Contribution Of RAP1Expression To RAP1Activity And Malignant Invasiveness Of Epithelial Ovarian Cancer

Objective:To investigate the significance of Rap1expression in Rapl-GTP activation and malignant feature of ovarian cancer, and to explore its underlying mechanism.Method:1. To validate the relationship between C3G and Dock180expression pattern in epithelial ovarian cancer (EOC) tissues through western blot and immunohistochemical analysis. The location of C3G/Dock180and their downstream proteins Rapl/Racl was demonstrated in ovarian cancer cell SKOV3by means of immunofluorescence to speculate their potential linkage in ovarian cancer cells.2. Rapl expression was evaluated through immunohistochemical analysis in EOC tissues, benign ovarian tumor tissues and normal ovarian tissues.3. Three Rapl shRNA eukaryotic expression vectors were constructed with RNA interference technology. Three recombinant plasmids, empty vector and negative plasmid were transfected into ovarian cancer cell line SKOV3through LipofectamineTM2000. The Rapl siRNA efficiency was proved through monoclonal screening with G418followed by immunoblotting. Two strains of SKOV3-Rapl-RNAi cell lines were established.4. To observe the contribution of Rapl expression to Rapl activity and to the biological behavior of SKOV3cells:focal adhesions and actin cytoskeleton were demonstrated in SKOV3cells through immuno-fluorescence and actin staining; malignant proliferation was evaluated by MTT; Cell motility and migration was reflected by wound healing assay and Matrigel Transwell invasion assay; Finally, Gelatinase activity and Rapl activity were detected by zymography and GST-pull down assay.Result:1. C3G expression appears inversely related with that of Dock180levels in EOC tissues. Subcellular distribution demonstrated that both C3G and Dock180are distributed around cytoplasm. Their downstream effectors, Rapl and Racl are distributed both in plasmalemma and cytoplasm, with Racl distribution extending to the membrane ruffling.2. EOC tissues presented with significantly higher Rapl expression than benign ovarian tumors and normal ovarian tissues (P<0.05).3. Three recombinant plasmids pSUPER.retro.neo/GFP-Rapli-#1.-Rap1i-#2and Rapli-#3were identified by restriction endonuclease digestion and DNA sequencing. pSUPER.retro.neo/GFP-Rapli-#2was proved the most efficiency through transient transfection. Accordingly, two monoclones of Rap1i cells (Rap1i-1and Rap1i-2) were established through pSUPER.retro.neo/GFP-Rapli-#2transfection followed by G418 screening. We selected Rapli-1cells to further verity the phenotype changes.4. Rap1-knockdown cells present with similar morphology, with similar focal adhesion formation and actin cytoskeleton to control cells. Unexpectedly, these cells did demonstrate increased proliferation rate and wound-healing ability, while decreased invasiveness, gelatinase production and Rap1activity as compared with control (P<0.05).Conclusion:Rap1expression and its contribution to Rap1activity might be implicated in the spread and metastasis of ovarian cancer on the one hand. On the other hand, its inhibitory role in cell proliferation would be considered when applying it to biotherapy of ovarian cancer.