Objective To investigate the cytidylyl phosphate guanosine(CpG) islands methylation status of E-cadherin (E-cad) promoter region in human ovarian carcinoma cell lines (ES-2,3 AO, SKOV3 ), and the effect of 5-azacytidine-2 '-deoxycytidines (5-Aza-CdR ) on the cell proliferative ability, invasion and the expression of E-cad protein. Methods Methylation specific PCR(MSP) was used to detect CpG islands methylation status of E-cad promoter region in ES-2,3AO and SKOV3 cell lines. After treated with different concentrations of 5-Aza-CdR, morphological changes of cell lines were observed under microscope. The proliferative ability was evaluated by methyl thiazolyl tetrazolium(MTT) assay. E-cad protein expression was detected by western-blot and cellular invasion was investigated by 24-well matrigel invasion chambers. Results Hypermethylatian status of CpG islands of E-cad promoter region was observed in ES-2 and SKOV3 cell lines, but not in 3AO cell lines. After treated with 5-Aza-CdR (0.1,1.0,10.0 μmol/L), ES-2 and SKOV3 cell lines displayed morphological evidence of differentiation. 5-Aza-CdR was found to decrease proliferation as evidenced by cell growth curve , to increase the level of E-cad protein expression (P ＜ 0.01 ), and effectively inhibit the ability of cell invasion(P ＜0.01 ). Conclusions CpG hypermethylation is an important mechanism of E-cad gene inactivation in ES-2 and SKOV3 cell lines. 5-Aza-CdR be found to inhibit proliferation and invasion, and increase the expression of E-cad probably by the inhibition of hypermethylation.

Objective To observe the effect of DNA methyltransferase 1 (DNMT1 ) gene silencing by RNA interfering technology on the proliferation and apoptosis of HeLa cells. Methods Recombinant plasmid pshRNA-DNMT1-A, B and C were respectively transfected into HeLa cells by lipofectamine 2000, while cells transfected plasmid vector pSilencer3.1-HI and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and western blotting was used to detected the mRNA and protein expression of DNMT1 in HeLa cells transfected for 24, 48 and 72 hours. Cell counting kit-8 (CCK-8 ) assay was used to investigate the proliferation of the HeLa cells after transfection, while apoptosis was detected by flowcytometry(FCM ) method. Results Three DNMT1-targeted short hairpin RNA (shRNA) A,B and C were successfully inserted into the plasmid vector PShRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragments. The results indicated that both recombinant plasmid pshRNA-DNMT1-A and B could effectively knock down the expression of DNMT1 gene in human cervical cancer cells, of which pshRNA-DNMTI-B was the better choice. While no effect of pshRNA-DNMTI-C was seen. BT-PCR results showed that the relative mRNA expression of DNMT1 gene in Helm cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were 0.406±0.057,0.191±0.036 and 0.104±0.015, which were significantly lower than that in Helm cells transfected by empty vector and non-transfected cells (0.520±0.020, 0.537±0.041, respectively, P ＜ 0.05 ). The western blotting analysis manifested that the relative expression of DNMT1 protein of Helm cells transfected by pshRNA-DNMT1 for 24, 48 and 72 hours were 0.197±0.024, 0.075±0.015, 0.040± 0.013, which were significantly lower than that in transfected cells by empty vector and non-transfected cells (0.273±0.010, 0.283±0.016, respectively, P ＜0.05). The CCK-8 results showed that the cell survival rates of HeLa cells transfected by pshRNA-DNMT1 for 24, 48, 72, 96 and 120 hours were 70.8%, 64.8%, 51.6%, 45.3% and 38.0%, there were statistically different compared with cells transfected by empty vector and non-transfected cells at different time-points (P ＜ 0.01 ). The results of FCM indicated that the apoptesis rate of HeLa cells trandected with pshRNA-DNMTI for 24, 48 and 72 hours were (17.7± 1.3 ) %, (35.3±1.3 ) %, (47.6±1.6 ) %, which were significantly higher than empty vector transfected cells and non-transfected cells [(4.9±0.5 ) %, (5.1±0.7 ) %, respectively, P ＜ 0.05]. Conclusions DNMT1 can be successfully silenced by RNA interfering in cervical Helm cells. Downregulation of DNMT1 can inhibit cervical cancer cells proliferation and induce cell apoptosis.

