miR424-5p functions as an anti-oncogene in cervical cancer cell growth by targeting KDM5B via the Notch signaling pathway.

AIMS: Aberrant expression of miRNAs exert the critical roles in carcinogenesis, including cervical cancer. Recent study corroborated the down-regulation of miR424-5p in uterine cervix adenocarcinoma. This research aimed to investigate the function and underlying mechanisms of miR424-5p in cervical cancer cell growth. MAIN METHODS: Tissues samples were collected from patients with cervical cancer and healthy control. The expression levels of miR424-5p were determined by qRT-PCR. After transfection with miR424-5p mimics or inhibitor, cervical cancer cell proliferation and apoptosis were evaluated by WST-1 and flow cytometry assay, respectively. The underlying mechanism involved in aforementioned processes was also explored. KEY FINDINGS: Expression of miR424-5p was notably decreased in cervical cancer tissues and cells. Overexpression of miR424-5p restrained cell proliferation and promoted cell apoptosis, but with little function in miR424-5p inhibitor-treated groups. Furthermore, KDM5B was identified as a direct target of miR424-5p as the evidence that miR-424-5p inhibited KDM5B expression and luciferase activity of KDM5B 3'-UTR. Here, KDM5B elevation majorly reversed miR424-5p-triggered inhibition in cell proliferation and increase in cell apoptosis. Moreover, silencing KDM5B expression also restrained cell growth. Additionally, miR424-5p overexpression inhibited the expression of Notch1 and Notch2, which was obviously rescued after KDM5B up-regulation. Simultaneously, blocking KDM5B also attenuated the activation of Notch pathway. Importantly, treatment with Notch agonist Jagged1 antagonized miR424-5p-mediated suppression on cell growth. SIGNIFICANCE: This research suggests that miR424-5p may act as a novel anti-oncogene in cervical cancer by blocking cell growth through targeting KDM5B-Notch pathway. Accordingly, our study will support a promising therapeutic strategy against cervical carcinoma.

[Effects of plasma from patients with preeclampsia on the proliferation and apoptosis of human umbilical vein endothelial cells and its relationship with lysophosphatidic acid receptors].

OBJECTIVES: To investigate the effects of plasma from the patients with preeclampsia on proliferation and apoptosis of human umbilical vein endothelial cells (HUVEC), and to explore the relationship between cell damage and lysophosphatidic acid (LPA) receptors. METHODS: Sixty patients with preeclampsia were recruited from October 2011 to June 2012 in the First Affiliated Hospital of Zhengzhou University. Among them, thirty cases were defined as the mild preeclampsia group and thirty cases were defined as the severe preeclampsia group. The other thirty healthy pregnant women were recruited in the healthy pregnant women group. The levels of plasma LPA in the three groups were measured. The HUVEC were cultured in vitro with plasma from the three groups, and a blank control group was set up as well. Proliferation and apoptosis of HUVEC were measured by MTT assay and flow cytometry. Immunohistochemistry of biotin streptomyces protein peroxidase (SP) method was used to measure the protein expression level of Edg 2, 4, 7. RESULTS: (1) The plasma LPA levels in the healthy pregnant woman group, mild preeclampsia group and severe preeclampsia group were (3.38 ± 2.08) µmol/L, (6.12 ± 0.22) µmol/L, (9.10 ± 0.17) µmol/L, respectively. The plasma levels of LPA in patients with preeclampsia were significantly higher than that in the healthy pregnant women (P < 0.01). (2) The proliferation rate of HUVEC in the mild and severe preeclampsia groups [(65.2 ± 2.7)% and (51.9 ± 2.8)%] were significantly lower than that in the healthy pregnant women group and the control group [(84.3 ± 3.1)% and (100.0 ± 0.0)%, P < 0.01]. (3) The early apoptosis rate, middle-late apoptosis rate and total apoptosis rate of HUVEC in the mild and severe preeclampsia groups [total apoptosis rate were (30.4 ± 2.0)% and (43.4 ± 2.5)%] were significantly higher than those in the healthy pregnant women group and the control group [total apoptosis rate were (18.6 ± 1.6)% and (8.0 ± 1.5)%, P < 0.01]. (4) The expression positive rates of Edg 2, 4, 7 proteins in the four groups were as following: mild preeclampsia group 83%, 80% and 73%; severe preeclampsia group 97%, 93% and 90%; healthy pregnant women group 40%, 40% and 37%, and the control group 10%, 10% and 7% respectively. The positive rates of HUVEC in the mild and severe preeclampsia groups were significantly higher than those in the healthy pregnant women group and the control group (P < 0.01). CONCLUSIONS: The plasma of patients with preeclampsia could inhibit proliferation and promote apoptosis of HUVEC, and induce the expression of Edg 2, 4, 7 proteins. It suggested that the increase of lysophosphatidic acid in plasma could be one of the reasons of endothelial cell damage in patients with preeclampsia.

