| Mortality of epithelial ovarian cancinoma is highest in gynecological malignant tumor. After the surgery, chemotherapy is the major treatment methods based on the combination of platinum chemotherapy. But ultimately, it is difficult to cure patients because of resistance to ovarian carcinoma.So its 5 years survival rate is always 30-50%. In order to better understand the mechanism of tumor suppressor genes in multi-drug resistance of ovarian cancinoma ,tumor suppressor genes related to carboplatin resistance were selected and verified from carboplatin resistance microarray. Promoter region methylation were detected and gene mutation were screened for tumor suppressor genes related to carbo-resistance.Clinical implication of tumor suppressor genes were futher explored in ovarian carcinoma tissue . Part two Verification of differential expression genes between carbo-resistance cell line and its parental cell line of ovarian carcinomaObjective: To select tumor suppressor gene from carboplatin resistance microarray , which its downregulation expression were verified between SKOV3 carboplatin resistance cell line and its parental SKOV3 cell line .Methods: tumor suppressor genes were selected from carboplatin resistance microarray ,which 1554 genes were analysized by bioinformation methods. differential expression of tumor suppressor gene which were two more folds in microarray between resistance SKOV3 cell line and SKOV3 cell lines were verified respectively using real-time RT-PCR。Results: . 17 tumor suppressor genes which were RNASET2,VHL,DLG1,COPS2,NOL7,GGNBP2, RBL1, S100A2, PARG1,PERP,TCF3,DNAJA3,PCGF2,ING1,CYLD,RARRES3,RBBP8 were selected from the microarray.Of those,differential expression of genes such as RNASET2,VHL, COPS2,NOL7, RBL1, S100A2, PARG1,PERP,TCF3,DNAJA3, ING1,CYLD,RARRES3,RBBP8 were downregulation whose folds were respectively 24.47,65.24,10.81,21.30,72.94,5.95,27.13,1.57,1.32,12.93,46.8,7.57,14.9,14. 00,23.98 .but differential expression of the three genes such as GGNBP2,PCGF2,DLG1 were upregulaion whose folds were 8.0,7.23,1.24 respectively. Accordance rate of the differential experssion downregulation result which were one more folds and two more folds between experimental verification to microarray and microarray respectively were 82.35%,64.70%。Conclusion:It is a effective method to analyse differential expression of gene which the results were verificated to microarray by real-time PCR technology and multiple tumor suppressor gene involved in the mechanism of resistance of ovarian carcinoma. Part three Methylation detection of differential expression gene between carbo- resistance cell line and parental cell line of ovarian carcinomaObjective: To observe the tumor suppressor gene promoter methylation status related to carboplatin resistance of ovarian carcinoma and explore its relationship with expression downregulation in carboplatin resistance of ovarian carcinoma.Methods: Using molecular bioinformatics methods, promoter CPG island of tumor suppressor gene were analysized to design primers for MS-PCR or bisulphite sequencing .And promoter methylation status of tumor suppressor genes associated with carboplatin resistance were detected between SKOV3-CB and SKOV3 cell line of ovarian carcinoma using methylation-specific PCR (MS-PCR) or bisulphite sequencing method.Results: 14 genes (VHL, COPS2, NOL7, GGNBP2, RBL1, S100A2, PARG1, PERP, TCF3, DNAJA3, ING1, RARRES3, RBBP8,CYLD) whose differential expressions were downregulation in SKOV3-CB cell line comparing with SKOV3 cell line. Of those ,10 genes (VHL, COPS2, NOL7, RBL1, PARG1, PERP, DNAJA3, ING1, RNASET2,CYLD) could be designed primers for MS-PCR in its promoter region. MS-PCR showed that PCR products of could be amplified by the ING1, DNAJA3, VHL methylation primers and PCR products could not be amplified by the unmethylated primers in both SKOV3-CB andSKOV3 cell line; PCR products could not be amplified by COPS2, PERP methylation primers and PCR products could be amplified by the unmethylated primers in both SKOV3-CB andSKOV3 cell line; PCR products could be amplified by both RNASET2,PARG1,NOL7,RBL1 methylation primers and unmethylated primers in the two