| BackgroudOvarian cancer is composed of a heterogeneous group of tumors that are derived from the surface epithelium of the ovary or from surface inclusions. They are the most lethal malignant tumors of female reproductive system and the mortality rate is first of all gynecological tumors. They are classified into serous, mucinous, endometrioid, clear cell, and Brenner (transitional) types corresponding to the different types of epithelia in the organs of the female reproductive tract. Among, serous carcinoma accounts for about 50% of all ovarian epithelial carcinomas. At present, the pathogenesis, molecular genetics, and epidemiology of ovarian epithelial carcinomas are still unclear. Moreover, a lot of differences of pathogenesis and prognosis are present among different ovarian epithelial carcinoma types.Recently, following the further understanding of clinical pathomorphology characteristic about ovarian serous carcinoma and the development of molecular genetics, we have a new recognition about the pathogenesis and pathomorphology characteristic of ovarian serous carcinoma, and propose a dualistic theory model for ovarian serous carcinogenesis. One pathway involves a stepwise progression from serours borderline tumor (SBT) to noninvasive and then invasive micropapillary serous carcinomas (MPSC). The other pathway is characterized by rapid progression from the ovarian surface epithelium or inclusion cysts to a conventional (high-grade) serous carcinoma. Despite considerable efforts aimed at elucidating the molecular mechanisms of ovarian serous carcinoma, its pathogenesis is still poorly understood and the dualistic theory model needs to be confirmed by moleclular genetics. In addition,we have focus on whether low and high grade serous carcinoma have a common moleclular base or not. Oncogenesis is a multistepwise process during which oncogenes are activated and the function of tumor suppressor genes is lost. Tumor suppressor genes play a important role in the process by down-regulation cell growth or promoting cell differentiation and death.ARHI is a maternally imprinted putative human tumor suppressor gene that maps to chromosome 1p31 and that encodes a 26-kDa small G protein with 60% homology to rap and ras. It inhibits cell growth, slows motility, prevents invasion, and induces apoptosis by signal transduction and cell cycle regulation. WWOX (WW domain-contaig oxidoreductase) gene, located on chromosome 16q 23.3-24.1 in the region recognized as the common fragile site FRA16D is considered to be a tumor suppressor gene. It can enhance many tumor suppressor genes and inhibit oncogenes.Many studies confirmed that loss expression of three tumor suppressor genes be very common in human carcinoma including ovarian cancer and breast caner. But the expression of three tumor suppressor genes in ovarian serous carcinoma, contribution to oncogenesis of it, and regulation mechanism were not explained.Ovarian epithelial carcinomas have a poor prognosis with high mortality. All the while, the studies on ovarian epithelial carcinomas prognosis were paied close attention by gynecology worker. Before, many studies about ovarian epithelial carcinomas prognosis were undertaked, which invoving in clinical stage, histological gade, pathological category, lymphatic metastasis, chemotherapy, and residual focal size. Owing to different histological types, it is difficult to more exactly evaluate ovarian epithelial carcinomas prognosis. At present, it has becamed a hot spot that searching for molecular targets which predicting prognosis besides traditional prognosis factors. The relationship between ARHI, WWOX, PTEN and prognosis of ovarian serous carcinoma was not reported.Objective The variance in ARHI, WWOX and PTEN expression were detected in ovarian serous benign tumor, borderline tumor and carcinoma with immunohistochemistry staining, RT-PCR, flow cytometry, and western blotting. The correlation between ARHI, WWOX, PTEN and STAT3, E2F1 were analysed. On the base, we explore that the rule of variance in ARHI, WWOX, PTEN expression and regulation mechanism in oncogenesis of ovarian serous carcinoma. Meanwhile, the prognosis factors were screen with Kaplan-Meier univariate survival analysis and COX multivariate survival analysis was used to determine the risk coefficient of each prognosis factor and different layers in each factor and analyze the relationship of ARHI expression and prognosis.Method1 Research object The 109 epithelial ovarian tumors investigated were collected from patients diagnosed between 2001 and 2003 at the Second Hospital of HeBei Medical University. 