Objective To investigate the relationship between raf kinase inhibitor protein (SKIP), a novel metastasis suppressor gene, and metastasis of ovarian carcinoma. Methods Immunohistochemistry, RT-PCR, and western blot analysis were performed to examine the expression of SKIP in clinical samples of ovarian tumors and five human ovarian carcinoma cell lines. Stable cell lines over-expressed or deleted of SKIP were cloned to investigate the function of SKIP in ovarian cancer cells. The recombinant plasmids expressing sense (ss) or antisense (as) SKIP cDNA or empty vector was transfected into ovarian cancer cell line SKOV3 by lipofectamine. The expression level of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) in ovarian cancer cells were detected by western blot analysis. Assays of cell proliferation, soft-agar colony formation, cell adhesion, and cell invasion in vitro were used to examine the malignant phenotypes of the transfected cells. Flow cytometric analysis was performed to observe the effect of SKIP on cell cycle distribution before and after transfection. Results (1 ) The expression levels of SKIP protein in ovarian carcinoma tissues from patients were found to be reduced than those in ovarian benign tumor and borderline tumor. SKOV3 clones stably expressing full-length recombinant ssRKIP, asRKIP, and their respective empty vector were obtained. (2)RKIP was able to block basal levels of MEK and ERK in ovarian cancer cells. The expression level of phosphorylation MEK in ssRKIP#1 and ssRKIP#4 cells were 0. 35, 0. 34; while the expression level of phosphorylation ERK in ssRKIP#1 and ssRKIP#4 cells were 0.48 and 0.46. (3) Abilities of cell proliferation in the ssRKIP vector-transfected cells were decreased compared with that in the non-transfected cells (P ＜0. 01 ). (4)Anchorage-independent growth in the ssRKIP#1 and ssRKIP#4 cells (83.7 ± 5.7, 106. 0±9. 2) were decreased compared with that in the empty vector-transfected cells (158.3 ± 14. 6, P＜ 0. 01). (5)Cell adhesion in the ssRKIP#1 and ssRKIP#4 cells [(68.3±0. 8)%, (64. 1±0. 9)%] were decreased compared with that in the non-transfected cells [(100. 0 ± 1.1 )%, P ＜ 0. 01]. (6) Cell invasion in the ssRKIP#1 and ssRKIP#4 cells (24 ± 5, 25±4) were decreased compared with that in the non-transfected cells (68 ± 5, P ＜ 0. 01 ). (7) ssRKIP cells had a significant increase in the G1 phase and decrease in the G2 + S phase. Conclusion RKIP could inhibits the metastasis, but also the growth of ovarian cancer cells.

Objective To evaluate accuracy of preoperative tumor grade and intracperative gross examination of myometrial invasion in patients with clinical stage Ⅰ endometriod adenocarcinoma for lymphadenectomy. Methods Clinic-pathological data were retrospectively collected from 687 patients with clinical stage Ⅰ endometriod adenocarcinoma who underwent operation in Women's Hospital, Zhejiang University School of Medicine from January 1999 to December 2008. According to postoperative histology diagnosis, accuracy of preoperative tumor grade by curettage and depth of myometrial invasion by intraoperative gross examination was evaluated, and clinic-pathological factors associated with accuracy were analyzed. Results Sensitivity, specificity, accuracy, false negative rate, false positive rate, and positive and negative predictive value for the prediction of needing for intraoperative lymphadenectomy in patients with clinical stage Ⅰ endometriod adenocarcinoma were 70. 4% ,80. 2% ,77.6% ,12.0%, 43.0%, 57.0% and 88.0%, respectively. Analysis of mutil-factors shown that patient age, tumor size, lymph node metastasis and extrauterine spread lesions were independent factors affected the accuracy of prediction(P ＜ 0. 05 ). Conclusion Prediction of needing for lymphadenectomy by preoperative tumor grade and intraoperative gross examination of myometrial invasion is reliable in clinical stage Ⅰ endometriod adenocarcinoma patients, while there is a highly false negative rate in prediction of not needing for lymphadenectomy, while other prognostic factors such as patient age, tumor size, lymph node metastasis and extrauterine spread lesion should be together considered.

