Screening And Identification For Lymphatic Metastasis-associated Differentially Expressed Genes And Proteins Between Ovarian Carcinoma Cell Lines With Directional And Non-directional Highly Lymphatic Metastasis

Objective The ovarian serous papillary adenocarcinoma cell line SKOV3 and its second subclone SKOV3-pm2,the third SKOV3-pm3 that institute with directional highly lymphatic metastasis in nude mice,which provides a cell model to investigate the molecular mechanism in the process of invasion and metastasis of ovarian cancers.Screening for lymphatic metastasis-associated genes and proteins can offer biomarkers and targets to clinical diagnosis and therapy.Methods The ovarian adenocarcinoma cell lines with directional (SKOV3-pm)and non-directional(SKOV3)highly lymphatic metastasis were examined by HE staining,series of cell growth curve,cell matrigel invasion assay and cloning assay to affirm the biological behaviors.Ovarian lymphatic metastasis-associated genes and proteins were screened by high-throughput and acuity gene chip method,SELDI-TOF-MS,PF-2D and ESI-MS.Results Compared to ovarian adenocarcinoma cell line with non-directional (SKOV3)lymphatic metastasis,the highly directional(SKOV3-pm)lymphatic cells are larger and anomalous in shape,whose caryocinesis phase can be detected more frequently.Meanwhile,the latter's growth rate is accelerated obviously and cell doubling time is shortened.The invasive power and proliferative ability of cell line(SKOV3-pm)are enhanced significantly as well.Among the 60 screened different expression genes,47 genes were expressed up-regulated(ratio＞3)and 13 genes down- regulated(ratio＜0.5).The coding productions refer to oncogenes,Metastasis Suppressors,Metastasis-Associated Proteases,Protease Inhibitors,Growth Factors and Receptors,Cell-Cell and Cell-Matrix Interaction Molecules,Signal Transduction Molecules and Cell apoptosis-related cysteine protease.The M/Z 6971,7475,9089,9453,10103,11655and 4746 of protein in endochylema and supernatant of SKOV3 and SKOV3-pm were expressed differentially.13 protein peaks,including H3 histone family 3A,Apolipoprotein E receptor 2, Huntingtin interacting protein HYPE,Similar to signal recognition particle 9kD, Adipocyte-derived leucine aminopeptidase variant,Beaded filament structural protein 1(filensin),Poly(A)binding protein cytoplasmic 4(Inducible form)and so on,were further identified by tryptic digestion,peptide mass fingerprinting and mass spectrometry.Nine proteins were up-regulated while four were down-regulated in SKOV3-pm cells.Conclusions To identify the key or candidate gene/pathway and proteinum production responsible for lymphatic metastasis might be used as diagnostic marker and therapeutic targets for tumor lymphatic metastasis.In addition,combining gene chips as well as proteome technology and cell models of ovarian lymphatic metastasis in vitro may provide a new approach to further research for tumor metastasis mechanisms. Objective The ovarian adenocarcinoma cell lines with directional highly lymphatic metastasis have been established effectively.In order to provide an efficient cell model for further investigation,to identify and verify the biological behaviors of cell invasive power and proliferative ability are available.Methods The ovarian adenocarcinoma cell lines with directional (SKOV3-pm)and non-directional(SKOV3)highly lymphatic metastasis were examined by HE staining to observe cell morphology and assay cell divisional index respectively;The growth rates of cell lines were described with series of growth curve;Cell matrigel invasion assay and cloning assay were used for detecting cell lines invasive power and proliferative ability.Results Compared to ovarian adenocarcinoma cell line with non-directional (SKOV3)lymphatic metastasis,the highly directional(SKOV3-pm)lymphatic cells are larger and anomalous in shape,whose caryocinesis phase can be detected more frequently.Meanwhile,the latter's growth rate is accelerated obviously and cell doubling time is shortened.The invasive power and proliferative ability of cell line(SKOV3-pm)are enhanced significantly as well.Conclusions The fiercely invasive power and proliferative ability which are possessed in ovarian adenocarcinoma cell line with directional(SKOV3-pm) highly lymphatic metastasis in vitro satisfied for further experiment.It is feasible to screen for lymphatic metastasis-associated genes and proteins.An effective experimental model for further investigation to the molecular mechanism in the process of invasion and metastasis of ovarian cancers. Objective The ovarian serous papillary adenocarcinoma cell line SKOV3 and its second su bclone SKOV3-pm2,the third SKOV3-pm3 that institute with directional highly lymphatic metastasis in nude mice,which provides a cell model to investigate the molecular mechanism in the process of invasion and metastasis of ovarian cancers.In order to screen for lymphatic metastasis-associated genes and offer biomarkers and target gene to clinical diagnosis and therapy,the gene expression profiles of the three ovarian carcinoma cell sublines with different metastatic capability were compared by cDNA microarray.Methods Total RNAs were isolated from cell line