancer are prone to bone and brain metastasis,which is the main reason for their low survival rate.Chemotherapy is the main method for the treatment of non-small cell lung cancer,especially the effect of cisplatin.At the beginning of the cisplatin treatment,the outcome is favorable,but later the chemoresistance develops and leads to failure.Therefore,it is particularly important to study the molecular mechanisms of non-small cell lung cancer metastasis and cisplatin resistance,seeking for new molecular markers or therapeutic targets.Canopy homolog2(CNPY2)belongs to the CNPY family and is a novel secreted protein widely distributed in various tissues such as human heart,liver and pancreas.The CNPY family also has CNPY1,CNPY3,and CNPY4 members.All of them share a similar domain structure,including a consensus signal peptide,a saposin-type B domain and an endoplasmic reticulum(ER)-retrieval sequence,but no hydrophobic transmembrane domain.The knowledge on CNPY2 is limited.The past researches reported that the upstream of CNPY2 promoter contains HIF-1α binding site,and CNPY2 is regulated by HIF-1α and can enhance proliferation,migration and tissue revascularization of human smooth muscle cells.And knockdown of CNPY2 inhibits tumor growth and angiogenesis and enhances cell apoptosis in human colorectal cancer.However,the research on CNPY2 is limited and its function in NSCLC is unclear.Thus,the main purpose of this study is to explore preliminarily the role of CNPY2 in invasion,metastasis and cisplatin resistance of nonsmall cell lung cancer in vitro,providing some experimental data about CNPY2 acted as oncogenen in non-small cell lung cancer,and further laying theoretical foundation of mechanism of CNPY2-induced malignant progress.In addition,we will test our hypothesis in animal experiments and clinical samples to provide potential molecular targets for early diagnosis and treatment of lung cancer.Methods 1.The database predicts the role of CNPY2 in non-small cell lung cancer: analyze the expression of CNPY2 in non-small cell lung cancer and adjacent normal tissues by TCGA.The association between overall survival and the expression of CNPY2 in NSCLC patients was drawn using the published Kaplan-Meier plotter database.2.To detect the expression of CNPY2 in non-small cell lung cancer tissues: 9 patients with nonsmall cell lung cancer and adjacent normal tissues were collected.The relationship of CNPY2 and E-Cadherin was determined by real-Time PCR and immunoblotting experiments.3.Construction of over-expressed CNPY2 cells and silenced CNPY2 cells : CNPY2 expression in primary bronchial epithelial BEAS-2B cells and 8 NSCLC cell lines(H2087,H810,H1563,A549,H2228,H2291,H358 and H1568)was detected by real-time PCR and immunoblotting,and then the overexpression of CNPY2 and knockdown of CNPY2 in A549 cell were established.4.In vitro detection of the effects of CNPY2 on migration and invasion and its mechanism: the effects of CNPY2 on the invasion and migration in A549 cells were observed by transwell assay and wound healing assays.The expression of EMT-related molecules and p-GSK3β,GSK3β,p-AKT and AKT proteins were detected by immunoblotting in CNPY2 overexpressed cells.In addidition,cells were treated with Perfosine(AKT /GSK3β inhibitor)and then the effects of CNPY2 on cell invasion and migration were further tested by transwell assay and wound healing assay.5.To detect the sensitivity to cisplatin in all cell lines: the IC50 and clone formation abilities in cisplatin-treated primary bronchial epithelial BEAS-2B cells and 8 NSCLC cell lines(H2087,H810,H1563,A549,H2228,H2291,H358 and H1568)were performed.6.In vitro detection of the effect of CNPY2 on cisplatin resistance and its mechanism: The effects of CNPY2 on the abilities of proliferation and apoptosis in cisplatin-treated A549 cells were examined by plate cloning assay,Annexin V-FITC assay and TUNEL assay.The expression of apoptosis regulators caspase-3,c-IAP1,c-IAP2,XIAP,Bcl-2 and Bcl-xl in A549-CNPY2 cells were detected by immunoblotting.Dual luciferase reporter assay was used to test the effect of CNPY2 on the transcriptional activity of NF-κB signaling pathway.The effect of CNPY2 on the phosphorylation levels of IκBα and IKK was detected by immunoblotting.Treatment of cells with NF-κB signaling pathway inhibitors IκBα-mut,NF-κB and IKK inhibitors,and broad-spectrum caspase inhibitors(Z-VAD-FMK),the effect of NF-κB signaling pathway on CNPY2-induced cisplatin resistance was examined by plate formation assay,Annexin V-FITC and TUNEL assay.Results 1.The results of TCGA analysis showed that the expression of CNPY2 m RNA in lung cancer was higher than that in adjacent normal tissues.The public platform Kmplot was used to analyze the relationship between CNPY2 m RNA expression and overall survival(OS)in patients with non-small cell lung cancer.It was found that the total survival of lung cancer patients with high CNPY2 expression was higher than that of patients with low CNPY2 expression.2.The expression of CNPY2 in non-small cell lung cancer was higher than that in normal adjacent normal tissues.CNPY2 was negatively correlated with EMT marker E-cadherin.3.A subcellular line expressing CNPY2,including Vetor and A549-CNPY2,was successfully constructed in A549 cell line.Simultaneously,subcellular line knockdown of CNPY2 was constructed in A549 cell line,including scramble and si CNPY2#1 and si CNPY2#2.4.In vitro cell experiments,we found that overexpression of CNPY2 promoted invasion and migration in lung cancer cells,decreased expression of E-cadherin,and increased expression of Vimentin and Snail.In addition,CNPY2 activates the AKT /GSK3β signaling pathway with the increased expression of p-GSK3β and p-AKT.After inhibition of the AKT /GSK3β signaling pathway,CNPY2-induced EMT and cell metastasis were attenuated.5.After treatment of primary bronchial epithelial BEAS-2B cells and 8 NSCLC cell lines with cisplatin,the IC50 was lower but the abilities of forming clones was higher in H1563 and H358 cells with higher CNPY2 expression than in H2291 cells.6.Overexpression of CNPY2 increased the number of plate clones and decreased the number of apoptotic cells,which contributing to the resistance to cisplatin.Silencing CNPY2 reduced the number of plate clones and increased the number of apoptotic cells,which leading to sensitivity to cisplatin.Moreover,CNPY2 promoted the apoptosis-related regulatory factors,including c-IAP1,c-IAP2,XIAP,Bcl-2 and Bcl-xl.CNPY2 activates NF-κB signaling pathway through significantly increasing NF-κB transcriptional activity.Upon activation of the NF-κB signaling pathway,CNPY2 inhibits the expression of NF-κB inhibitory factors and enhances anti-apoptotic effects,thereby promoting cisplatin resistance.Conclusion 1.CNPY2 is significantly correlated with the clinicopathological grade and survival prognosis of patients with non-small cell lung cancer.CNPY2 is expected to be a protein marker that determines diagnosis and clinical prognosis of non-small cell lung cancer.2.CNPY2 promotes the malignant progression of non-small cell lung cancer through activating the AKT/GSK3β pathway,inducing EMT,and promoting cell migration and invasion.3.CNPY2 activates NF-κB signaling pathway,promotes transcription of downstream genes of NF-κB,enhances resistance to cisplatin via increasing survival and anti-apoptosis of NSCLC cells. |
PDF Full Text Request