Objective To survey age of menarehe in Pudong district in Shanghai. Methods Data in this study were derived from 56 924 women at age of 20 -81 years in screening for cervical cancer between January 2007 and July 2008 in Pudong district. The age of menarche were recorded in a questionnaire. To investigate the trends in age at menarehe in different socioeconomic status, the subjects were divided into 12 groups in 5-year birth cohorts. The mean menarche age in each group was analyzed by analysis of variance(ANOVA). The percentage of menarche age at 10- 12 years and more than 18 years was analyzed by χ2 method. Results (1 ) The minimum age of menarcbe recorded is 10 years old, and the maximum is 28 years old, with average age of menarche at 15.7 years. In all groups, the smallest average age of menarcbe is 14. 6 years in 26 - 30 years old age group, while the biggest average age of menarche age is 16. 5 years in ＞ 75 years old group; The difference showed statistical significance (P ＜ 0. 01 ). (2) The percentages for participants with early menarehe age (10 - 12 years old) or late menarehe age (＞ 18-year-old menarche) were 1.82% (1034/56 924 ) and 5.20 % (2959/56 924 ) respectively. However, the maximum percentage for early menarche was recorded in 31 -35 years old group (4. 45% ,197/4431 ), only 0. 84% (10/1191 ) of participants in ＞75 years old group was classified as early menarebe. Meanwhile, the lowest percentage for late menarehe was 0. 38% (17/4431 ) in 31 - 35 years old group, and the highest percentage was 14. 70% (91/619) in ＞ 75 years old group. The changes in the percentages for early menarche or late menarche are significantly associated with age differences (P ＜ 0. 01 ). Conclusion The study suggested that the average of onset age of menarche in Pudong district has declined over the past decades in an age-based way, accompanied with the increase of the percentage for early menarche and the decrease of percentage for late menarehe.

Objective To evaluate the application of domestic probe fluorescence in situ hybridization (FISH) in prenatal diagnosis on uncultured amniocytes aneuploid. Methods One thousand three hundred and sixty-nine uncultured amniocytes (16-24 gestational weeks) from 37 hospitals in China were selected for prenatal diagnosis. 5 chromosomes (21, 13, 18, X and Y) were detected with muhicolor FISH. In the mean time, cytogenetic karyotype analysis was performed as control. Results Of all the samples, 1361 samples were successfully tested by FISH, the rate of successful detection was 99.42% (1361/1369). Thirty-five samples were shown with abnormal karyotypes by domestic FISH probe, the abnormal rate is 2. 57% (35/1361 ), including trisomy 21 (22 samples), trisomy 13 (4 samples), trisomy 18(6 samples), X0 (1 sample) and XXY (2 samples). Results of both FISH and cytogenetic karyotype analysis exhibited extreme concordance. Conclusion Domestic FISH probe used in prenatal diagnosis on uncultured aminiocytes showed the following advantages, such as highly efficient, low cost, small amounts of samples needed and reliable results.

Objective To investigate the effects of adjuvant chemotherapy for patients with high-risk stage Ⅰ and Ⅱ (early stage)endometrial cancer. Methods From Jan. 1994 to Jun. 2007,106 eases with early stage high-risk endometrial cancer were treated in Peking University First Hospital and were divided into two groups based with postoperative adjuvant chemotherapy (ACT group,66 eases) and without adjuvant chemotherapy( control group,40 cases ). The 5-year survival rates was calculated by Kaplan-Meier method. Prognosis factors were further determined by univariate analysis and Cox proportional hazards models. Results The 5-year survival rate in the ACT group was significantly higher than that in control group (94% and 81%, P <0.05). On the univariate analysis, the 5-year survival rate of patients received four or more cycles combined chemotherapy was higher than that of cases less than four cycles chemotherapy (100% and 86%, P<0.05). While, it were not significant difference in age, stage, histology, grade,radiotherapy alone, chemotherapy combined radiotherapy or progestin hormonal therapy(P>0.05). On the multivariate analysis, adjuvant chemotherapy was found to affect independent prognostic covariates on early stage cases (P<0.05). Conclusion Postoperative adjuvant chemotherapy maybe improve the prognosis of patients with high-risk early stage endometrial cancer, which need to be further study by prospective randomized trials.