[Influence of pertussis toxin on GPER-mediated activation of phosphatidylinositol 3-kinase/protein kinase B signaling induced by 17ß-estradiol in endometrial carcinoma cells].

OBJECTIVE: To investigate the influence of pertussis toxin (PTX) on G protein-coupled estrogen receptor (GPER)-mediated activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling activated by 17ß-estradiol (17ß-E2) in endometrial carcinoma cells. METHODS: Expressions of GPER protein were detected by immunohistochemical SP method in Ishikawa and HEC-1A cells. Changes of levels of GPER, ERα and ERß protein and the activation of Akt protein were observed by western blot in the two cells after they were treated by PTX for 30 minutes at different concentrations (0, 0.1, 0.5, 1.0 µg/ml), and then co-stimulated with with 1×10(-6) mol/L 17ß-E2 respectively at different time (Ishikawa 30 minutes, HEC-1A 15 minutes). RESULTS: (1) Immunohistochemical SP method showed that GPER was positive stained in cell cytoplasm of Ishikawa and HEC-1A cell. (2) After co-treated with PTX at different concentrations (0, 0.1, 0.5, 1.0 µg/ml) and 10(-6) mol/L 17ß-E2, in Ishikawa cell, the ratio of p-Akt/Akt was 0.74 ± 0.54, 0.34 ± 0.06, 0.18 ± 0.03, 0.07 ± 0.15, the gray values of GPER was 0.872 ± 0.490, 0.395 ± 0.054, 0.145 ± 0.014, 0.034 ± 0.008, and with increasing concentration of PTX, the ratio of p-Akt/Akt and the expression of GPER decreased gradually (P < 0.05), which was most obviously when the concentration was 1.0 µg/ml (F = 63.729, P = 0.0001; F = 160.284, P = 0.0001); ERα and ERß protein had no significant change among different groups (P > 0.05). In HEC-1A cell, the ratio of p-Akt/Akt was 0.73 ± 0.09, 0.26 ± 0.14, 0.11 ± 0.03, 0, the Gray values of GPER is 0.927 ± 0.134, 0.485 ± 0.022, 0.194 ± 0.004, 0, and with increasing concentration of PTX, the ratio of p-Akt/Akt and the expression of GPER decreased gradually (P < 0.05), which were also completely inhibited when the concentration was 1 µg/ml (F = 1039.321, P = 0.0001; F = 109.646, P = 0.0001), ERα protein had no significant differences (P > 0.05) among different groups. ERß was negatively expressed. CONCLUSION: The results proposed that the activation of PI3K/Akt signaling in Ishikawa and HEC-1A cells could be inhibited after blocking the role of GPER by PTX.

HDAC6 siRNA inhibits proliferation and induces apoptosis of HeLa cells and its related molecular mechanism.

OBJECTIVE: To investigate the effects of histone deacetylase 6 (HDAC6) siRNA on cell proliferation and cell apoptosis of the HeLa cervical carcinoma cell line and the molecular mechanisms involved. METHODS: Division was into three groups: A, the untreated group; B, the control siRNA group; and C, the HDAC6 siRNA group. Lipofectamine 2000 was used for siRNA transfection, and Western blot analysis was used to determine the protein levels. Cell proliferation and apoptosis were characterized using a CCK-8 assay and flow cytometry, respectively. RESULTS: HDAC6 protein expression in the HDAC6 siRNA-transfection group was significantly lower (P < 0.05) than in the untreated and control siRNA groups. The CCK-8 kit results demonstrated that the proliferation of HeLa cells was clearly inhibited in the HDAC6 siRNA transfection group (P < 0.05). In addition, flow cytometry revealed that the early apoptotic rate (26.0% ± 0.87%) was significantly elevated (P < 0.05) as compared with the untreated group (10.6% ± 1.19%) and control siRNA group (8.61% ± 0.98%). Furthermore, Western blot analysis indicated that bcl-2 protein expression in the HDAC6 siRNA-transfection group was down-regulated, whereas the expression of p21 and bax was up-regulated. CONCLUSION: HDAC6 plays an essential role in the occurrence and development of cervical carcinoma, and the down-regulation of HDAC6 expression may be useful molecular therapeutic method.

