| Part1Objective:To investigate the inhibitory effects of Raf kinase inhibitor protein (RKIP) on epithelial-mesenchymal transition (EMT) of human epithelial ovarian cancer (OVCA) and explore its possible mechanism.Methods:1.The recombinant plasmids expressing sense (Ss) or antisense (As) RKIP cDNA or empty vector was transfected into human OVCA cell line SKOV-3, the positive cell clone was selected by G418. The expression level of RKIP protein in OVCA cells were detected by Western blot analysis. The stable cell lines overexpressing or silencing of RKIP were cloned to investigate the function of RKIP in OVCA cells.2.Western blot assay was used to detect the protein expression of the mesenchymal markers Vimentin and Fibronectin in overexpressing or silencing of RKIP OVCA cells.3.Real-time PCR was used to detect the mRNA expression of the mesenchymal markers Vimentin and Fibronectin in overexpressing or silencing of RKIP OVCA cells.Results:1. SKOV-3clones stably expressing full-length recombinant pcDNA3.1(+)-ssRKIP, pcDNA3.1(-)-asRKIP, and their respective empty vector were obtained.2. The protein expression of Vimentin and Fibronectin in the ssRKIP vector-transfected cells were decreased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected cells, whereas those in the as RKIP vector-transfected cells were increased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected cells.3. The mRNA expression of Vimentin and Fibronectin in the ssRKIP vector-transfected cells were decreased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected cells, whereas those in the asRKIP vector-transfected cells were increased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected cells.Conclusion:RKIP inhibited the epithelial-mesenchymal transition of human epithelial ovarian cancer by down-regulating the mRNA and protein expression of Fibronectin and Vimentin in OVCA cells.Part2Objective:To investigate the inhibitory effects of Raf kinase inhibitor protein (RKIP) on vasculogenic mimicry (VM) of human epithelial ovarian cancer and explore its possible mechanism.Methods:1.Three-dimensional matrigel culture was used to analyz the effect of RKIP on angiogenic remodeling of OVCA cells.2.Western blot assay was used to detect the protein expression of MMP-2/9, VE-cadherin and EphA2in overexpressing or silencing of RKIP OVCA cells.3.Real-time PCR was used to detect the mRNA expression of MMP-2/9, VE-cadherin and EphA2in overexpressing or silencing of RKIP OVCA cells.4.Gelatin zymography assay was applied for MMP-2/9activities from supernatant of overexpressing or silencing of RKIP OVCA cells in vitro.Results:1.In three-dimensional matrigel culture, the capability of angiogenic remodeling in the ssRKIP vector-transfected cells was decreased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected cells, whereas that in the asRKIP vector-transfected cells was increased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected cells.2.The protein expression of MMP-2/9, VE-cadherin and EphA2in the ssRKIP vector-transfected cells were decreased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected cells, whereas those in the asRKIP vector-transfected cells were increased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected cells.3.The mRNA expression of MMP-2/9, VE-cadherin and EphA2in the ssRKIP vector-transfected cells were decreased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected cells, whereas those in the asRKIP vector-transfected cells were increased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected cells.4. The MMP-2/9activities from supernatant of ssRKIP vector-transfected cells were decreased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected cells, whereas that in the asRKIP vector-transfected cells were increased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected cells.Conclusion:RKIP inhibited the vasculogenic mimicry of human epithelial ovarian cancer by down-regulating the mRNA and protein expression of MMP-2/9,VE-cadherin and EphA2, and the MMP-2/9activities in OVCA cells.Part3Objective:To investigate the inhibitory effects of Raf kinase inhibitor protein (RKIP) on the regulatory factor of epithelial-mesenchymal transition and angiogenesis of human epithelial ovarian cancer and explore its possible mechanism.Methods:l.ELISA assay was used to detect the expression of VEGF, bFGF, TGF-β and IL-8in overexpressing