35 of the tumors were serous benign tumors, 18 of the tumors were serous borderline tumors, and 56 of the tumors were ovarian serous carcinomas with complete clinical information and follow-up data. 25 normal ovarian tissues were selected as control. Formalin-fixed and paraffin-embedded tissue blocks from all samples were used in this study with immunohistochemistry staining and survival analysis. 93 fresh ovarian tumor tissues were abtained from patients at The Fouth Hospital of HeBei Medical University. 32 of the tumors were serous benign tumors, 16 of the tumors were serous borderline tumors, and 45 of the tumors were ovarian serous carcinomas. 15 normal ovarian tissues were selected as control. All tissues were fresh-frozen and stored at -80℃for RT-PCR, FCM, and Western Blotting.2 ImmunohistochemistryThe protein expressin of ARHI, WWOX, PTEN, STAT3 and E2F1 were detected in 25 normal ovarian tissues and 109 ovarian serous tumors with immnohistochemisty ElivisionTM plus two-step. The immunocytochemistry procession was also carried out according to manufacturer's instructions. Hematoxylin stsining, grad alcohol dehydration, dimethyl benzene transparence, neutral gum mounting, observed under light microscope. Negative control was carried out in identical condition excluding PBS insteading antibody.3 Flow cytometryThe fresh frozen tissues were fixed with 4% formaldehyde and The protein expressin of ARHI, WWOX, PTEN, STAT3 and E2F1 were detected with Flow cytometry according to manufacturer's instructions.4 Protein extraction and quantitationThe fresh frozen tissues were splited in total cell lysates. The protein concentration was determined using Coomassie brilliant blue (CBB) reagent in spectrophotometer.5 Western BlottingFifty micrograms of protein were electrophoretically separated using a 12% SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with primery monoclone antibodies and secondary monoclone antibodies. Immunoreactive bands were visualized using a ECL system. Band density was quantified using Snygene-Image Systems and normalized toβ-actin.6 RNA extraction, identify and quantitationTotal RNA from fresh ovarian serous tumors was isolated with guanidinium isothiocyanate (GITC). The integrity of total RNA was identfied at 90V on 1% agarose gels containing EB (0.5μg/ml). The UV Spectro- photometer was used for the quantitation of the total RNA.7 RT-PCRTotal RNA was reverse transcribed using reverse transcriptase with oligo dT at 43°C for 1.5 h to synthesize cDNA.Then the PCR was carried out in a complex mixture such as cDNA,dNTP,Taq DNA polymerase,primers and so on, denaturation for 5 mins, 94℃50s, 56℃30s, 72℃30s, 30 circulations . After amplification, each sample was analyzed by 1.5% agarose gel electrophoresis for 35 mins and visualized by ethidium bromide staining. The fragment for glyceraldehydes -3-phosphate dehydrogenase(GAPDH)cDNA gene was used as an internal control and the relative expression was quantitated as standardization on GAPDH mRNA content by BIO-LD densitometric image analyzer.8 Survival analysis of 56 ovarian serous carcinoma patiens by Kaplan-Meier univariate survival analysis and COX multivariate survival analysis56 ovarian serous carcinoma patiens with follow-up were analysed by Kaplan-Meier univariate survival analysis, the sum survival rates in 3 and 5 years were calculated by means of 36 months and 60 months as cut-off point. The correlation between tumor suppressor genes and prognosis was analysed, prognosic factors were screened, and the risk coefficient of independent factors were determined by COX multivariate survival analysis.9 Statistics analysisThe statistics analysises in the study were carried out by SPSS13.0 statistical package. The measurement datas were expressed as means±SD. x2 test, one-way ANOVA, SNK, and Spearman rank correlation were used for numeration and measurement datas respectively. Kaplan-Meier univariate survival analysis was used to calculate the survival rate; the difference was test by Log-Rank test. COX multivariate survival analysis was used to determine the risk coefficient of each prognosis factor and different layers in each factor.