Effect of coincubation time of sperm-oocytes on fertilization, embryonic development, and subsequent pregnancy outcome.

Several studies have reported improved IVF by shortening the time of sperm-oocyte coincubation from 16-18 hours to 1-4 hours. The objective of this study was to examine the advantages and disadvantages of a shortened sperm-oocyte coincubation time in order to assess the effects of this insemination method for clinical IVF practice. Two insemination methods, the shortened method (4 hours) and the standard method (16-18 hours) of coincubation of sperm-oocytes for two groups of patients based on the quality of sperm were compared. Group I, was composed of couples without male factor; Group II, involved couples with mild male factor. Fertilization, good quality embryos, clinical pregnancy, and implantation rates were compared by two different insemination methods. In Group I, fertilization, clinical pregnancy, and implantation rates were not different between the two insemination methods. However, the polyspermy rate was significantly higher (P < 0.05) in the shortened (7.3%) than in the standard (4.1%) insemination method. In Group II, the fertilization rate was significantly lower (P < 0.05) using the shortened insemination method (62.6%) compared to the standard insemination method (68.7%). When fertilization failed with the shortened insemination method, the clinical pregnancy and implantation rates were 34.7% and 24.1%, respectively, from the rescue intracytoplasmic sperm injection (ICSI). The live birth rate from the rescue ICSI was 32.0% with normal infants. The duration of sperm-oocyte coincubation does not affect fertilization, embryo quality, clinical pregnancy, and implantation rates. However, fertilization rates will decrease with the shortened insemination method when the sperm parameters are poor. From the results of the present study we suggest that the combination of the shortened sperm-oocyte coincubation and rescue ICSI method may be an efficient method for IVF treatment in order to prevent fertilization failure when sperm parameters were poor as mild male factor.

[Effect of GPER on the activation of PI3K/Akt induced by 17ß-estradiol in endometrial carcinoma cells].

OBJECTIVE: To investigate the expression of G protein-coupled ER (GPER) and ER in the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) induced by 17ß-estradiol (17ß-E(2))in endometrial carcinoma cells, Ishikawa and HEC-1A. METHODS: Expressions of GPER, ERα and ERß protein in Ishikawa and HEC-1A cells were detected by immunohistochemical SP method. Levels of GPER, ERα and ERß were examined by western blot in Ishikawa and HEC-1A cells after treated with 1×10(-6) mol/L 17ß-E(2) at different time (0, 15, 30, 60, 120 minutes). RESULTS: GPER was positive expressed in Ishikawa and HEC-1A cells. ERα and ERß were both positive expressed in Ishikawa cells. While, ERα was weakly expressed and ERß was almost negatively expressed in HEC-1A cells. Western blot analysis showed that 1×10(-6) mol/L 17ß-E(2) treatment, the Ishikawa and HEC-1A cells GPER protein level for 15 minutes markedly increased (P < 0.05), which Ishikawa 30 minutes, when cells reached the highest level (0.192 ± 0.004), HEC-1A cells for 15 minutes and reached the highest level (0.184 ± 0.006); Ishikawa and HEC-1A cells, Akt, activation of 15 minutes from the treatment start was significantly increased (P < 0.05), which Ishikawa cells for 30 minutes and reached the highest level (0.666 ± 0.021), HEC-1A cells for 15 minutes and reached maximum (0.788 ± 0.035); Ishikawa and HEC-1A cells, ERα and ERß protein expression did not change significantly (P > 0.05). CONCLUSION: GPER likely involved in non-nuclear activation of PI3K/Akt signaling pathways in endometrial carcinoma cells, Ishikawa and HEC-1A.

[Effects of PD98059 and LY294002 on subcutaneous xenograft of human endometrial carcinoma in nude mice].

