Molecular Mechanism Of Rap1GAP Down-regulating Cell-matrix Adhesion In Cervical Carcinoma Cells

Objective: by discussing the effect of tumor suppressor gene Rap1GAP activatingSrc pathway in Hela cells to clarifying the molecular mechanism of Rap1GAPinhibiting cell-matrix adhesion, and for further to provide the theory basis for clarifyingthe molecular biology mechanism of cervical cancer generation and development.Cervical cancer is one of women’s common malignant tumors. New cases ofcervical cancer are about460000globally each year by WHO report. Among them,there are about100000, which is one-fifth of all, every year in china. Cervical cancer isthe second cause of death in women malignant tumors, the main reason is tumor cellsmetastasis. It is one of the prerequisite conditions for cancer cells transferring to distanttissues that the reduction of cell-cell adhesion and the enhancement of cell-matrixadhesion. Small G protein Rap1plays key role in regulating cell adhesion. Tumorsuppressor gene Rap1GAP is an inactivator of Rap1, and was found to inhibit tumorgrowth, invasion and metastasis by blocking Rap1activation. It is closely related withthe development of a variety of tumors. Src, non-receptor protein tyrosine kinase,induce activating of Rap1by CRK and C3G. Knockout of Rap1GAP expressionweakens cell-cell adhesion and enhances cell-matrix adhesion to promoting tumor cellmetastasis.Our early results show that Rap1GAP may inhibit migration and invasion of Helacells in vitro by inhibiting cell-matrix adhesion and Rap1GAP may dimerize. Dimmerplays an important role in cellular signal transduction. To investigation the molecularmechanism of Rap1GAP inhibits cell-matrix adhesion and the biological effects ofRap1GAP dimmer in this paper, wild type Rap1GAP and dimerization deleted mutantsof Rap1GAP90and992(mutants at90or99site respectively, which are key sites forthe dimerization of Rap1GAP) were transfected into Hela cells and HEK293cells toobserve the change of Src phosphorylation, in order to investigate the mechanism Rap1GAP on cancer generation and progression.Methods：1. The plasmids of wild type and mutants of M90, M992of Rap1GAPwere isolated.2. Hela cells and HEK293cells were transfected with GFP, Rap1GAP ormutants of M90and M992respectively by lipofection method.3. Transfectionefficiency was observed by fluorescence microscopy and the expression level ofexogenous gene in the cell was analyzed by Western blot.4. Western blot was used tocheck the phosphorylation of Src protein to observe the effects of Rap1GAP on Srcactivity.Results：1. The plasmids of wild type and mutants of Rap1GAP were acquired.2.Hela cells and HEK293cells over-expression GFP, wild type and mutants of Rap1GAPwere achieved.3. Western blot showed: Src-pY416expression quantity are(1.036±0.033),(0.975±0.133) and (0.669±0.172) in Hela cells over expressing wild typeand mutants of M90and M992of respectively, there are no statistical difference(P>0.05) compared with the control group of Hela-GFP (0.884±0.123) or Hela(0.865±0.146); the expression level of Src-pY416are (1.193±0.200),(1.277±0.173) and(1.267±0.290) in over-expression of wild type and mutants of M90, M992of Rap1GAPin HEK293cells respectively. There are no significant differences also (P>0.05).Compared with the control group293-GFP (1.193±0.179) and293(1.227±0.265).Conclusion：1. Over-expression of Rap1GAP in Hela cells and HWK293cellsdoes not affect the level of Src phosphorylation (Y416).2. Rap1GAP dimer mutants donot affect the level of Src phosphorylation.3. Rap1GAP does not regulating cervicalcancer cell-matrix adhesion by activating Src.4. Maybe there is cell specificity forRap1GAP regulating Src activity.