α=0.05.ReultsPart one Tumor suppressor gene ARHI,WWOX,and PTEN expression in ovarin seruous tumors1 ARHI mRNA and protein expression in ovarin seruous tumors1.1 ARHI mRNA expression in ovarin seruous tumorsThe result of RT-PCR showed that ARHI mRNA expression were detected in 28 of 32 ovarian serous benign tumors, 7 of 16 ovarian serous borderline tumors, and 18 of 45 ovarian serous carcinomas respectively. The positive expression rates of ARHI mRNA in ovarian serous carcinomas and ovarian serous borderline tumors were 40% and 43.8% respectively, significantly lower than that in ovarian serous benign tumors and normal ovaries (P<0.05), but the statistical differences were not found between borderline tumors and serous carcinomas, benign tumors and normal ovaries. Quantitative analysis of ARHI mRNA demonstrated that the relative quantities of ARHI mRNA were 0.12±0.06 in serous carcinomas, 0.15±0.04 in serous borderline tumors, statistically decreased than that in benign tumors and normal ovaries (P<0.05).1.2 ARHI protein expression in ovarin seruous tumors1.2.1 Western BlottingWestern Blotting showed that positive bands appeared in 26kd location for 26 of 32 benign tumors, 8 of 16 borderline tumors, 19 of 45 serous carcinomas. The positive expression rates of ARHI protein were 50%, 42.2% in borderline tumors and serous carcinomas respectively, obviously lower that in benign tumors and normal ovaries (P<0.05). Quantitative analysis of positive band density scanning diplay that the relative quantitiy of ARHI protein in serous carcinomas was 0.17±0.12, significantly decreased than that in borderline tumors, benign tumors, and normal ovaris (P<0.05).1.2.2 FCMThe FCM revealed that the FI in serous carcinomas and borderline tumors were 0.894±0.008 and 0.914±0.06 respectively, obviously decreased compare to benign tumors and normal ovaries.1.2.3 ImmunohistochemistryThe result of ICH showed that ARHI protein expression was detected in 22 serous carcinomas, 10 borderline tumors, and 27 benign tumors samples. The positve rates in serous carcinomas and borderline tumors were 39.3% and 55.6%, significantly lower than that in benign tumors and normal ovaries (P<0.05), but the statistical differences were not found between borderline tumors and serous carcinomas, benign tumors and normal ovaries.2 WWOX mRNA and protein expression in ovarin seruous tumors2.1 WWOX mRNA expression in ovarin seruous tumors The result of RT-PCR showed that WWOX mRNA expression were detected in 29 of 32 ovarian serous benign tumors, 12 of 16 ovarian serous borderline tumors, and 21 of 45 ovarian serous carcinomas respectively. The positive expression rates of WWOX mRNA in ovarian serous carcinomas was 46.7%, significantly lower than that in ovarian serous borderline tumors, benign tumors and normal ovaries (P<0.05). But the statistical differences were not found among borderline tumors, benign tumors, and normal ovaries. Quantitative analysis of WWOX mRNA demonstrated that the relative quantities were 0.25±0.08 in serous carcinomas, statistically decreased compare to borderline tumors, benign tumors and normal ovaries (P<0.05).2.2 WWOX protein expression in ovarin seruous tumors2.2.1 Western BlottingWestern Blotting showed that the positive expression rates of WWOX protein in serous carcinomas was 44.4%, significantly lower than 68.8% in borderline tumors, 78.1% in benign tumors, and 86.7% in normal ovaries (P<0.05). Quantitative analysis of positive band density scanning diplaied that the relative quantitiy in serous carcinomas was 0.11±0.04, significantly decreased compare to borderline tumors, benign tumors, and normal ovaris (P<0.05).2.2.2 FCMThe FCM revealed that the FI in serous carcinomas was 0.930±0.02, obviously decreased compare to borderline tumors, benign tumors, and normal ovaries.2.2.3 ImmunohistochemistryThe result of IHC showed that WWOX protein expression was detected in 27 serous carcinomas, 16 