The Establishment And Function Of LCN2 Overexpression In Ovarian Cancer Cells

Objective:Epithelial ovarian cancer is the most lethal and aggressive gynaecological cancer and the third common cause of female cancer death in the world. Although the diagnostic methods have been improved, the recurrence, metastasis and multi-drug resistance affect ovarian cancer therapeutic effects seriously. The high mortality rate of ovarian cancer results from the high percentage of cases diagnosed at an advanced stage, which is because of the relatively asymptomatic nature of early-stage disease and the lack of adequate screening tests. Use of serum markers for early detection of ovarian cancer has largely focused on CA125, or CA199. According to different types of cancer, they have different changes. Especially, Some serum markers has increased in advanced stage. Not universal, but it was already serves for early detection and prognosis of ovarian cancer patients.Lipocalin-2(LCN-2), also known as neutrophil gelatinaseassociated lipocalin (NGAL), is secreted glycoprotein belonging to the gelatinaseassociated lipocalin 2 members of the family. Elevated LCN2 expression has been observed in multiple human cancers including HCC, breast, color-ectal, and ovarian cancers. However, the biological roles of elevated LCN2 in cancer cells are not yet clear. Our previous study demonstrated that over-expression of LCN-2 could promote cell proliferation. in order to further study the role of LCN-2 in ovarian cancer cells mechanism provides the information platform, and provide more valuable information for therapeutic target in ovarian cancer.Methods:The expressions of LCN-2 in ovarian cancer cell lines(CAOV3, ES2, OVCAR3, SKOV3) were evaluated by Western blot analysis; Specific immunoreactivity of LCN-2 was tested by transiently transfection in ES-2 cells; The best concentration of G418 was selected by dose depended manner.LCN-2 stable expression cell line was established in ES-2 cells, and expression of LCN-2 was detected by Western blotting analysis. MTT assay vas used to examine the proliferation of LCN-2-expressing and empty control ransfectants.Results:1 We detected the expression of LCN-2 in four ovarian cancer cell lines (CAOV3, ES2, OVCAR3, SKOV3) by Western Blot analysis, discovering that CAOV3 and OVCAR3 have a higher expression of LCN-2, ES-2 have no expression, and SKOV3 have a lower expression.To examine the protein level of LCN-2 in ovarian cancer cell lines, we performed western blot analysis in four cell lines (CAOV3, ES2, OVCAR3, SKOV3), the results showed that CAOV3 and OVCAR3 have a higher expression of LCN-2, ES-2 have no expression, and SKOV3 have a lower expression. After cell culture, we also observed morphological characteristics in four cell lines.2 LCN-2 plasmid and antibody were tested by transient transfection in ES-2 cells.To clarify whether LCN-2 plasmid we established is succeed. ES-2 transfected with pcDNA3.1/mycHis-LACZ, pcDNA3.1/mycHis-LCN-2, PE GFPN3, PEGFPN3-LCN-2.24 hours later western blot analysis was perl-ormed to detect LCN-2 expression. We found that in cells where LCN-2 was detected by transfecting pcDNA3.1/mycHis-LCN-2 and PEGFPN3-L-CN-2, LCN-2 specific immunoreactivity was over-expression. This resu-It indicated that LCN-2 plasmid we established is succeed.3 The best concentration (700 μg/ml) of G418 treatment in ES-2 cells.ES-2 cells were treated with G418 by dose-depended manner (200 μg/ml, 400 μg/ml,600 μg/ml,900 μg/ml,1200 μg/ml,1500 μg/ml). Microscope was used to detect the best concentration of G418 treatment in ES-2 cells.4 LCN-2 stable over-expression cell lines were established in ES-2.To further examine LCN-2 function in ovarian cancer cells. We established ES-2 cells stably expressing LCN-2 (#2,#3,#12,#14,#21,#22, #23 and #31) and control cells (V1, V2, V3 and V7) by Western blot analysis. Furthermore, western blot analysis was re-performed to confirm LCN-2 over-expression and similar results were obtained in these cell lines (#2;#3; #31 and #23). Microscopically observed ES2 cell lines and the expression ector pcDNA/mycHis-LCN2 stable transfection into ES2 cell line,the norphology shows no significant change.5 Determined by MTT cell proliferation test found that over-expression of LCN-2 promotes ovarian cancer cell proliferation.In order to test the effects of LCN-2 on cell growth, the proliferation of stable transfectants of LCN-2 was measured by MTT assay and compared with parental and vector control cells. This result indicated that cell proliferation was more increased in the LCN-2 transfectants (#2 and #3) than n the parental and control cells (V1 and V2).Conclusions:1 LCN-2 plasmid we established is succeed.2 Varying levels of LCN-2 expression were observed among four ovarian cancer cell lines.3 LCN-2 antibody is working well.4 LCN-2 stable over-expression cell lines were established in ES-2.5 Over-expression of LCN-2 can promote ovarian cancer cell proliferation.