OBJECTIVE: To investigate the effects of signal pathway inhibitors PD98059 and LY294002 on cell proliferation, apoptosis, expressions of phosphorylated extracellular signal-regulared kinase (p-ERK) and phosphorylated protein kinase B (p-Akt) in endometrial carcinoma xenografts. METHODS: Human endometrial carcinoma Ishikawa cells were cultured in vitro. The effects of PD98059 and LY294002 on proliferation, apoptosis, and cell cycle distribution of endometrial cancer cells were detected by monotetrazolium (MTT) assay and fluorescence-activated cell sorting technique. The models of xenografted tumor were established by the subcutaneous inoculation in 24 nude mice, and then they were randomly divided into 4 groups (n = 6), normal saline group, PD98059 group (PD group), LY294002 group (LY group) or PD98059 + LY294002 group (PD + LY group) by intraperitoneal injections, respectively. The anti-tumor efficacy was evaluated by measuring tumor volume and tumor growth status. The histopathological change of tumor specimens was observed using HE staining and terminal deoxynucleotidyl transferase-mediated dUTP-digoxigen in nick and labeling method (TUNEL) testing and the expression levels of p-ERK and p-Akt were detected by immunohistochemistry method. RESULTS: (1) The proliferation of Ishikawa cells were suppressed after treated by PD98059 and(or) Y294002, in which A(570) values of cells decreased showing both time-dependent and concentration-dependent manner (LY294002: F(group) = 9.801, P = 0.002; F(time) = 10.398, P = 0.001. PD98059: F(group) = 8.213, P = 0.015; F(time) = 6.839, P = 0.036). Cell cycle distribution analysis revealed that percentage of Ishikawa cells at G(0)/G(1) phase (F(time) = 35.049, P = 0.004; F(group) = 32.024, P < 0.01) increased and percentage of S phase cells (F(time) = 7.789, P = 0.049; F(group) = 30.132, P < 0.01) decreased significantly. The percentage of apoptotic cells increased significantly among PD group, LY group and PD + LY group, in which there were significant difference [(63.3 ± 0.5)% vs (30.7 ± 20.1)% vs (40.8 ± 1.3)%; F = 621.059, P < 0.01]. (2) Compared with the control group, the increasing of transplanting tumor volume in the treated groups were obviously (F = 23.545, P < 0.01), and the inhibited rate of the tumor was higher in PD + LY group than that in PD group or LY group [(68 ± 9)% vs (32 ± 16)% or (38 ± 17)%; F = 10.283, P < 0.05]. (3) HE staining shown that there were different degrees of necrosis for endometrial carcinoma cell in different groups. The apoptosis of tumor cells were significantly increased in treated groups by TUNEL testing [(13.7 ± 1.5)%, (14.1 ± 1.2)%, (29.0 ± 1.8)%; F = 320.344, P < 0.01]. Immunohistochemistry results demonstrated that the expressions of p-ERK and p-Akt in treated groups were lower than that in control group, of which LY + PD group was the lowest one. CONCLUSION: The signal pathway inhibitors PD98059 and LY294002 could inhibit the growth of human endometrial carcinoma in vivo and in vitro, in which may induce cell apoptosis.

Homo-binding character of LMO2 isoforms and their both synergic and antagonistic functions in regulating hematopoietic-related target genes.

BACKGROUND: The human lmo2 gene plays important roles in hematopoiesis and is associated with acute T lymphocyte leukemia. The gene encodes two protein isoforms, a longer form LMO2-L and a shorter form LMO2-S. Both isoforms function as bridge molecules to assemble their partners together to regulate their target genes. A typical LMO2 binding site consists of two elements, a GATA site and an E-box, with an interval of 9 approximately 12 bp. METHODS: In this study, the combination of MBP pulldown assay and mammalian two hybrid assay were used to confirm the homo-binding character of LMO2-L/-S isoforms. Luciferase reporter assay and Real-time PCR assay were used to detect expression levels and relative promoter activities of LMO2-L/-S isoforms. Co-transfection and Luciferase reporter assay were used to reveal the detailed regulatory pattern of LMO2-L/-S isoforms on their targets. RESULTS: Herein we report the homo-interaction character of LMO2-L and LMO2-S and their major difference in manner of regulating their target genes. Our results showed that LMO2-L and LMO2-S could only bind to themselves but not each other. It was also demonstrated that LMO2-L could either positively or negatively regulate the transcription of its different target genes, depending on the arrangement and strand location of the two elements GATA site and E-box, LMO2-S, however, performed constitutively transcriptional inhibiting function on all target genes. CONCLUSION: These results suggest that LMO2 isoforms have independent functions while there is no interaction between each other and they could play synergetic or antagonistic roles precisely in regulating their different genes involved in normal and aberrant hematopoiesis.

[Effect of DNA methyltransferase 1 gene silencing on proliferation and apoptosis of HeLa cells].

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-H1 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-DNMT1-B was the better choice. While no effect of pshRNA-DNMT1-C was seen. RT-PCR results showed that the relative mRNA expression of DNMT1 gene in HeLa 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 HeLa 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 HeLa 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 apoptosis rate of HeLa cells transfected with pshRNA-DNMT1 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 HeLa cells. Downregulation of DNMT1 can inhibit cervical cancer cells proliferation and induce cell apoptosis.

[Reconstruction of adenovirus vector contained canstatin gene and hTERT gene core promoter and its anti-tumor effect in human ovarian cancer].

