Involement Of LCN2 In Regulation Of Proliferation In Human Ovarian Carcinoma And The Implicated Mechanisms

Objective: As ovarian carcinoma is insidious in onset,most ovarian cancer presents as stage III or IV disease,its five-year survival rate is less than30% after initial diagnosis.Therefore,it has the highest fatality-to-case ratio.Lipocalin-2(LCN2)is a kind of 25 kDa secreted glycoprotein.NGAL(Neutrophil Gelatinase-Associated Lipocalin)protein discovered in human Neutrophilic granulocyte has a high homology with 24p3 protein.24p3 in mice has proved to be an oncogene,so LCN2 may be a new oncogene of human beings.It has shown that the higher degree of malignant ovarian cancer,the higher level of LCN2 expression.it's not clear about function or molecular mechanism of LCN2 in the ovarian cancer.This article is aimed at describing the relation of LCN2 expression with clinicopathologic characteristics of ovarian cancer,determing the biological roles of LCN2 in carcinomagenisis and progress of ovarian cancer,studying the interrated regulators and signallings,providing new free radical biological data can offer biomarker and targets to clinical diagnosis and therapy.Recently,neutrophil gelatinaseassociated lipocalin(LCN2)has been suggested as a key player in different cancer types.Methods: The LCN2 expression in ovarian cancer tissues and normal ovarian tissues were evaluated by immunohistochemistry;the impact of LCN2 on cell growth was measured by MTT and colony formation analysis;LCN2stable expression cell line was established in SKOV3 cells.Protein lysates from the treated and non-treated cells were applied to Human Phospho-Kinase Array to determine up-regulated or down-regulated protein expression pattern after treatment;Proteins were validated by Western Blot and immunofluorescence assay.Results:1 The IHC results showed that the expression of LCN2 in the normal tissues was negative expression,in the malignant tumor tissues was different positive expression.Statistical analysis that the expression of LCN2 increased significantly in the malignant tumor tissues when compared with normal tissues after positive intensity was analysised quantitatively.2 In order to test the effects of LCN2 on cell growth,the proliferation of transient transfectants of LCN2 was measured by MTT assay and compared with parental and vector control cells.MTT results showed that overexpression of LCN2 promotes ovarian cancer cell proliferation.3 Colony generation ability of LCN2 transfectanted were assessed.LCN2 transfectants conspicuously formed more and larger colonies than the parental cells or the control cells in ES2 cell line and SKOV3 cell line.These results suggest that over-expression of LCN2 can promote cell growth.4 The Phospho-kinase Array showed that in SKOV3 cells where LCN2 was detected by stable transfecting pcDNA3.1/MycHis-LCN2,LCN2 specific immunoreactivity was over-expression.After transfecting pcDNA3.1/MycHisLCN2,p-ERK and p-GSK3? were shown to be up regulated.5 The expression of LCN2,p-ERK,p-GSK3? and ?-catenin were analysised by Western Blot.In CAOV3 and OVCAR3 cells p-ERK,p-GSK3?and ?-catenin expression were significantly higher than ES2 and SKOV3 cells;Stably transfection of LCN2 up-regulated p-ERK,p-GSK3? and ?-catenin in ES2 and SKOV3 cell lines.6 Immunofluorescence assay showed that LCN2 induced p-ERK ?p-GSK3? and ?-catenin expression by transfecting pEGFPN3-LCN2 in SKOV3 cells.Conclusions:1 LCN2 expression was increased in the malignant tumor tissues when compared with normal tissues.2 LCN2 over-expression increased cell proliferation ability.3 LCN2 promoted cell proliferation may through ERK/GSK3? signaling pathway.