The Study On Relationship Between The Expression Of Cathepsin And Tissue Inhibitors And Invasion And Metastasis In Ovarian Carcinoma

Ovarian cancer is the msot lethal of the gynecolog,malignancies due to early metastastic progression.Most patients of ovarian cancer are diagnosed in their advanced stage and the relative 5-year survival rate is low.Cathepsins invovled in basement membrane and extracelluar matrix degradation may effect tumor invasiveness and metastastic potential.The activity of cathepsins is regulated by the tissue inhibitor(cystatin C).The imblance between cathepsins and cystatin C has significant relevance with the invasion and metastasis of ovarian cancer.This study demonstrates cathepsins expression and clinic-pathologic patterns in ovarian cancer and the role of cathepsin L and cystatin C in diagnosis of ovarian cancer,the cathepsin L and invasion and metastasis in ovarian cancer cells in vitro,in order to find the posibility for therapeuptic ovarian cancer.The relationship between the expression of cathepsins and cystatin C mRNA and clinicopathological characteristics in ovarian carcinomaObjective:This study investigates the cathepsins and cystatin C mRNA expression and the association of their expreesion with clinicopathological parameters in ovarian cancer.Methods:The cathepsins and cystatin C mRNA were determined by RT-PCR in 47 ovarian carcinoma,20 benign ovarian tumor and 21 normal control.Results were correlated with clinicopathological characteristics.Results:ovarian carcinoma cathepsin B,L and cystatin C expression were significantly higher(p＜0.01)than benign ovarian tumor and normal ovarian tissue,the mRNA expression of cathepsin D was no significant different in malignant,benign ovarian tumor and normal ovarian tissue(p＞0.05).Cathepsin B expression was associated with ascites and histological types(p＜0.05).Cathepsin L expression was associated with clinical stages,differentiation degree of cancer cells and lymph node metastasis(p＜0.05).Cystatin C expression was associated with differentiation degree of cancer cells and distant metastasis(p＜0.05). Cathepsin D expression was associated with ascites and liver metastasis(p＜0.05).Cathepsin L expression was an independent prognostic marker for ovarian carcinoma.Conclusion:Cahtepsins overexpression is linked to invasion and metastasis of ovarian cancer Cathepsin L may be an independent maker for prognosis.The serum levels of cathepsin L and cystatin C in ovarian carcinoma and their values in diagnoseObjective:The aim of this study was to investagate the contribution of cathepsin L and cystatin C to the mechanisms of invasion by ovarian cancer.Methods:Serum levels of cathepsin L and cystatin C were determined by ELISA in 64 ovarian cancinomas,23 cases of benign tumors and 20 healthy women as controls,17 matched ovarian cancers from preoperation and postoperation.Serum levels of cathepsin L and cystatin C were associated with clinicopathological characteristics,the diagnosis of ovarian cancer.Result:The serum levels of cathepsin L were significantly higher in ovarian carcinoma than that in benign ovarian tumor and normal ovarian(p＜0.05).The serum levels of cathepsin L were significantly dropped than those in postoperative.The serum levels of cathepsin L and cystatin C of postoperative recurrence were significantly higher than that in preoperative nulli-recurrence(p＜0.01).Serum levels of cathepsin L was associated with advanced stage and poorly differentiated compared with early stage and well differentiated(p＜0.05),but cathepsin L levels is no significant difference in histological types,distant metastasis,ascetic fluid.Serum cystatin C levels was associated with lymph node metastasis(p＜0.05).As threshold value of 100 ng/mL,sentivity and specificity of serum cathepsin L levels for diagnosing ovarian carcinoma was 72.7%and 76.7%respectively,positive predictive value 70.58%,negative predictive value 76.7%and accurate ratio 75.0%。