or silencing of RKIP OVCA cells.3. Reporter gene assay was used to detect the trahscriptional activity of NF-κB and AP-1in overexpressing or silencing of RKIP OVCA cells.Results:1.The expression of VEGF, bFGF, TGF-β and IL-8in the ssRKIP vector-transfected cells were decreased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected cells, whereas those in the asRKIP vector-transfected cells were increased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected cells.2.The transcriptional activity of NF-κB and AP-1in the ssRKIP vector-transfected cells were decreased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected cells, whereas those in the asRKIP vector-transfected cells were increased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected cells.Conclusion:RKIP inhibited the expression of VEGF, bFGF, TGF-β and IL-8of human epithelial ovarian cancer by down-regulating the transcriptional activity of NF-κB and AP-1in OVCA cells.Part4Objective:To investigate the inhibitory effects of Raf kinase inhibitor protein (RKIP) on the capability of metastasis and invasion in vivo of abdominal cavity metastasis nude mice models of human ovary carcinoma SKOV-3and explore its possible mechanism.Methods:1. Abdominal cavity metastasis nude mice models of human ovary carcinoma SKOV-3were established. Anti-metastasis effect of RKIP was evaluated.2.In vivo imaging technology was used to detect the capability of invasion of overexpressing or silencing of RKIP OVCA cells in nude mice models.3.1mmunohistochemical staining assay used to detect the expression of Vimentin, Fibronectin, E-cadherin. CD44, ICAM-1, MMP-2, MMP-9, p65(NF-icB) and c-Jun (AP-1) in nude mice models.4. EL1SA assay was used to detect the secretion of VEGF, bFGF, TGF-β and IL-8in blood serum of nude mice models.Results:1. In abdominal cavity nude mice models of human ovary carcinoma SKOV-3, the capability of capability for tumor formation and metastasis in vivo in the ssRKIP vector-transfected nude mice models were decreased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected nude mice models, whereas those in the asRKIP vector-transfected nude mice models were increased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected nude mice models.2.In abdominal cavity nude mice models of human ovary carcinoma SKOV-3, less fluorescence intensity in the ssRKIP vector-transfected nude mice models was observed compared with the empty vector pcDNA3.1(+)-transfected and non-transfected nude mice models, whereas more fluorescence intensity in the asRKIP vector-transfected nude mice models was observed compared with the empty vector pcDNA3.1(-)-transfected and non-transfected nude mice models.3.1n abdominal cavity nude mice models of human ovary carcinoma SKOV-3, the expression of E-cadherin in the ssRKIP vector-transfected nude mice models was increased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected nude mice models, whereas that in the asRKIP vector-transfected nude mice models was decreased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected nude mice models. The expression of Vimentin, Fibronectin, CD44, ICAM-1, MMP-2, MMP-9, p65(NF-KB) and c-Jun (AP-1) in the ssRKIP vector-transfected nude mice models were decreased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected nude mice models, whereas those in the asRKIP vector-transfected nude mice models were increased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected nude mice models.4.In abdominal cavity nude mice models blood serum of human ovary carcinoma SKOV-3, the secretion of VEGF, bFGF, TGF-p and IL-8in the ssRKIP vector-transfected nude mice models were decreased compared with the empty vector pcDNA3.1(+)-transfected and non-transfected nude mice models, whereas those in the asRKIP vector-transfected nude mice models were increased compared with the empty vector pcDNA3.1(-)-transfected and non-transfected nude mice models.Conclusion:RKIP inhibited the capability of metastasis and invasion in vivo of abdominal cavity metastasis nude mice models of human ovary carcinoma SKOV-3by up-regulating the protein expression of E-cadherin and down-regulating the protein expression of Vimentin, Fibronectin, CD44, ICAM-1, MMP-2, MMP-9, p65(NF-KB) and c-Jun (AP-1), and down-regulating secretion of VEGF, bFGF, TGF-β and IL-8in nude mice models. |
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