Mechanism Study Of Nck1 Overexpression Promotes Malignant Behavior In Epithelial Overrian Cancer

Ovarian cancer is the fifth most common cause of cancer death in women and the most deadly gynecological malignancy.The overall five-year survival rate for ovarian cancer patients is approximately 47%.However,the five-year survival rate for EOC patients with advanced stage,presenting with intraperitoneal metastasis,is a mere 29%.Approximately90% of ovarian cancers are classified as malignant epithelial ovarian carcinomas(EOCs),and the subtypes include serous,mucinous,clear cell and endometrioid.The noncatalytic region of the tyrosine kinase(Nck)family of proteins are ubiquitously expressed adapter proteins and thus potentially interact with proline-rich sequence(PRS)–bearing proteins and tyrosine phosphorylated proteins.Previous studies have indicated that Nck plays vital roles in the progression and metastasis of carcinomas and is also involved in angiogenesis.The association between Nck1 expression and prognosis has not been adequately analyzed in EOC.PART ? THE CLINICAL SIGNIFICANCE OF NCK1 EXPRESSION IN EPITHELIAL OVERRIAN CANCERObjectivesTo detect the expression of Nck1 protein in ovarian tumors of different pathological types,and analyze the relationship between the expression of Nck1 and the prognosis of EOC patients.MethodsTo evaluate Nck1 protein expression,289 paraffin-embedded(176EOC)and 95 fresh surgical(42 EOC)samples were examined by immunohistochemical staining and Western blot,respectively.Survival analysis and Cox regression were performed to assess the correlation between Nck1 expression and survival.The localization of Nck1 in ovarian cancer cells was observed by immunofluorescence staining by a laser scanning confocal microscopy.ResultsBy immunohistochemical staining and Western blot,Nck1 protein expression was significantly higher in EOCs(P < 0.05).Nck1 overexpression was significantly correlated with shorter overall and disease-free survival(P<0.05).Nck1 overexpression was an independent prognostic factor of an unfavorable outcome,together with advanced stage,serum CA125 level?900UI/ml and suboptimal surgical debulking(P <0.05).Nck1 is overexpressed in three human ovarian cancer cell lines(SKOV3,OVCAR-3 and ES-2).Nck1 was mainly localized in the cytoplasm and membrane of SKOV3 and OVCAR-3 cells by immunofluorescence assay.ConclusionsNck1 is overexpressed in EOC tissues and cells.Nck1 overexpression was an independent prognostic factor of an unfavorable outcome,together with advanced stage,serum CA125 level?900UI/ml and suboptimal surgical debulking.PART ? CONSTRUCTION OF LENTIVIRAL-MEDIATED NCK1 INHIBITION VECTOR AND SELECTION OF STABLY TRANSFECTED EPITHELIAL OVARIAN CANCER CELL LINESObjectives To construct lentiviral vector-mediated Nck1 inhibition vector and to select stable transfected EOC cells for subsequent experiments.Methods(1)The sh RNA sequences targeting Nck1 and a negative control sequence were designed.p HB-U6-MCS-zsgreen-puro or p HBLV-CMV-MCS-EF1-NEO vectors were digested by double restriction digestion.And DNA sequencing were used to confirm the constructed vectors whether or not successful recombinant vector plasmid.(2)The correct recombinant vector plasmids were propagated and co-transformed into the 293 T cells by a lentivirus packaging system.The virus supernatant was collected for obtainin lentivirus supernatant by ultracentrifugation.The virus titers were determined by fluorescence intensity.(3)The lentiviral vector was transfected into SKOV3 and OVCAR3 cells,and stable transfected cell lines were screened by fluorescence microscopy and purinomycin or G-418.(4)Western bolt and RT-PCR were used to screen the stably transfected EOC cell line with low protein and m RNA expression levels of Nck1.Results(1)Three pairs of sh RNA sequences targeting Nck1 and a negative control sequence were successfully designed.The constructed vectors were confirmed by DNA sequencing.And successful recombinant lentiviral vector plasmids were obtained,with a tilter of 2×108TU/ml.