Expression And Clinical Significance Of AQP3 And AQP5 In Tirple-negative Breast Cancer

Part Ⅰ:Expression of AQP3 and AQP5 in Triple-negative Breast CancerObjective:The aim of this study was to investigate the expression of aquaporin(AQP)3 and AQP5 in triple-negative breast cancer(TNBC)tissues and in normal tissues adjacent to the carcinoma and to explore their clinical significance in patients with TNBC.Methods:The quantitative real-time polymerase chain reaction(RT-PCR)and western blot(WB)were used to detect the mRNA and protein expression levels of AQP3 and AQP5 in TNBC and adjacent normal tissues of patients who underwent surgery at Jiangsu Taizhou People’s Hospital between 2011 and 2012.Written informed consent was obtained from all patients.Results:1.The expression level of AQP3 in TNBC tissues was significantly higher than that in adjacent normal tissues in both the mRNA and protein levels(P<0.01).2.The expression level of AQP5 in TNBC tissues was significantly higher than that in adjacent normal tissues in both the mRNA and protein levels(P<0.01).Conclusions:The expression level of AQP3 and AQP5 in TNBC tissues was significantly higher than that in adjacent normal tissues.Hence,the results suggested that the increased expressions of AQP3 and AQP5 may be related to the tumorigenesis and development of TNBC.Part Ⅱ:The Expression and Distribution of AQP3 and AQP5 in TNBC Patients and Its Association With Patients’ PrognosisObjective:The aims of this study were to investigate the expression of AQP3 and AQP5,analyzing their associations with clinicopathological parameters,and to explore their clinical significance in TNBC patients.Methods:1.Immunohistochemistry was performed to detect the expressions and localizations of AQP3 and AQP5 in carcinoma tissues and matched adjacent normal tissues of TNBC patients.2.The relationship between clinicopathological features and AQP3/AQP5 expression in TNBC patients was investigated.3.Kaplan-Meier method was used for analyzing the association of AQP3 and AQP5 expression levels with 5-year disease-free survival and overall survival in TNBC patients.Results:1.AQP3 and AQP5 were expressed mainly in the membrane and cytoplasm of tumor cells in TNBC tissues,and the positive cells were stained brown.2.The expression levels of AQP3 and AQP5 were remarkably higher in the carcinoma tissues than that in the matched adjacent normal tissues(P<0.01).Moreover,in some of the TNBC tissues,AQP5 was more prominent on the invasive front,and AQP5 staining was decreased in the central areas adjacent to necrosis.3.Overexpression of AQP3 and AQP5 were significantly associated with tumor size,lymph node status,and distant metastasis(P<0.05).There was no significant difference in age,menopausal status,surgical method,or histological grade between the groups(P>0.05).In addition,aberrant overexpression of AQP5 was observed more frequently in TNBC tissues with high Ki-67 expression than in those with low Ki-67 expression(P<0.05).As analyzed by the Spearman’s rank correlation analysis,the expression level of AQP5 was closely associated with the expression of Ki-67(r=0.255,P<0.05).A similar correlation was not found for AQP3(P>0.05).4.5-year DFS and OS in the group with high AQP5 expression were poorer than those in the group with low AQP5 expression(P<0.05).Pairwise comparison showed that patients with AQP3-high/AQP5-high expression had the poorest DFS and OS of all the groups(P<0.05).5.Cox model statistical analysis shows that nodal status,distant metastasis,and increased the expression of both AQP3 and AQP5 were independent poor prognostic factors for OS and DFS in TNBC patients.Conclusions:We firstly reported the expression and distribution of AQP3 and AQP5 in TNBC tissues and its association with patients’ prognosis.The patients with AQP3-high/AQP5-high expression had the poorest 5-year DFS and OS of all the groups.In summary,the present study demonstrated for the first time that increased co-expression of AQP3 and AQP5 may be associated with tumor clinicopathological characteristics and could serve as the independent prognostic factors in TNBC patients.Combined expression of the two proteins may be a potential promising marker in patients with TNBC and could be a novel target for anti-cancer treatment.Part Ⅲ: AQP5 Regulates Proliferation,Apoptosis,Invasion and Migration of Triple-negative Breast Cancer Cells through Wnt/β-catenin Signaling PathwayObjective:The present study was to explore the mechanism of AQP5 and its role in the tumorigenesis and development of TNBC,and to find potential effective therapeutic targets.Methods:1.MDA-MB-231 cells were transfected with siRNA-AQP5 and AQP5 overexpression vector to establish a differential expression system for AQP5.2.CCK-8 assays were performed to determine the effect of the differential expression level of AQP5 in the proliferation of TNBC cells.3.The flow cytometry was conducted to evaluate the apoptosis of TNBC cells concerning the differential expression of AQP5.4.Wound healing assays were applied to examine the role of AQP5 in the migratory ability of TNBC cells.5.Transwell assays were performed to study the role of AQP5 in the invasive ability of TNBC cells.6.The qRT-PCR and western blot assays were used to study the effect of AQP5 expression level on the expression of EMT related molecules.7.The effects of ICG-001,a Wnt/β-catenin signaling pathway inhibitor,on the invasive and migratory capabilities of overexpressed AQP5 cells and downstream molecules were measured.Results:1.The expression of AQP5 in the MDA-MB-231 cells was significantly higher than that in the MCF-10A cells.2.Up-regulation of AQP5 significantly promoted the proliferation,migration and invasion,while inhibited the cell apoptosis of TNBC cells;in addition,up-regulation of AQP5 increased the expression of Bcl-2 and decreased the expression of Caspase-3.However,knockdown of AQP5 presented the adverse effects of AQP5 overexpression.3.Overexpressed AQP5 induced the overexpression of EMT-related factors,which further promoted the migration and invasion of cells.4.Overexpression of AQP5 could up-regulate the expression of β-catenin in the nucleus followed by increasing the expression levels of downstream genes in Wnt/β-catenin signaling pathway.Moreover,ICG-001,the inhibitor of Wnt/β-catenin signaling pathway,could significantly attenuate the effect of overexpression of AQP5 on cells,further confirming that AQP5 may promote the proliferation,migration and invasion of TNBC cells by activating Wnt/β-catenin signaling pathway.Conclusion:In the TNBC cells,AQP5 modulates the expression levels of EMT-related proteins through activation of Wnt/β-catenin signaling pathway,thus enhancing the cell proliferation,migration and invasion while inhibiting the cell apoptosis.