Differential Expression Analysis Of Ubiquitin-proteasome In Epithelial Ovarian Cancer And The Mechanism Of Apoptosis Induced By Blocking Ubiquitin-proteasome Pathway

Objective:To establish the differentially expression gene profile of ovariancancer tissue and normal tissue by the technique Restriction FragmentDifferential Display—Polymerase Chained Reaction(RFDD-PCR) ; Toscreen differential expression genes which are related to ovarian carcinomapathogenesis to study the relation of ovarian cancer andubiquitin-proteasome, which is favor of elucidating the underlyingmechanism and searching for useful targets related to clinic for htLrnanovarian cancer ; to study the effect of ubiquitin-proteasome, inhibited byMG132, on the regulation of cell cycle and apoptosis in human ovariancancer cell line SKOV3, and to demonstrate the its mechanism.Methods: Collecting tissues of ovarian cancer (T) and normal ovary(N)from one epithelial ovarian carcinoma case, gene expression profile of eachkind of tissue has been constructed using RFDD-PCR technique at equalpace according to operating manual of Qbio-gene company to establish the differentially expression gene profile of the two tissues. Bioinformaticsanalysis was applied for identifying ovarian carcinoma pathogenesis relatedgenes/ESTs.To investigate the differential expression of genes of USPs andPSs in ovarian cancer tissue compared normal tissue, and some of themwere further verified as over-expression by semi-quantitative RT-PCR andimmunohistochemical stain; The expression of genes of USPs and PSs,p53,caspase3,bcl-2 was investigated by immunhistochemistry, and theirbundances of mRNA tested by semi-quantitative RT-PCR. SKOV3 wastreated with various levels of MG132, its proliferation evaluated by MTTassay, apoptosis determined by TUNEL-POD method and PI staining flowcytometry, and cell cycle analyzed by flow cytometry, respectively.Results:1. Electrophoresis of RFDD-PCR products efficiently separated5642 significant bands altogether, including 2227 in ovarian cancer tissueand 3384 in normal tissue. There were 2082 bands (36.9ï¼…) alteredaccording to software GeneTools. The differential expression of thirty-sixgenes in the two tissues were concerned with oncogene and anti-oncogene,cyclins, proliferation and apoptosis related genes, vascular growth factor,metabolism enzymes, etc.Among them, ubiquitin-proteasome system genesshowed a significant difference. Expression ofPSMB5, PSMD2, PSMD8,USP9X, USP9Y, USP2, USP14å’ŒUBE4A was observed up-regulated inovarian cancer, while PSMA7, PSMB2, PSMB9, PSMD1, PSMD11,USP4, USP5, USP7, USP8, USP11, USP16, USP21, USP25, USP26å’ŒUBE2B was observed down-regulated in ovarian cancer, PSMA1 showedno obvious difference between 2 profiles. As to oncogene and anti-oncogenefamily, ras and c-myc showed up-regulated in ovarian cancer, p53, PTEN and BRCA1 showed down-regulated in ovarian cancer; cyclinsP27 was observed down-regulated in ovarian cancer, while CDK4, CDK6 CDK8 showed up-regulated in ovarian cancer; apoptosis related gene bcl-2showed up-regulated in ovarian cancer; vascular growth factor VEGFEGF, FGF showed up-regulated, in ovarian cancer. 2.The results ofSemi-quantitative PCR and immunohistochemical stain also showed ahigher expression amount ofPSMB5, PSMD2,PSMD8,usP9x,usP9Y,USP2,USP14,UBE4A in ovarian cancer among 24 PSs and USPs genesin expression profile we constructed.3. MTT assay indicated that MG132could inhibit the proliferation of SKOV3 in a concentration-dependent andtime-dependent pattern.4.PI staining flow cytometry data showed theapoptosis of SKOV3 and cell cycle was arrested at G₁ stage induced byMG132 in a concentration-dependent manner.5.TUNEL-POD methoddemonstrated that the apoptosis bodies were found in the SKOV3 treatedwith MG132.6.Immunohistochemistry and semi-quantitative PCR indicatedthat SKOV3 overexpressed some of PSs and USPs genes. The expression ofbcl-2 and some of PSs and USPs genes mRNA and protein reduced, whilethe expression of p53 and caspase-3 was increased in SKOV3 treated withMG132 in a concentration-dependent manner(P＜0.05).Conclusions: 1.It is favor of elucidating the underlying mechanism andsearching for useful targets related to clinic for human ovarian cancer toestablish the differentially expression gene profile of ovarian cancer tissueand normal tissue by the technique Restriction Fragment DifferentialDisplay- Polymerase Chained Reaction(RFDD-PCR). The differentialexpression of thirty-six genes in the two tissues were concerned with oncogene and anti- oncogene, cyclins, proliferation and apoptosis relatedgenes, vascular growth factor, metabolism enzymes, etc. Among them,someof ubiquitin-proteasome system genes was observed up-regulated in ovariancancer, and the results were also proved by Semi-quantitative PCR andimmunohistochemical, stain. The results suggested the activity ofubiquitin-proteasome system were obviously enhanced inovarian cancer andthe pathological precess of ovarian cancer may involved in. change offunction of ubiquitin-proteasome system. This results provid some newideas related to treat ovarian cancer. 2. MG132 significantly inhibitsSKOV3 survival and induces its apoptosis by stoppage in G₁ phase.3.Thegrowth inhibition is associated with downregulation of bcl-2 whileupregulation of P53 and caspase-3 via inhibited of PSs and USPs.These findings suggest that It is favor of elucidating the underlyingmechanism and searching for useful targets related to clinic for humanovarian cancer to establish the differentially expression gene profile ofovarian cancer tissue and normal tissue by the technique RFDD-PCR; thechange of function of ubiquitin-proteasome system was related with ovariancancer pathogenesis; it might be one of the ideal strategies for gene therapyof ovarian cancer, and data exhibit that the inducing cell apoptosis may playa pivotal role in the progression of ovarian cancer.Keyword:Ovariancancer;differentialexpression;RFDD-PCR;ubiquitin-proteasomesystem;ubiquitin-proteasomeinhibitorMG132;apoptosis;p53;caspase3;bcl-2...