(2)After transfecting the lentivirus into SKOV3 and OVCAR3 cells,we established and screened the stably transfected cells by fluorescence microscopy and purinomycin or G-418.(3)Nck1 si RNA transfection of SKOV3 and OVCAR3 cells resulted in decreased Nck1 protein and m RNA levels(P<0.05).Therefore,SKOV3-sh RNA3 and OVCAR3-sh RNA1 were selected for subsequent experiments.Conclusions:The lentiviral vector-mediated Nck1 inhibition vectors were successfully constructed.By lentivirus vector,we screened and established the stably transfected EOC cell line with the more inhibited effect of Nck1,which laid the foundation for subsequent experiments.PART ? EFFECTS OF NCK1 GENE SILENCING VIA RNA INTERFERENCE ON MALIGNANT BIOLOGICAL BEHAVIOR OF EPITHELIAL OVARIAN CANCER CELLSObjectives To determined the effect of Nck1 silencing in EOC cell invasion,migration,proliferation and adhesion.Methods The morphology of SKOV3/Nck1-and SKOV3-NC cells were observed by confocal immunofluorescence microscopy.The transwell invasion and migration assays were used to determine whether Nck1 was critical to EOC cell invasion and migration.The cell adhesion assay was used to investigate the change of cell adhesion ability after Nck1 gene silencing or over-expression.In addition,CCK-8 assay and soft agar assay were used to detect the change of EOC cell proliferation after Nck1 gene silencing.To examine whether Nck1 silencing in human EOC cells confers oncogenic transformation in vivo,SKOV3/Nck1-and SKOV3-NC cells cells were injected subcutaneously and intraperitoneally in female BABL/c nude mice.Results(1)By confocal immunofluorescence microscopy,the morphology of SKOV3/Nck1-cells was reshaped.Compared with SKOV3-NC cells,SKOV3/Nck1-cells showed loose polarization,missing lamellipodia and filopodia and reduced cellular protrusions.(2)By transwell invasion and migration assays,Nck1 silencing significantly reduced the number of invaded and migrated SKOV3 and OVCAR3 cells.Furthermore,Nck1 silencing also reduced the colonogenicity and decreased the number of formed colonies significantly in SKOV3 and OVCAR3 cells.Nck1 silencing significantly inhibited the proliferation of SKOV3 and OVCAR3 cells.In comparison with the controls,Nck1 over-expression significantly increased the invasion,migration,colonogenicity and proliferation of SKOV3 and OVCAR3 cells.(3)Nck1 silencing retarded the growth of implanted OC tumors in mice.Furthermore,we injected SKOV3-NC or SKOV3/Nck1-cells intraperitoneally into individual mice and on day 28,we found that SKOV3/Nck1-cells formed fewer(59.83±4.91)nodules than SKOV3-NC cells(89.83±3.26,P<0.05)in mice.Conclusions(1)Nck1 gene silencing can change the morphology of ovarian cancer cells and inhibit the migration,invasion and proliferation of ovarian cancer cells in vitro.(2)Nck1 silencing limited the migration and metastasis of OC tumors in vivo.PART ? THE MECHANISM OF NCK1 PROMOTES MALIGNANT BEHAVIOR THOUGH THE MODULATION OF DOWNSTREAM KINASES IN EPITHELIAL OVARIAN CANCERObjectives To determined the mechanism of Nck1 promotes malignant behavior in epithelial ovarian cancer.Methods The morphology of Nck1-silencing xenografts was observed by HE staining.Western blot was used to detect the changes of VEGF,MMP-2and MMP-9 in xenografts after Nck1 silencing.The human phosphokinase antibody array was used to detect the effects of Nck1 silencing on downstream phosphorylated kinase signaling pathways.Results(1)Morphologic changes of Nck1 silencing xenografts were observed by HE staining.(2)The downregulation of Nck1,MMP-2,MMP-9 and VEGF expression was observed in xenografts formed by SKOV3-Nck1-compared with those formed by SKOV3-NC(all P< 0.05).(3)By human phosphokinase antibody array,a total of 11phospho-kinase-related proteins were detected in decreasing activation.(4)Nck1 promotes the progression of OC predominantly by enhancing the PI3K/AKT/p70S6 K signaling.Conclusion(1)Nck1 may participate in angiogenesis and promote the invasion and metastasis of ovarian cancer cells by regulating the expression of MMP2,MMP-9 and VEGF.(2)Nck1 may be involved in regulating malignant behavior of EOC cells through PI3K/AKT/p70S6 K signaling.