SKOV3,SKOV3-pm2,SKOV3-pm3,and reversely transcribed to the cDNA with the incorporation of fluorescent dUTP labeled by AMPOLABELING-LPR after the detection of concentration and purity,prepare the hybridization probes and mix to hybridized to the cDNA microarray.Though high-stringent washing,the cDNA microarray was scanned for fluorescent signals and analyzed for different expression of the target genes between three cell lines.Then two of the different expression genes were further validated by RT-PCR technique.Results Among the 60 screened different expression genes,47 genes were expressed up-regulated(ratio＞3)and 13 genes down-regulated(ratio＜0.5).The coding productions refer to oncogenes,Metastasis Suppressors,Metastasis-Associated Proteases,Protease Inhibitors,Growth Factors and Receptors,Cell-Cell and Cell-Matrix Interaction Molecules,Signal Transduction Molecules and Cell apoptosis-related cysteine protease.Conclusions The ovarian carcinoma with directional highly lymphatic metastasis is a complex process involving multiple factors and genetic changes, many lymphatic metastasis-associated genes were screened by high-throughput gene chip method;validating their cellular function will help to identify the key or candidate gene/pathway responsible for lymphatic metastasis,which might be used as diagnostic marker and therapeutic targets for tumor lymphatic metastasis. Objective To screen relatively lymphatic metastasis-associated differentially expressed protein in ovarian carcinoma cell lines with directional and nondirectional highly lymphatic metastasis using protein chips and time of flight mass spectrometry technology.Methods The ovarian serous papillary adenocarcinoma cell line SKOV3 and its second subclone SKOV3-pm2,the third SKOV3-pm3 that institute with directional highly lymphatic metastasis in nude mice,which performed surface-enhanced laser desorption and ionization-time of flight-mass spectrometry(SELDI-TOF-MS)to compare the proteins in endochylema and supernatant of the three types of cell lines with differentially directional lymphatic metastasis.Each sample was detected using weak cation exchange protein chip CM-10 and metal affinity chromatography protein chip IMAC-3,which can specifically bind the metal-combining-proteins.The detected peaks were filtrated and analyzed by Ciphergen proteinchip software 3.2.0,then we classified the contrasted ratio of height from(＞0.5)as differentially expressed protein.Results Proteomic spectra of the three cell lines using protein chip CM-10 and IMAC3 were generated by mass spectrometry,which shows that:The M/Z 6971,7475,9089,9453,10103,11655and 4746 of protein in endochylema and supernatant of SKOV3,SKOV3-pm2 and SKOV3-pm3 were expressed differentially.Conclusions With surface-enhanced laser desorption and ionization-time of flight-mass spectrometry(SELDI-TOF-MS)technology,we effectively screened and identified for lymphatic metastasis-associated differentially expressed protein in ovarian carcinoma cell lines with directional and non-directional highly lymphatic metastasis.These distinct proteins were closely correlated with directional highly lymphatic invasive potential. Objective To screen,purify and identify directional highly lymphatic metastasis-associated differentially expressed protein by two-dimensional chromatographic liquid-phase separation method and electrospray ionization mass spectrometry(ESI-MS).Methods Proteome analysis of ovarian carcinoma cell lines with directional(SKOV3-pm)and non- directional(SKOV3)highly lymphatic metastasis was enforced with two-dimensional liquid chromatography(PROTEMELAB^TM PF2D)and matched kit(Backman company).Cell lysates of Skov3 and Skov3-pm were fractionated according to PI using chromatofocusing with analytical columns in the first dimension,followed by separation of the proteins in each PI fraction using nonporous reversed-phase high performance liquid chromatography(HPLC), the two steps above-mentioned were detected twice respectively.A two-dimensional map of the protein content for each cell line based upon PI versus hydrophobicity as detected by UV absorption was generated and a differential display map indicating the presence of up- or down-regulated proteins was exhibited using ProteoVue and DeltaVue software.The differentially expressed proteins were digested by trypsin and identified with electrospray ionization mass spectrometry(ESI-MS).Results Image analysis of two-dimensional map revealed 22 differentially expressed proteins between Skov3 and Skov3-pm cells,and 13 protein peaks, including H3 histone family 3A,Apolipoprotein E receptor 2,Huntingtin interacting protein HYPE,SH2D1A protein,Adipocyte-derived leucine aminopeptidase variant,Beaded filament structural protein 1(filensin),Poly(A) binding protein cytoplasmic 4(Inducible form)and so on,were further identified by tryptic digestion,peptide mass fingerprinting and mass spectrometry.Nine proteins were up-regulated while four were down-regulated in SKOV3-pm cells.Conclusion The identified differentially expressed proteins may play important roles in the lymphatic metastasis of ovarian carcinoma,and offer biomarkers and target to ovarian clinical early diagnosis and drug treatment.