Impact Of Downregulated Rap1GAP Expression Upon The Malignant Behavior Of Ovarian Carcinoma

Objective:To analyse the differential expression of Rap 1 GAP in human ovarian cancer and its potential reason. To explore the role of Rap 1 GAP in the malignant behaviors of ovarian carcinoma, and the possible mechanism.Method:1.Rap1GAP、C3G、Rap1-GTP were detected by Western blotting and GST-Pull down in ovarian cancer tissues with different types of histopathology.2. The mRNA and protein level of Rap 1 GAP were measured by RT-PCR and Western blotting in both tissues and cells.3.Endogenous Rap 1 GAP expression in human ovarian cancer cells were interfered by lentivirus, and screened by puromycin.4.To observe the biological behavior of OVCAR3 cell with Rap1GAP silenced and Hey cell with Rap 1 GAP over expressed:malignant proliferation was analysed by MTT and plate clone assay; Transwell assay was utilized to detect cell motility and migration; The change of cell morphology demonstrated through immmunofluorescence. To address the potential reason of decreased Rap1GAP level in those with high metastatic potential, Rap 1 GAP transcriptive level in ovarian cancer cell lines was detected by RT-PCR before and after treated with aza and tsz, Finally, to investigate the underlying mechanism which Rap1GAP was implicated in regulating the malignant events of ovarian cancer, associated proteins possibly involved in cell proliferation were assessed.Result:1. Compared with normal tissues and benign tumors, the malignant tumor with high Rap1 activity were regulated by both Rap1 GAP and C3G.2. Rap1 GAP expression levels (mRNA and protein) was lower in ovarian cancer than in normal controls.3.We successfully established two cell lines with different Rap1 GAP expression level, including high Rap1 GAP level OVCAR3 cell with its Rap1 GAP silenced and low Rap1GAP level Hey cell with forced Rap1GAP expression.4. Decreased Rap1 GAP expression in OVCAR3 brought to accelerated proliferation and gained metastatic potential. F-actin staining demonstrates that cells with Rap1GAP knockdown have gained ability of forming filopodia and amellipodia at the leading edge.5.The key proteins in Wnt signal pathway including GSK3pser9、β-catenin、c-myc、cyclinD1, were down-regulated in Rap1GAP knockdown OVCAR3 cells, while up-regulated in forced Rap1GAP expression HEY cells, respectively.6. After treated with Aza or TSA, the Rap1GAP mRNA level were upregulated.Conclusion:1.Low Rap1GAP expression in ovarian cancer with high metastatic potential might arise from methylation.2.Rap1GAP might suppress the outgrowth of ovarian cancer through inhibiting canonical Wnt signal, while might reduce the invasiveness and metastasis through activating the hydrolysis of Rap1-GTP.