Lentiviral Mediated Down-regulation Of Mesothelin Inhibit Cell Growth And Metastasis Of Epithelial Ovarian Cancer

Ovarian cancer is the most common female malignancy and is the leading cause of death from gynecological malignancies. The majority of patients are diagnosed with advanced epithelial ovarian cancer. only 30% of patients with advanced-stage ovarian cancer survive 5 years after initial diagnosis. But the survival rate of patients diagnosed in early stage can reach up to 90%.Additional serum markers will be required to detect all patients in an initial phase by screening, and effective treatment will be required for patients with advanced-stage ovarian cancer.Mesothelin is a differentiation antigen whose expression in normal human tissues is limited to mesothelial cells lining of the pleura, pericardium and peritoneum. However, mesothelin is highly expressed in several human cancers, including mesotheliomas and pancreatic adenocarcinomas, and ovarian cancers. The mesothelin gene encodes a precursor protein of 69 kDa that is processed to a 32 kDa shed protein called megakaryocyte potentiating factor (MPF) and a 40 kDa fragment, mesothelin, that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Results of recent studies suggest that the mesothelin may play a role in ovarian cancer metastasis by binding to MLSN/CA125. The binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors. CA125 was reported to be a ligand of mesothelin . Blocking MLSN/CA125-dependent cell attachment may prevent or delay peritoneal metastatic recurrence.A small amount of cell bound mesothelin is shed into the serum and has been shown to be elevated in patients with mesothelioma and ovarian cancer. Studies suggest that serum mesothelin(soluble mesothelin-related protein SMRP) could be useful for diagnosis and follow-up of these patients. Mesothelin is a new target for immunotherapy.SS1P,a recombinant anti-mesothelin immunotoxin, its potent antitumor efficacy in animal models was confirmed, given to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers is well tolerated.Results of our recent studies suggested that mesothelin was a significantly up-regulated gene in epithelial ovarian carcer by cDNA microarray analysis. The expression of mesothelin and CA125 is high expressed in epithelial ovarian cancer at protein level.There is significant correlation between mesothelin,CA125 expression and grade,histologic type of epithelial ovarian cancer.The anti-SMRP antibody 2H10 were obtained with the compound peptide. The antibody could react with native MSLN and had high specificity.The function of mesothelin is not known clearly. In this study,firstly,the co-located expression of MSLN and CA125 were detected in human ovarian cancer cells and tissue. Secondly, Mesothelin'function was investsigated in the process of epithelial ovarian cancer's growth and metastasis in vitro and vivo.Thirdly, Lentivirus was given to animal models of ovarian cancer for gene therapy . Finally,a ELISA kit of SMRP was developped.Part I Expressions of mesothelin and CA125 in human epithelial ovarian cancer and its significanceObjective: To investigate the expressions of mesothelin and CA125 and their biological significance in human ovarian cancer cell lines and in human epithelial ovarian neoplasms.Methods: Mesothelin and CA125 protein expressions and locations in human epithelial ovarian cancer tissues and cell lines were detected by indirect immunofluorescence double maker and western blotting. EDTA-induced cell detachment and cell adhesive ability to matrigel were performed.Results: Mesothelin and CA125 specifically bound to cytomembrane of human epithelial ovarian cancer with indirect immunofluorescence double labelling. Mesothelin was red and CA125 was green,they were located together as yellow flourescence. Expressions of mesothelin in epithelial ovarian cancer(1639.2±181.92) was significantly higher than that in borderline adenoma(590.8±126.9) ,benign ovarian tumor(227.69±26.54) and normal ovary(213.0±24.37) (p<0.05).The expression of pathology grade in G2,G3(1642.63±44.58; 1701.57±54.73) was significantly higher than that in G1(1553.99±57.2)( F=10.01, p<0.05).The expression of histologic type in serous,endometrioid(1723.81±33.20; 1673.81±70.27) was significantly higher than that in mucinous(1466.12±94.93) (F=18.61,p<0.05). In age,stage and CA125 in serum,the expression of MSLN have no significant differences(p>0.05). Expressions of mesothelin and CA125 was correlated highly(r=0.95, p<0.05). The expression of mesothelin in the SKOV3(1.33±0.04 ) and 3AO(1.03±0.08) cells were weaker than OVCAR-3(1.57±0.07).Cell adhesion tests showed that the SKOV3 (47.3±3.9)%,and 3AO(45.7±2.1)% adhered less effectively to matrigel than OVCAR-3(70.4±2.5)% (p<0.01).Conclusions: Mesothelin and CA125 is high expressed in human epithelial ovarian cancer tissues and cell lines,and co-located .Mesothelin may be related with cell adhesive ability and gradeing,histologic type of epithelial ovarian cancer.Part II Construction and identification of recombinant lentiviral vectors and cell lines of cDNA and RNA interference of mesothelin geneObjective: To construst recombinant Lentivirus vectors of cDNA and RNA interference of mesothelin gene . To construst cell lines of down- regulated and up-regulated of mesothelin by gene transfer for function study and gene therapy.Methods: According to the Genebank information of mesothelin, 4 interfering sequence and a negative sequence were