OBJECTIVE: To study anti-tumor effects on human ovarian cancer xenografted tumors in mice by constructing adenoviral expression vector containing canstatin gene and hTERT gene core promoter (AdhTERT-Can). METHODS: AdhTERT-Can vector was constructed and identified by means of enzyme cutting, electrophoresis and sequencing. Then, transfected into HO8910PM cells by means of lipofectamine and confirmed by green fluorescence protein (GFP) expression under laser confocus microscope. The mRNA expression of canstatin gene was tested by RT-PCR. Human ovarian cancer xenografted tumor models in nude mice were established and randomly divided into AdhTERT-Can group received viral supernatant solution of AdhTERT-Can by tail vein injection, Ad-Can groups received viral supernatant solution of Ad-Can by tail vein injection, and control groups received phosphate buffer solution (PBS). The volume of tumors were measured and compared in each group to evaluated the anti-tumor efficacy. RESULTS: All the constructed vectors of AdhTERT-Can were verified by enzymed digestion. There were green fluorescence from 60% of HO8910PM cells transfected by AdhTERT-Can under laser confocus microscope, and the mRNA expression of canstatin gene in HO8910PM cells were also verified by RT-PCR. The growth of tumor in AdhTERT-Can group was significantly inhibited compared with those in Ad-Can groups and control groups from the 8th day (P<0.01). However, there were not significant difference between Ad-Can group and PBS group (P>0.05). On the 30th day, the tumors showed liquefaction necrosis and cystic degeneration in each group, especially in AdhTERT-Can group. CONCLUSIONS: The recombinant adenovirus vector of AdhTERT-Can has been constructed successfully and could steady express in ovarian cancer cell lines HO8910PM. The results shown that it could inhibit significantly the growth of human ovarian cancer xenografted tumors in mice and shown to be the target of gene therapy.

Increased staining for phosphorylated AKT and nuclear factor-kappaB p65 and their relationship with prognosis in epithelial ovarian cancer.

AKT plays an important role in malignant behavior of tumors. The purpose of the present study was to determine the expression of phosphorylated AKT (P-AKT) and nuclear factor-kappaB (NF-kappaB) p65 and their association with clinicopathological parameters and prognosis in epithelial ovarian tumor. On immunohistochemistry 115 samples of ovarian tissue that included 68 specimens of epithelial ovarian cancer, 12 of borderline tumor, 24 of epithelial benign tumor and 11 of normal ovary, were evaluated. Sixty-three patients with ovarian cancer were followed up from 7 to 68 months. The positive expression rate of P-AKT and NF-kappaB p65 were higher in epithelial ovarian cancer than in normal ovarian tissue (P<0.01). Elevated P-AKT or NF-kappaB p65 expression was significantly correlated with late clinical stage (P<0.05 and P<0.01) and poor histological differentiation (both P<0.01). P-AKT expression was significantly correlated with NF-kappaB p65 immunostaining (phi=0.272, P<0.05). Elevated expression of P-AKT was negatively correlated with the survival of ovarian cancer patients, but it was not an independent prognostic factor after multivariate analysis. Overexpression of P-AKT and NF-kappaB p65 were involved in the carcinogenesis and metastasis of ovarian cancer. P-AKT might contribute to the malignant transformation through NF-kappaBp65 upregulation.

[Promoter methylation of DAPK gene in cervical carcinoma].

BACKGROUND & OBJECTIVE: Aberrant DNA methylation plays important roles during multistage carcinogenesis in various human organs. This study was to explore the relationship between the promoter methylation and inactivation of DAPK gene in cervical cancer. METHODS: The promoter methylation of DAPK was investigated with methylation-specific polymerase chain reaction (MSP) in 52 specimens of cervical cancer, 60 specimens of cervical intraepithelial neoplasia (CIN) and 20 specimens of normal cervical squamous epithelial tissues. Its correlation to clinicopathologic features of cervical cancer was analyzed. The protein expression of DAPK was detected by immunohistochemistry. RESULTS: The methylation rate of DAPK gene promoter was significantly higher in cervical cancer tissues than in CIN (65.4% vs. 18.3%, P<0.05); while no methylation of DAPK gene was found in normal cervical tissues. The methylation rate of DAPK gene was significantly higher in cervical squamous cell carcinomas than in adenocarcinomas (80.0% vs. 16.7%, P<0.001). Promoter methylation of DAPK was negatively correlated to its protein expression (r=-0.849, P<0.001). CONCLUSION: The promoter methylation may lead to inactivation of DAPK gene, and may be related with tumorigenesis of cervical cancer.