borderline tumors, and 30 benign tumors samples. The positve rates in serous carcinomas was 48.2%, significantly lower than that in borderline tumors, benign tumors, and normal ovaries (P<0.05), but the statistical differences were not found among borderline tumors, benign tumors, and normal ovaries.3 PTEN mRNA and protein expression in ovarin seruous tumors 3.1 PTEN mRNA expression in ovarin seruous tumorsThe positive expression rates of PTEN mRNA in ovarian serous carcinomas was 51.1%, significantly lower than 81.3% in borderline tumors, 90.6% in benign tumors, and 100% in normal ovaries (P<0.05). But the statistical differences were not found among borderline tumors, benign tumors, and normal ovaries. Quantitative analysis of PTEN mRNA demonstrated that the relative quantities were 0.31±0.08 in serous carcinomas, statistically decreased compare to borderline tumors, benign tumors and normal ovaries (P<0.05).3.2 PTEN protein expression in ovarin seruous tumors3.2.1 Western BlottingWestern Blotting showed that the positive expression rates of PTEN protein in serous carcinomas was 53.3%, significantly lower than 75.0% in borderline tumors, 84.4% in benign tumors, and 93.3% in normal ovaries (P<0.05). Quantitative analysis of positive band density scanning diplaied that the relative quantitiy in serous carcinomas was 0.34±0.12, significantly decreased compare to borderline tumors, benign tumors, and normal ovaris (P<0.05).3.2.2 FCMThe FCM revealed that the FI in serous carcinomas was 0.908±0.023, obviously decreased compare to borderline tumors, benign tumors, and normal ovaries.3.2.3 ImmunohistochemistryThe result of IHC showed that PTEN protein expression was detected in 29 serous carcinomas, 15 borderline tumors, and 31 benign tumors samples. The positve rates in serous carcinomas was 51.8%, significantly lower than that 83.3% in borderline tumors, 88.6% in benign tumors, and 100% in normal ovaries (P<0.05), but the statistical differences were not found among borderline tumors, benign tumors, and normal ovaries.4 Protein expressions of ARHI, WWOX, and PTEN in low grade and high grade ovarian serous carcinoma The results showed that the differnce of protein expressions of ARHI was not found between low and high grade. But the positive rates of WWOX and PTEN protein expressions in high grade were significantly lower than that in low grade (P<0.05).Part two Expression of STAT3 and E2F1 protein in ovarian serous tumors1 Expression of STAT3 protein in ovarian serous tumors1.1 ImmunohistochemistryThe result of ICH showed that the positve rates in serous carcinomas and borderline tumors were 87.5% (49/56) and 77.8% (14/18), significantly higher than that in benign tumors and normal ovaries (P<0.05), but the statistical differences were not found between borderline tumors and serous carcinomas, benign tumors and normal ovaries.1.2 FCMThe FCM revealed that the FI in serous carcinomas was 1.192±0.031, obviously increased compare to borderline tumors, benign tumors, and normal ovaries.2 Expression of E2F1 protein in ovarian serous tumors2.1 ImmunohistochemistryThe result of ICH showed that the positve rates in serous carcinomas was 82.1%, significantly higher than that in borderline tumors, benign tumors, and normal ovaries (P<0.05), but the statistical differences were not found among borderline tumors, benign tumors, and normal ovaries.2.2 FCMThe FCM revealed that the FI in serous carcinomas was 1.139±0.023, obviously increased compare to borderline tumors, benign tumors, and normal ovaries.3 The relationship between STAT3, E2F1 and clinicpathological characteristics in serous carcinomasThe statistical differences of STAT3, E2F1 protein expression were not found in different grades, stages, lymph node metastasis, and peritoneum metastasis.4 The correlation between ARHI protein and STAT3, E2F1 protein in in serous carcinomasStatistically, negtive correlations were found between ARHI protein expression and STAT3, E2F1 protein expression in serous carcinomas.5 The correlation between WWOX protein and STAT3, E2F1 protein in in serous carcinomasStatistically, negtive correlations were found between WWOX protein expression and STAT3, E2F1 protein expression in serous carcinomas.6 The