Objective To investigate the effect of anti-Mlllerian hormone (AMH) on hormone secretion and P450 aromatase mRNA expression from cultured human luteinized granulosa cells. Methods Human luteinized granulose cells were derived from 10 patients treated by in vitro fertilization-embryo transplantation (IVF-ET) in the Second Affiliated Hospital of Sun Yat-sen University from June to December 2006. Granulose cells were divided into group A, B, C, D, E depending on different concentration of AMH,testosterone group and blank group. 1×10-7moL/L testosterone and 1,5,10,20,50 μg/L AMH were added into the culture medium of group A,B,C,D and E. 1×10-7mol/L testosterone was added into the culture medium of testosterone group while no other ingredient was added into the medium of blank group. Estrogen levels in supernates were measured at 24,48,72 hours after cell incubation. RT-PCR was performed to detect the P450 aromatase mRNA expression in group B, C, D, E and testosterone group at 72 hours after cell incubation. Results (1) Estrogen levels in supernates of granulose cell culture at 24,48,72 hours were (8.529±0.381)×104, (10.977±0.436)×104, (13.309±0.506)×104 pmol/L in group A, (7.027±0.276)×104, (9.167±0.300)×104, (10.794±0.555)×104 pmol/L in group B, (6.039±0.226)×104,(7.585±0.548)×104, (8.797±0.518)×104 pmol/L in group C, (5.118±0.460)×104, (5.716±0.496)×104, (6.205±0.667)×104 pmol/L in group D, (4.932±0.148)×104, (5.323±0.184)×104,(5.629±0.212)×104 pmol/L in group E. When compared with blank group [(0.001±0.001)×104,(0.006±0.003)×104, (0.029±0.011)×104 pmol/L], the statistical differences were observed in group A,B,C,D,E(P<0.01) ; when compared with testosterone group [ (8.418±0.569)×104, (10.841±0.689)×104, (13.301±0.637)×104 pmol/L], the statistical differences were observed in group B,C,D and E(P<0.01) ; statistical differences were also observed in group C, D and E when compared with group B, and also group D and E when compared with group C(P<0.01). No significant difference was observed between group D and E (P>0.05). In group A, B, C, D, E and testosterone group, the estrogen levels at 24 hours after cell culture were significantly lower than those at 48 and 72 hours (P<0.01) ; statistical difference was observed between estrogen levels at 48 and 72 hours(P<0.01). No significant difference was observed among 24,48 and 72 hours in blank group (P>0.05). (2) Relative ratios of intensity of P450 aromatase/β-actin at72 hours of cell culture in group B,C,D and E were 0.6148±0.0046, 0.5156±0.0012, 0.4698±0.0027 and 0.4282±0.0017, respectively, which were statistically lower than that in testosterone group (0.8224±0.0021, P<0.01) ;statistical differences were also observed in group C, D and E when compared with group B, and also group D and E when compared with group C(P<0.01). No significant difference was observed between group D and E (P>0.05). Conclusion It is suggested that AMH might affect estrogen synthesis by inhibiting P450 aromatose activity so that lead to hyperandrogenism microenvironment in local ovary.