[Correlation of DPPIV expression with clinicopathological features and prognosis in epithelial ovarian carcinoma].

OBJECTIVE: To investigate the expression of dipeptidyl peptidase IV (DPPIV) in patients with epithelial ovarian carcinoma (EOC) and its clinical significance. METHODS: Immunohistochemistry (IHC) was used to detect the expression of DPPIV protein in 378 formalin-fixed paraffin-embedded EOC tissue samples. The expression of DPPIV mRNA in 86 EOC tissue samples were examined by in situ hybridization (ISH) using specific FITC-labelled RNA probes. Forty-two samples of normal ovarian tissues were used as control. Statistical analyses were carried out by Chi-square test, Spearman rank correlation and Kaplan-Meier method. RESULTS: Among the 378 epithelial ovarian carcinomas, 351 (92.9%) showed a positive expression of DPPIV protein, while only 25/42 (59.5%) of normal ovaries had a positive expression by semi-quantitative IHC analysis. The expression level of DPPIV protein was significantly lower in the normal ovaries than that in ovarian carcinomas (chi(2) = 18.4, P = 0.001). There was no significant correlation between the expression of DPPIV protein and age, FIGO stage and histological grade (P > 0.05). However, the expression of DPPIV protein was significantly associated with histological type (chi(2) = 28.5, P = 0.005). The patients with high level expression of DPPIV protein likely had a poor prognosis in terms of overall survival (P = 0.02). Of the 86 patients, 84 (97.7%)showed positive expression of DPPIV mRNA, also higher than that in normal ovarian tissues (P < 0.05). A statistically significant correlation between DPPIV mRNA and protein expression was observed (r(s) = 0.66, P = 0.001). CONCLUSION: DPPIV may be involved in the carcinogenesis of ovarian cancer, and may become a potential prognostic marker for epithelial ovarian carcinoma.

[Effect of estradiol supplementation during the luteal phase on mouse endometrial expression of leukaemia inhibitory factor and pinopodes in controlled ovarian stimulation cycles].

OBJECTIVE: To study the effect of estradiol supplementation during the luteal phase on mouse endometrial expression of leukaemia inhibitory factor and pinopodes in controlled ovarian stimulation cycles. METHODS: Female mice were randomly divided into four groups: group A [controlled ovarian stimulation (COS) group], group B (COS group with progesterone for luteal-phase-support), group C (COS group with progesterone and estradiol for luteal-phase-support), and group D of natural cycle group. Pinopodes were investigated by scanning electronic microscopy (SEM) in the uterine endometrium of pregnant mice on pregnancy days (pd) 3 - 5. Leukaemia inhibitory factor (LIF) protein was determined by immunohistochemistry in the uterine endometrium of pregnant mice on pd 3 - 5. RESULTS: (1) In groups B, C, and D, there were small developed pinopodes in the endometrial surface of pregnant mouse on day 3; there were large fully developed pinopodes in endometrial surface, which was smooth with well defined borders resembling a mushroom on day 4. The regressing pinopodes were observed on day 5. In group A, there were small developed pinopodes in endometrial surface of pregnant mouse on day 3. The regressing pinopodes were seen on day 4. (2) In the pregnant mice of groups C and D, the level of LIF protein on days 3 - 5 (138.5 +/- 20.3, 143.1 +/- 19.0) was significantly higher than group A (103.2 +/- 5.0, P < 0.05), and strong immunostaining of LIF protein was found on day 4 of gestation. In group B, the level of LIF protein on days 3 - 5 (123.5 +/- 10.8) was significantly higher than group A (P < 0.05), but significantly lower than groups C and D (P < 0.05). Strong immunostaining of LIF protein was found on day 4 of gestation. In group A, weak immunostaining of LIF protein peaked on day 3 of gestation. In groups B, C, and D, the level of LIF protein on day 4 was significantly higher than group A on day 3 (F = 55.76, P < 0.01). CONCLUSIONS: Estradiol supplementation during the luteal phase can improve the expression of LIF and pinopodes in mouse endometrium in controlled ovarian stimulation cycles and redress the harmful effect on implantation window by COS. Therefore, estradiol supplementation can improve the endometrial receptivity.

[Expression and significance of Edg4 and Edg7 in the placentas of patients with hypertensive disorder complicating pregnancy].

