Hlec Effect On The Secreted Proteins Of Epithelial Ovarian Cancer Cells With Proning To Metastasize To Lymph Nodes And Verification Of Candidate Proteins

Chapter one: HLEC（human lymphatic endothelial cells） effect on thesecreted proteins of epithelial ovarian cancer cells with directionalmetastasize to lymph nodesObjective: To explore the effect of HLEC（human lymphatic endothelialcells） on the secreted proteins of epithelial ovarian cancer (EOC) cells(SKOV3-pm4) with directional highly lymphatic metastasis. A co-culturesystem consisting of human lymphatic endothelial cells（HLEC）,SKOV3cellline and its sub-clone SKOV3-pm4was built. Methods: The supernatants offour groups of cultured cells(A：SKOV3，B：SKOV3+HLEC，C：SKOV3-PM4， D：SKOV3-PM4+HLEC) were collected and their proteins were detected byantibody arrays and iTRAQ-2D-LC-MALDI-TOF/TOF/MS, respectively. Thebioinformatics analysis of the differential proteins among those groups wasperformed by Gene ontology and DAVID database，String, KEGG PATHWAYand Coremine database were used to analyze molecular function, location andbiological process of different proteins. Results:(1) The results of antibodyarrays demonstrated that there were39up-regulated and5down-regulatedproteins between group A and C,22up-regulated and19down-regulatedproteins between group D and C, respectively.(2) Total200varieties secretoryproteins in SKOV3cells,SKOV3-PM4cells, SKOV3+HLEC cells andSKOV3-PM4+HLEC cells were identified by iTRAQ (confidence level greaterthan95%).The difference secretory proteins between SKOV3-PM4andSKOV3cells were36varieties. There were22proteins up-regulated and14proteins down-regulated. The difference secretory proteins betweenSKOV3-PM4+HLEC and SKOV3-PM4cells were65varieties. There were37proteins up-regulated and28proteins down-regulated.(3)The biological processof the different proteins involved in substance metabolism, enzymatic activityregulation, DNA synthesis, protein translation, ion transport and so on.Candidate proteins of SPARC,VEGFA、GRN may be relate to ovarian carcinomalymphatic metastasis. Conclusion: Directional metastasis to lymph nodes ofovarian cancer cells was closely associated with the HLEC microenvironment which included cell stromal proteins, adhesion factors, interleukins, chemokinesand signal transduction molecular. Chapter two: Verification of candidate proteins effected by HLEC on thesecreted proteins of epithelial ovarian cancer cells with directional highlylymphatic metastasisObjective: Differential secretory proteins related to the lymph node metastasiswere GO cluster analyzed, their network was mapped by bioinformatics，theexpression candidate proteins of VEGFA, IGFBP7and SPARC were validatedin clinical serum samples and different lymph node metastasis potential ovariancancer cells. Methods: The candidate screened proteins of VEGFA and IGFBP7were validated in human serums and cell mediums by ELISA; the expression ofSPARC in skov3cell and SKOV3-PM4cell was validated by RT-PCR andimmunofluorescence technique. Results: there are statistical significance of theVEGFA and IGFBP7among three groups of patients with ovarian cancers,benign tumors and control.VEGFA specifically up-regulated in the ovariancancers group and down-regulated in the control.IGFBP7up-regulated in thecontrol and down-regulated in the ovarian cancers group. The results of RT-PCRand immunofluorescence shows that the expression of SPARC in SKOV3-PM4cells were lower than that of SKOV3cells. Conclusion: up-regulation ofVEGFA and down-regulation of SPARC and IGFBP7are closely associated withdirectional metastasis to lymph nodes in ovarian cancer cells. Chapter three: The latest research on the relationship between tumormicroenvironment and lymphatic metastasisNormal cells are in a relatively stable internal environment. Cellproliferation, differentiation, and apoptosis, as well as secretion and expressionof related factors, occur in accordance with the normal process. However, thebalance is broken with tumor growth and progression. A gradual formation of asuitable external environment for the tumor growth, namely, the tumormicroenvironment, occurs. And the pathological and physiologicalcharacteristics of tumor microenvironment, which contains numerouslymphangiogenic growth factors and inflammatory conditions, hypoxia, acidicmicroenvironment, and high interstitial fluid pressure, promote tumorlymphangiogenesis and formation of tumor lymph node metastasis. This articlewill summarize the relationship between tumor microenvironment,lymphangiogenesis and lymphatic metastasis in ovarian cancer.