designed and inserted into plasmid pRNAT-U6.2/Lenti. Mesothelin'cDNA sequence was inserted into plasmid pWPXL-MOD.After packaging,SKOV3 cell was transfected and detected by Western blotting.The plasmid with the highest interfering efficiency was packaged by packaging plasmid mix.LV-MSLN-cDNA , LV-MSLN-shRNA, LV-MSLN-neg were transfered into ovarian cancer cells lines SKOV3,and screened.The expression of MSLN was confirmed by the means of Western blotting and immunofluorescence. Results: DNA sequencing showed that the sequence of recombinant Lentivirus plasmids were correct. The lentivirus with the highest interfering efficiency was LV-MSLN-shRNA4,The interfering efficiency was 90%. MSLN of SKOV3 cells transfered by LV-MSLN-cDNA was up-regulated 50%. It was confirmed that the shRNA,cDNA and negative sequence had been stably integrated into SKOV3 cells lines. Mesothelin specifically bound to cytomembrane of these cells. The expressions of mesothelin in the interfered cell (SKOV3-MSLN-shRNA) was weaker than the control cells (SKOV3-MSLN- neg,SKOV3) and up-regulated cell(SKOV3-MSLN-cDNA).Conclusion: Recombinant lentiviral vectors and cell lines of down- regulated and up-regulated of mesothelin gene were constructed successfully. It can be a useful toll to study the function and gene therapy of mesothelin.PartⅢEffects of mesothelin on cell growth and adhesion,invasion ability in human ovarian cancerObjective: To evalute the effect of mesothelin on the cell growth, adhesion and invasion ability in human ovarian cancer cell line with different expressions of mesothelin.Methods: The cell growth ability was evaluated by proliferation and clone forming experiments. EDTA-induced cell detachment and cell adhesive ability to matrigel were performed. Invasion and migration assay was performed by the Transwell. Mesothelial cells were cultured for adhesive assay.Results: The proliferation of SKOV3-MSLN-shRNA((9.89±2.0)×105) was lower than SKOV3((19.81±2.5)×105),SKOV3-MSLN-neg((18.9±2.24)×105) and SKOV3-MSLN-cDNA((23.68±2.35)×105) (p<0.01).Cell adhesion tests showed that the SKOV3-MSLN-shRNA adhered less effectively to plastic substance and matrigel than SKOV3,SKOV3-MSLN-neg and SKOV3-MSLN-cDNA(p<0.01).In invasion and migration assay, the invasion cells and migration cells of SKOV3-MSLN-shRNA were lower than that of respective control groups and up-regulated group(p<0.01). Mesothelial cells were cultured successfully. SKOV3-MSLN-shRNA cells adhered less than other groups with mesothelial cells(p<0.01).Conclusions: Down-regulation of mesothelin led to inhibition of epithelial ovarian cancer cell growth and adhesive,invasion and migration ability,and may prevent or delay peritoneal metastatic recurrence.PartⅣEffects of mesothelin on transplanted tumor growth of human epithelium ovarian cancer and gene therapy in nude miceObjective: To study the effects of mesothelin in epithelium ovarian cancer and gene therapy in vivo.Methods: Test one: SKOV3,SKOV3-MSLN-cDNA, SKOV3-MSLN-neg, SKOV3-MSLN-shRNA cells were injected into the peritoneal cavity of nude mice; Test two:tumor cells SKOV3 and LV-MSLN-cDNA,LV-MSLN-neg, LV-MSLN-shRNA,PBS were injected at the same time; Test three:SKOV3 and Lentivirus were injected at the frist day, Lentivirus were injected every other day; Test four: only lentivirus were injected. Weight,distribution and ascites of transplanted tumor were measured after 14 days.Results: Transplanted tumor of SKOV3-MSLN-shRNA cells were suppressed obviously in nude mice compared with the control cells (p<0.01).In test two, the growth of SKOV3 were not suppressed obviously by LV-MSLN-shRNA;But in test three, suppression was obvious by LV-MSLN-shRNA, compared with the control cells; Three kinds of lentivirus have no toxic effec on nude mice.Conclusions: Lentivirus mediated RNA interference of mesothelin suppressed the transplanted tumor growth of human epithelium ovarian cancer.It will be a powerful strategy for cancer gene therapy. PartⅤDevelopment of enzyme-linked immunosorbent assay for SMRPObjective: To develop a indirect ELISA assay with the prokaryotically expressed gene product of SMRP.Methods: The complete length of mesothelin gene was cloned into prokaryotic expression vector pMLSN-cDNAY from eukaryotic expression vector pMLSN-cDNA. The target gene was then expressed in the E.coli cells,the target protein in total bacterial proteins was purified.BALB/c mice were immunized with the recombinant protein.The splenic cells of the mice were fused with SP2/0 mouse myeloma cells.The positive clones were screened.The stable hybridoma cell lines which can excrete antibody were expand cultivation.The titer of antibody were detected with ELISA.BALB/c mice were immunized with some hybridoma cells.Ascites were taken after two weeks. High titer antibodys of anti-MSLN were obtained. Some matched antibodys were selected.Precoating microplate with one anti-MSLN mAb, detecting method includes another anti-MSLN mAb with HRP,and was used in the detection of the recombinant protein at different levels.Results: Recombinant prokaryotic expression vector pMLSN-cDNAY of mesothelin gene was constructed successfully. The concentration of the purified protein was 82%.The stable hybridoma cell lines(21) were screened. Some matched antibodys were selected and the best was detected. This assay had good reproducibility and steadiness,the sensitivity reached to 10 ng/ml.Conclusions: The indirect ELISA assay for the detection of SMRP was established.