correlation between PTEN protein and STAT3, E2F1 protein in in serous carcinomasStatistically, negtive correlations were not found between PTEN protein expression and STAT3, E2F1 protein expression in serous carcinomas.Part three The effect of ARHI, WWOX and PTEN protein expression on prognosis in ovarain serous carcinomas1 Correlation between ARHI protein expression and prognosis, clinicpathological characteristics in ovarain serous carcinomasThe result of Kaplan-Meier univariate survival analysis showed that the expression of ARHI was a related factor of prognosis in ovarian serous carcinoma patients. The survival rates of 3 and 5 years were 68.2% and 54.5% in ovarian serous carcinoma patients with ARHI positive expression, significantly higher than 32.4% and 17.6% in patients with ARHI negitive expression.The statistical differences of ARHI protein expression were not found in different grades, stages, lymph node metastasis, and peritoneum metastasis.2 Correlation between WWOX protein expression and prognosis, clinicpathological characteristics in ovarain serous carcinomasThe result of Kaplan-Meier univariate survival analysis showed that the expression of WWOX was a related factor of prognosis in ovarian serous carcinoma patients. The survival rates of 3 and 5 years were 63.0% and 44.4% in ovarian serous carcinoma patients with WWOX positive expression, significantly higher than 31.0% and 20.7% in patients with WWOX negitive expression.The statistical differences of WWOX protein expression were found in different grades, but not in lymph node metastasis, peritoneum metastasis, and stages.3 Correlation between PTEN protein expression and prognosis, clinicpathological characteristics in ovarain serous carcinomasThe result of Kaplan-Meier univariate survival analysis showed that the expression of PTEN was a related factor of prognosis in ovarian serous carcinoma patients. The survival rates of 3 and 5 years were 62.1% and 44.8% in ovarian serous carcinoma patients with PTEN positive expression, significantly higher than 29.6% and 18.5% in patients with PTEN negitive expression.The statistical differences of PTEN protein expression were found in different grades, but not in lymph node metastasis, peritoneum metastasis, and stages.4 Survival analyses of ovarain serous carcinoma patients4.1 Screening relative prognosis factors of ovarain serous carcinomaKaplan-Meier univariate survival analysis showed clinical stage, pathology grades, residual disease and chemotherapy had effects on survival, were prognosis factors of patients with serous ovarian carcinoma.4.2 COX multivariate survival analysis of ovarain serous carcinoma patientsCOX multivariate survival analysis showed that clinical stage was the important independent prognosis factor, followed by pathology grades, chemotherapy, and ARHI expression (the independent risk coefficients were 2.810, 1.966, 0.547 and 0.428 respectively), but residual disease, WWOX, and PTEN expression were not.Conclusion1 Protein and mRNA expression of ARHI, WWOX and PTEN were decreased in ovarain serous carcinoma, suggesting that loss of function of tumor suppressor gene may contribute to oncogenesis of ovarian serous carcinoma.2 Proteins expression of STAT3 and E2F1 in ovarain serous carcinoma were increased, suggesting that E2F1 overexpression inhibited ARHI expression and down-regulation of STAT3 by ARHI were losed and many down stream genes promoting proliferation and inducing apoptosis were actived.3 The differnces of tumor suppressor genes exppression between low and high grade confirmed a dualistic theory model for ovarian serous carcinogenesis.4 Protein and mRNA expression of ARHI was significantly decreased compared to WWOX and PTEN, suggestting that ARHI may play a main role in oncogenesis of ovarian serous carcinoma.5 Beside conventional prognosis factors, expression of ARHI, WWOX and PTEN had effects on survival of ovarain serous carcinoma patients, moreover ARHI protein expression was an independant prognosis factor.6 It was very useful for more exactly evaluating prognosis and guiding efficient therapy that analyzing independent prognosis factors including ARHI, WWOX, and PTEN expression.
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