Objective To analyze the association of serum CA125 level at the different phases with recurrence and survival, for providing simple and efficient methods about predicting recurrence and prognosis in epithelial ovarian cancer.Methods The clinical-pathological data from 151 patients were collected, who were histologically confirmed as primary ovarian cancer between Jan 2002 and Dec 2005.All the patients were followed up.The relationship between serum CA125 level at different phases and clinical-pathological data were analyzed, including prognostic associated factors, 2-year or 5-year recurrent rate, 5-year survival rate, progression-free survival times, and overall survival times.Results Serum CA125 level at pre-surgery and the end of 3-course chemotherapy were associated with most of the clinical-pathological parameters,included stage, pathological grade, amount of ascites, residual tumor size, type of recurrence, 2-year and 5-year recurrent rate, and 5-year survival rate ( all P ＜ 0.05 ).Progression-free survival and overall survival times were shorter in the patients with higher CA125 level at pre-surgery or abnormal CA125 level at the end of 3-course chemotherapy (P ＜0.01 ).There was no relationship between the ratio of CA125 level at pre- and post-surgery and recurrence or prognosis ( all P ＞ 0.05).Conclusion Serum CA125 level at pre-surgery and the end of 3-course chemotherapy can be used for predicting the recurrence and prognosis of epithelial ovarian cancer.

Objective To study the influence of survivin mutant-T34A ( survivinT34A) and survivin deletant-N-terminal 8 amino acids residues ( survivinN-8AA ) on the cell cycle distribution and chemosensitivity in human ovarian cancer HO-8910 cells for explorating the roles of modified survivin-mediated apoptosis induced by chemotherapeutic agents and possible signaling pathways involved. Methods pcDNA3.1 plasmid contained wild-type, survivinT34A and survivinN-8AA genes were transfected into HO-8910 cells,respectively, the control groups were HO-8910 cells transfected with pcDNA3. 1 plasmids. The expression of mRNA was examined by reverse transcription(RT) PCR and identified by DNA sequencing; the cell cycles were determined by flow cytometer analysis ( FCM ); the growth inhibitions rate of cisplatin ( DDP),paclitaxel (PTX) and LY294002 on the transfected cells were determined using methyl thiazolyl tetrazolium (MTT) assay. Results (1) The RT-PCR procedures and genome sequences showed that the survivin mRNA were expressed stable in the transfected HO-8910 cells. (2) There was lower percent of G0/G1 phase cells in SN-HO-8910 cells than that in PC-HO-8910 cells (44. 72% vs. 49.64%, P ＜0. 05) ;while higher percentage of G2/M phase and S phase cells( 1.06% and 54. 22% vs. 0. 56% and 49. 80%, P ＜ 0. 05 ).There was lower the G2/M phase and S phase cells in M-HO-8910 cells 0. 16% and 36. 33%, than that in PC-HO-8910 cells( P ＜ 0. 05 ); while higher percentage of G0/G1 phase cells(63. 51% ,P ＜ 0. 05 ). G0/G1 ,G2/M and S phase cells in Sur-HO-8910 cells were 54. 46%, 0. 62% and 44. 92%, and there were not significantly difference ( P ＞ 0. 05 ), compared to those in PC-HO-8910 cells. ( 3 ) The inhibitory concentration ( IC50 ) of DDP and PTX were higher in Sur-HO-8910 cells than those in control cells [(20. 4 ±6. 1)vs. (14.4 ±3.9)μmol/L,(36.7 ±4.0) vs. (28.6 ±3.6) μmol/L;all P＜0.05]. The IC50 of DDP and LY294002 in SN-HO-8910 cells were lower than those in control cells[(7. 6 ± 1.0) vs. ( 14. 4 ± 3.9)μmol/L, ( 13.2 ± 4. 0) vs. (41.0 ± 7. 9 ) μmol/L; all P ＜ 0. 01]. The IC50 of PTX [( 37. 9 ± 4. 8 ) μmol/L]in SN-HO-8910 cells were higher than that in control cells(P ＜0. 05). The IC50 of DDP in M-HO-8910 cells [(9.9 ± 1.2) μmol/L] were lower than that in control cells(P ＜0. 05) ,and the IC50 of LY294002 in M-HO-8910 cells [(66. 9 ± 4. 8) μ mol/L] higher than that in control cells ( P ＜ 0. 01 ). Conclusions The changes of cells cycle distribution caused by survivinT34A or survivinN-8AA enhanced the G2/M cell cycle-dependent chemosensitivity of PTX. Compared to survivinT34A, survivinN-8AA preferentially to mediate the cytotoxicity of DDP and LY294002, suggesting that it may be related to the cell cycle-dependence of survivin function and to blockage of the formation of its active dimer.