cell lines,CYLD which could not be amplified by MS-PCR primers were amplified by bisulphite sequencing and its promoter region were detected to be unmethylated in the two cell lines.Conclusion: No hypermethylation were detected in promoter region of the tumor suppressor genes related to carboplatin-resistance of ovarian carcinoma ,which differential expression downregulation of the tumor suppressor genes is not caused by methylation mechanism . Part four Mutation detection of differential expression gene between carboplation resistance and parental cell line of ovarian carcinomaObjective: Screening of possible genetic mutations, to explore relationship with carboplatin resistance of ovarian and expression downregulation of related tumor suppressor genesMethods: The parental cell line (SKOV3) as control, in the carboplatin-resistant ovarian cancer cell line (SKOV3-CB), single-strand conformation polymorphism analysis (SSCP) were applied to detect RT-PCR amplified fragments of RNASET2, VHL, COPS2, NOL7, PARG1, RBL1 , PERP, DNAJA3, ING1, CYLD10 genes so as to find genetic mutation.Results: In the SKOV3-CB cell lines, differential types in polyacrylamide gel electrophoresis of DNAJA3, PERP gene were detected by SSCP in contrast to SKOV3 cell lines, NO differential types of RNASET2, VHL, COPS2, NOL7, PARG1, RBL1, ING1, CYLD genes were detected in polyacrylamide gel electrophoresis between SKOV3-CB and SKOV3 . Respectively there were A→G pot mutation in 1093,1160,1222 locous and T,C,C loss in 1083,1085,1086 locous of the third exon of PERP.There was A→T pot mutation in 87 locous of the sequence of the eighth exon of DNAJA3.Conclusion: DNAJA3, PERP gene mutation may be related to carboplatin resistance of ovarian carcinoma and to expression downregulation of related tumor suppressor genes . Should be further investigated Part five The clinical research of expression of related carbo-resistance tumor suppressor genes of ovarian carcinomaObjective:To investigate the clinical implications of expression of ovarian carcinoma carboplatin resistance-related tumor suppressor gene.Methods: The total RNA of the ovarian tissues was extracted by one-step Trizol extraction method. Expressions of genes which were RNASET2,VHL,PARG1,CYLD,COPS2,PERP,DNAJA3,ING1,NOL7,RBL1 were detected in ovarian tissues of 85 patients using RT-PCR,which included 49 patients with ovarian carcinoma, 21 patients with benign ovarian tumors,15 patients with normal ovary.Results:RT-PCR results show that there were expressions of RNASET2,ING1,PARG 1,RBL1,NOL7 gene in ovarian carcinoma tissues ,ovarian benign tumor tissues and normal ovarian tissues and there were expression of VHL,PERP,COPS2,CYLD gene in normal ovarian tissues ,ovarian carcinoma tissues and ovarian benign tumor tissues except expression silence in some ovarian carcinoma tissues which there were 4(4/49),3(3/49),2(2/49),32(32/49)cases respectively.There are no difference for positive ratio of expression silence of RNASET2,ING1,PARG 1,RBL1,NOL7,VHL,PERP,COPS2 gene in between ovarian carcinoma tissues and NOT ovarian carcinoma tissues(P were 1.00,1.00,1.00,1.00,1.00,0.11,0.19,0.33 respectively)and there were difference for Positive ratio of expression silence of CYLD,DNAJA3 gene in between ovarian carcinoma tissues and NOT ovarian carcinoma tissues(P were 0.00,0.00 respectively) with chi square test. There were no difference for survival time between positive expression and negative experrsion of VHL,PERP,CYLD gene (P were 0.183,0.493,0.271 respectively) and there was difference for survival time between positive expression and negative experrsion of COPS2 gene in ovarian carcinoma patients with Log Rank(P=0.005). COX regression show that FIGO stage,differentiation involved in the model(P were 0.045,0.028 respectively)and Lymph node metastasis and expression silence of COPS2,PERP,CYLD,ING1 gene did not involved in the model(P were 0.907,0.476,0.754,0.728,0.304 respectively).Conclusion:Expression silence of CYLD,DNAJA3 gene maybe were potential marker for differential diagnosis in ovarian tumors and expression silence of COPS2,PERP,CYLD, ING1 gene were not independent prognostic fators for ovarian carcinoma. |