OBJECTIVE: To study Edg4 and Edg7 expression in placenta of women with hypertensive disorder complicating pregnancy, and to investigate the relation between the expression of lysophosphatidic acid (LPA) and hypertensive disorder complicating pregnancy. METHODS: Immunohistochemical SP method was used to measure the expressions of Edg4 and Edg7 in placenta of women with normal pregnancy, 20 women with gestational hypertension, 20 with mild preeclampsia, and with severe preeclampsia. RESULTS: (1) LOCATION: immunohistochemical staining for Edg4 and Edg7 protein were located at the membrane and endochylema of cytotrophoblast as well as decidua cells. (2) The positive expression of Edg4 protein and Edg7 protein on membrane and endochylema of cytotrophoblast was 25% and 20% (normal women), 60% and 40% (gestational hypertension), 80% and 65% (mild preeclampsia), and 83.3% and 86.7% (severe preeclampsia). The expression of Edg4 and Edg7 protein in mild preeclampsia and severe preeclampsia was significantly correlated with the degree of differentiation (P < 0.05). The expression of Edg4 and Edg7 protein showed an insignificant difference in normal pregnant women and gestational hypertension (P > 0.05). (3) The positive expression of Edg4 protein and Edg7 protein on membrane and endochylema of decidua was 20% and 25% (normal pregnancy), 55% and 50% (gestational hypertension), 70% and 55% (mild preeclampsia), and 83.3% and 73.3% (severe preeclampsia) respectively. The expression of Edg4 and Edg7 protein in mild preeclampsia and severe preeclampsia showed a significant correlation with the degree of differentiation (P < 0.05). The expression of Edg4 and Edg7 protein showed an insignificant difference in normal pregnancy and gestational hypertension (P > 0.05). CONCLUSIONS: The high expression of Edg4 and Edg7 protein in the placentas of patients with hypertensive disorder complicating pregnancy indicates that LPA combines with Edg4 and Edg7, inducing the occurrence of hypertensive disorder complicating pregnancy.

[Expression of phosphorylated protein kinase B and PTEN protein in ovarian epithelial cancer].

OBJECTIVE: To analyze the expression of phosphorylated protein kinase B (pAKT) and PTEN protein in ovarian epithelial cancer and to investigate the correlations between their expression and prognosis of ovarian epithelial cancers. METHODS: Expression of pAKT and PTEN in 12 normal ovarian tissues, 20 benign tumors, 12 borderline tumors and 80 cases of ovarian epithelial cancers were detected by immunohistochemical method, and their correlations were analyzed. RESULTS: The positive expression of pAKT in normal ovarian and benign tumor tissues were significantly lower than that in ovarian epithelial cancers (8%, 10% vs 55%; P < 0.01), respectively. However, loss of PTEN expression in ovarian epithelial cancers was significantly higher than that in normal ovarian and benign tumor tissues, and the positive rate of PTEN expression were respectively, 45%, 100%, and 80% (P < 0.01). The expression of pAKT and PTEN were correlated with clinical stages, differentiation degree of cancer cells, and metastasis (including lymph node; P < 0.05). But there was no difference with regard to age, histological type and ascites. In ovarian cancers, a negative correlation between expression of pAKT and PTEN was observed (r = -0.444, P < 0.01). A univariate analysis revealed that clinical stage, differentiation degree, lymph node involvement, distant metastasis, pAKT and PTEN expression were correlative factors with prognosis (P < 0.05). Multivariate Cox analysis showed that PTEN and clinical stage were the independent risk factors of prognosis (P < 0.05). CONCLUSIONS: The overexpression of pAKT and absence of PTEN are related to ovarian carcinogenesis and development. Loss of PTEN expression is the independent risk factor of poor prognosis in patients with ovarian epithelial cancers.

Expression of seprase in effusions from patients with epithelial ovarian carcinoma.

BACKGROUND: Seprase plays an important role in malignant cell invasion and metastasis by degrading the extracellular matrix. However, its clinical significance remains largely unknown. The objective of the current study was to evaluate the expression of seprase in effusions from patients with epithelial ovarian carcinoma and its clinical values. METHODS: Immunohistochemistry was used to examine the expression of seprase protein in a series of 74 malignant peritoneal (n = 64) and pleural (n = 10) effusions from Norwegian patients with epithelial ovarian carcinoma. Additionally, 34 effusions were evaluated using the Western blotting. Nine reactive effusions, obtained from patients with benign lesions, served as a control group. Statistical analyses were carried out by Chi-square test and Kaplan-Meier method. RESULTS: In the 74 malignant effusion specimens, 57 (77.02%) were positive for seprase, while only 2 (22.22%) of the control group were positively stained (P = 0.001). In the malignant effusions, 17 (22.97%), 22 (29.73%), 22 (29.73%), 13 (17.57%) had negative, weak, moderate and strong seprase protein expression, respectively. The expression of seprase protein was predominant in cytoplasm of carcinoma cells. Increased seprase protein was negatively associated with the overall survival rate of the patients (P = 0.03). However, there was no significant correlation between protein expression and FIGO stage, age, histology, and histological grade. By Western blotting, 27 of the 34 effusions showed the presence of both 170-kD dimeric form and 97-KD monomeric form of seprase while only 1 of the 34 had 170-KD dimeric form, which was consistent with the results of immunohistochemistry (P = 0.05). CONCLUSIONS: Seprase may be involved in the development of ovarian cancer, and is a potential predictive marker for the disease.

