| Context:Advanced ovarian cancer is a devastating disease. Gaining biomarkers of early detection during ovarian tumor genesis may lead to earlier diagnosis and better therapeutic strategies. Cystatin B (CSTB) functions as an inhibitor to suppress intracellular cytokine proteases and has been implicated in several types of cancers. Cytokines and growth factors, including transforming growth factor-β (TGF-β), regulate a variety of cellular functions and have been implicated in tumor genesis.Objective:The present study is to explore the expression of CSTB in human ovarian tumors associated with clinic pathological features, to examine the effect of TGF-β on CSTB expression in ovarian cancer cells, and to investigate the role of CSTB on cell proliferation in ovarian cancer cells.Materials and methods:Patients and ovarian tissue samples. The ovarian tissue specimens were collected from 27 patients with ovarian tumor and 6 patients without ovarian tumor at Jinshan Hospital, Fusan University from January,2005 to December,2012.Immunohistochemical staining and analysis. The expression of CSTB was detected by immunohistochemical staining. A final immunoreactive score was determined by the sum of the positive extent and staining intensity.Cell culture and TGF-β1 treatment. Human serous ovarian cancer cell lines OVCAR-3 cells and SK-OV-3 cells were treated with recombinant human TGF-β1. A TβRI inhibitor, SB-431542, was applied for blocking the TGF-β signaling pathway.RNA extraction and quantitative real-time PCR. Total RNA in the cells was extracted using TRIzol. Western blot analysis. Equal amount of protein was separated on 15% SDS-PAGE and transferred to a PVDF membrane. The different primary antibodies were used and the signals were detected subsequently.siRNA transfection and cell proliferation. OVCAR-3 cells were transfected with CSTB-siRNA. The cell proliferation was detected by WST-1 cell proliferation assay. Cell cycle was measured and analyzed by flow cytometry.Statistical analyses. All analyses were performed with SPSS Statistics 19.0.Significant difference was considered at the value of P< 0.05.Results:1. Cystatin B was a progression marker of human epithelial ovarian tumors. By immunohistochemistry staining, the overexpression of CSTB was observed in human ovarian tumors, including benign, borderline and malignant tumors, albeit at different levels compared with the normal ovarian tissues that showed negative staining. The positive rate of CSTB expression was significantly higher in tumors, including benign, borderline and malignant tumors, than that in normal tissue.2. No correlation of CSTB expression with clinicopathological features of ovarian cancer patients was observed. Overall, there is no correlation of CSTB expression with clinicopathological features of ovarian cancer patients.3. The regulation of CSTB expression in ovarian cancer cells was mediated by the TGF-β signaling pathway. TGF-β1 significantly decreased CSTB mRNA and protein in OVCAR-3 and SK-OV-3 cells. This effect of TGF-β1 was abolished in the presence of 10 μM SB-431542, indicating that the expression of CSTB in ovarian cancer cells was mediated by the TGF-β signialing pathway.4. CSTB promoted the proliferation of ovarian cancer. Downregulation of CSTB expression in ovarian cancer cells using CSTB-siRNA led to the inhibition of proliferation. A G2/M phase arrest in the cell cycle was found after CSTB-siRNA transfection.Discussion and conclusions:CSTB is an ovarian tumor marker and an increase in the expression of CSTB in ovarian tissue represents tumor progression. The regulation of CSTB expression in ovarian cancer cells is mediated by the TGF-β signaling pathway. CSTB promotes ovarian cancer cell proliferation. |
