GEF Molecule C3G Facilitate The Malignant Behavior Of Human Ovarian Cancer Cell SKOV3 Through Activating RAP1

Objective: To identify the role of C3G in malignant behavior of human ovarian cancer and investigate the molecular mechanism, thus uncover new target for ovarian cancer biotherapy.Method: Three pairs of shRNA specific to C3G and one pair as negative control were subcloned into pSUPER.retro.neo/GFP. Among these three, those with high inhibition efficiency were chosen for further experiment, which was confirmed through transient transfection into SKOV3. C3G-knockdown cells were established through screening with G418. Malignant behavior of C3G-knockdown SKOV3 cells were evaluated by MTT, plate clone formation assay and invasive assay, gelatinase activity and Rap1 activity were measured by zymography and GST-pull down assay.Result: Three recombinant plasmids pSUPER. retro.neo/GFP -C3Gi1, -C3Gi2 and -C3Gi3 and non-target pSUPER.retro.neo/GFP-NT were identified by enzyme digestion and DNA sequencing. Through transient transfection, pSUPER. retro.neo/GFP -C3Gi2 and - C3Gi3 were proved to knock down the endogenous C3G expression in SKOV3 cells efficiently( by 31% and 69%, respectively, P<0.05), Stable C3G-knockdown SKOV3 cells were established successfully and designated as C3Gi-1 and C3Gi-2, and their growth rate, invasion, and thus Rap1 activity and gelatinase were demonstrated to be reduced as compared with those in controls(P<0.05).Conclusion: Guanine-nucleotide exchange factor C3G facilitate the proliferation, migration and invasion of human ovarian cancer cell SKOV3 by activating Rap1. Knocking down its expression may reverse malignant behavior of human ovarian cancer through decreasing the activity Rap1 and gelatinase.