[Vector construction and silencing effect of Edg4 gene targeted small interfering RNA in ovarian cancer cell line].

OBJECTIVE: To construct the recombinant eukaryotic expression vector pRNAT-U6.1-siEdg4 which carries small interfering RNA (siRNA) of Edg4 and observe the silencing effect of Edg4 gene targeted siRNA in ovarian cancer cell line SKOV3. METHODS: The Edg4 gene-targeted hairpin siRNA sequence was designed according to the Edg4 sequence in Genbank, and the two complementary oligo nucleotide strands were synthesized and annealed and inserted into the pRNAT-U6.1 plasmid to build a recombinant Edg4 siRNA eukaryotic expression vector, which was sequenced and identified to contain the correct Edg4 siRNA sequence. The human ovarian carcinoma cell lines SKOV3 were transfected with the vector using lipofectamine method. The efficiency of transfecting cells was observed with fluorescent microscope and the mRNA expression level of Edg4 gene was detected by real time quantitative PCR. The LPA levels in cell supernatants were detected using a biochemical method. And the apoptosis of SKOV3 cells induced by the vector was evaluated by flow cytometry. RESULTS: The recombinant eukaryotic expression vector was confirmed to contain correct Edg4 siRNA sequence by PCR and sequencing. After transfection large amounts of green fluorescence were seen in plasma and nuclei of SKOV3 cells and the positive cell rates were 64%. The expression level of Edg4 mRNA in transfected SKOV3 cell line was significantly decreased (0.05 +/- 0.01vs 0.29 +/- 0.04, P < 0.05). The decrease in LPA level in the cell supernatants was revealed [(3.0 +/- 1.0) vs (7.5 +/- 2.2)micromol/L, P < 0.05]. The apoptosis rate of transfected SKOV3 was increased obviously (53.38% vs 0.51%, P < 0.05). CONCLUSIONS: We have successfully constructed the recombinant eukaryotic expression vector containing Edg4 gene targeted siRNA (pRNAT-U6.1-siEdg4). The vector could effectively transfect SKOV3 cell line, and obviously suppress the Edg4 mRNA expression and induce cell apoptosis in ovarian cancer cell line SKOV3.

[Expressions of estrogen receptor subtypes in epithelial ovarian carcinomas].

OBJECTIVE: To investigate the expressions of estrogen receptor(ER) subtypes ERa and ERP in epithelial ovarian carcinomas. METHODS: One hundred and eighteen Norwegian patients with epithelial ovarian carcinoma were included in this study. The expressions of ERalpha and ERbeta were examined by means of immunohistochemistry. The relationships between protein expressions and clinicopathological features and survival were analyzed. Frozen tissues from 10 cases in which the tumors showed variable ERalpha and ERbeta protein expressions were used for Laser capture microdissection (LCM). Cancer cells in each frozen section were captured with the LCM method and processed for Western blot analysis. RESULTS: Of the 118 tumours, 32 (27.1%), 20 (16.9%), 17 (14.4%), 49 (41.5%) demonstrated negative, weak, moderate and strong expression of ERalpha protein, respectively; 1 (0.8%), 7 (5.9%), 13 (11%), 97 (82.2%) showed negative, weak, moderate and strong expressions of ERbeta protein, respectively. There was no significant association between ERalpha expression and the clinicopathological features such as age, histological type, FIGO stage, histological grade and residual tumour size. ERbeta expression was not associated with age, histological type,FIGO stage and residual tumour size, but it was significantly associated with higher histological grade. In addition, Kaplan-meier analysis revealed that high levels of ERbeta expression was significantly associated with a shorter overall survival (P = 0.03). CONCLUSION: There are ERalpha and ERbeta expressions in epithelial ovarian carcinomas. Higher level of ERbeta protein expression is associated with higher histological grade and poorer clinical outcome in the cases of ovarian carcinoma. ERbeta may be a useful marker of ovarian carcinogenesis and could predict the efficacy of endocrinotherapy and the prognosis for patients with ovarian carcinoma.