The Spop Effect Of Pro-apoptpsis And Its Possible Mechanism In Ovarian Cancer

Objective:The thesis aims at studying the status of Speckle-type POZ(pox virus and zinc finger protein) protein(SPOP) gene and analyzing the correlation between the status of SPOP gene in ovarian cancer(OC) and the histological subtypes and grade. Then it further studies the role of SPOP gene and its possible mechanism in OC.Methods:1. To detect the status of SPOP gene and to evaluate correlation between the status of SPOP gene in OC and the histological subtypes and grade in a Tissue Microarray(TMA) through Fluorescence In Situ Hybridization(FISH) technique;2. To detect the expression of SPOP protein through the immunohistochemical(IHC)technique;3. To detect the expression of SPOP protein through Western Blotting in serous epithelial ovarian cancer(EOC) cell lines SKOV3 and OVCAR-3;4. To construct the si RNA-SPOP models of SKOV3 and to evaluatem RNA of SPOP and SPOP protein expression through RT-PCR and Western Blotting respectively;5. To detectcell cycle and apoptosis through flow cytometry method;6. To seek the potential substrate proteins of SPOP through the technique of protein mass spectrometry analysis and bioinformation analysis and then analyze the possible mechanism in OC.Results:1.The status of SPOP gene was significantly different between the OC tissue and normal ovarian tissue(P<0.01).2.The deletion of SPOP gene has been found in 52.27%(46/88)of the OC tissue, but has not been found in normal ovarian tissue(0/10).3. Monosomy 17 frequently appeared in OC tissue but never appeared in normal ovarian tissue.4. The results showed that the histological subtypes and grade were significantly correlated with the deletion of SPOP gene(resulted from the appearance of monosomy 17) in OC.5. The deletion of SPOP gene appeared at a large proportion in EOC tissue(67.21%,41/61), particular in grade 3(83.78%,31/37).6. The result of IHC has suggested that positive expression of SPOP protein appears in OC tissue(45.45%,40/88), but expression of SPOP protein has not been found in normal ovarian tissue(0%,0/10).7. The results from FISH and IHC have showed inconformity(Kappa=0.036,P=0.501).8. Compared with the empty virus group, the apoptosis decreased in si RNA-SPOP group.9. Protein mass spectrometry analysis has found 4347 distinct proteins which 110 proteins have significant differences. Among the 110 proteins,16 are up-regulated and 94 are down-regulated.10. Bioinformatics analysis of 110 proteins has found DUSP10 is the potential substrate of SPOP. Meanwhile, Bcl2l13 and Bak1 are key downstream factors in SPOP regulatory pathway.Conclusion:The deletion of SPOP gene appears at a large proportion in the OC tissue, particular in grade 3 of EOC tissue(83.78%,31/37). SPOP may play a cancer suppressive role by promoting cell apoptosis.