Conclusion:Serum level of cathepsin L was higher in malignant tumors and preoperative. Serum cathepsin L level was a potential marker for diagnosing and monitoring relapse in ovarian carcinoma.Construction of cathepsin L eukaryotic expressive plasmidObjective:In this study we amplification cDNA of cathepsin L and construct the eukaryotic expression plasmid pcDNA3.1-CTSL.Methods:The intact cDNA of cathepsin L was amplified from the ovarian cancer tissue by RT-PCR,then the product of RT-PCR was cloned to PMD18-T vector,and subcloned to pcDNA3.1 plasmid.It was identified by the double enzyme digests and DNA sequencing. Cationic lipid transfection reagent lipofectamine 2000 was used to transfect pcDNA3.1-CTSL recombinant plasmid and control pcDNA3.1 plasmid to HO8910 cells.Expression of cathepsin L in transfected cells was determined by RT-PCR and Western-blot.Results:The cathepsin L gene was successfully cloned from the ovarian cancer tissue.Results of restrictive enzyme analysis and sequencing showed that the cathepsin L gene was successfully inserted into pcDNA3.1 plasmid.Results of RT-PCR and Western-blot showed that the recombinant plasmid could express gene and protein of cathepsin L in HO8910 cells.Conclusion:The successfully constructed eukaryotic expression plasmid of cathepsin L gene could provide an experimental basis for the research of its value in ovarian cancer.The study of relationship between cathepsin L and invasion and metastasis in ovarian carcinoma cell in vitroObjective:To study the relationship between cathepsin L and invasion and metastasis in ovarian cancer cells in vitro.Methods:The eukaryotic expression plasmid of cathepsin L was introduced into HO8910 cells by liposome transfection reagent.Positive clones were selected with G418.RT-PCR was used to confirm the recombinant plasmid DNA integrated with the genomic DNA of HO8910 cells.Western-blot was used to confirm the CTSL protein expression in positive clones cells.The cell growth curves,clonogenicity efficiency were observed.The cell cycles were measured by flow cytometer.The ability of invasion and adhesion of ovarian cancer cells also were detected by transwell inserts.Results:The positive clones HO8910-CTSL and HO8910-pcDNA3.1 were confirm by RT-PCR and Western-blot.The proliferation and adhesion of the cells were no diference in HO8910-CTSL and HO8910-pcDNA3.1.Martrigel invasion assay and transwell migration assay revealed cathepsin L may play an important role in invasion and metastasis of ovarian cancer.Conclusion:Cathepsin L was assiociated with invasion and metastasis of ovarian cancer in vitro.Construction of suppression of cathepsin L gene siRNA expression vectorObjective:The aim of this study was to construct the cathepsin L siRNA eukaryotic expression vector.Methods:The specific siRNA sequence was designed according to the cathepsin L sequence in genbank.The sequence was cloned into psilencer^TM4.1-CMV-neo and the sequence analysis was performed.Results:It was verified by partical nucleotide sequencing and retriction endinuclease digestion that the constructed eukaryotic vector expressing siRNA of cathepsin L were correct.Conclusion:The eukaryotic vector expressing siRNA of cathepsin L was constructed successfully.Effects of RNA interference on cathepsin L Expression in ovarian carcinoma cells in vitroobjective:To investigate whether RNA interference(RNAi)could induce cathepsin L gene silencing in ovarian cells and its effect on functional outcome.Methods:The small interference RNA(siRNA)of cathepsin L expression vector was established and transfected into ovarian carcinoma cells(A2780).The silence effect is decteted by RT-PCR and Western-blot.The cell growth curves,clonogenicity efficiency were observed and the cell cycles were measured by flow cytometer.The ability of invasion and adhesion of ovarian cancer cells also were detected by transwell inserts.Results:RT-PCR and Western-blot assay show that the expression of cathepsin L in stably transfection cells were significantly lower than untransfection cells.The proliferation and adhesion of transfect cells were no significant diference in A2780-CTSL,A2780-psilencer and A2780-Control.Martrigel invasion assay and transwell migration assay revealed that cathepsin L may play an important role in invasion and metastasis of ovarian carcinoma.Conclusion:RNA interference represents an exciting technology for functional geomics by selective targeting of genes.Inhibit the expression of cathepsin L can suppress invasion and metastasis of ovarian carcinoma cells.Since metastasis depends upon both the invasiveness and migration of tumor cells,cathepsin L may be a therapeutic target of strong clinical interest.