APOBEC3 deletion polymorphism is associated with epithelial ovarian cancer risk among Chinese women.

Ovarian cancer remains the leading cause of death from gynecological malignancies and the second most common gynecological malignancy among women worldwide. However, the etiology still remains largely unknown. Previous studies identified APOBEC3 gene deletions were significantly associated with higher breast cancer risk in both European and Chinese women. Considering both breast and ovarian cancers being hormonally driven and sharing multiple risk factors, we performed this case-control study to evaluate the association between APOBEC3 deletion and epithelial ovarian cancer (EOC) risk. We analyzed the APOBEC3 deletion in a case-control study of 2,938 Chinese women (including 1,374 EOC cases and 1,564 healthy controls). All participants were genotyped using real-time qualitative PCR (qPCR). APOBEC3 deletion was significant associated with EOC risk, with ORs (95 % CIs) of 1.46 (1.14-1.86) associated with one copy deletion and 2.53 (0.91-7.06) associated with two copy deletion compared with subjects with no deletion (P for trend =1.50 × 10(-3)). Additional adjustments and stratified analyses did not change the results materially. Our data suggests that the loss genotypes of APOBEC3 deletion predispose their carriers to EOC.

The involvement of osteopontin and ß3 integrin in implantation and endometrial receptivity in an early mouse pregnancy model.

OBJECTIVE: To explore the roles of osteopontin and ß3 integrin in successful implantation. STUDY DESIGN: In this study, an early pregnant mouse model was established by peritoneal injection of pregnant mare serum gonadotropin and human chorionic gonadotropin (PMSG+hCG). The expression of osteopontin (OPN) and ß3 integrin on the endometrium was measured by immunohistochemistry, RT-PCR, and western blot. The function of OPN and ß3 integrin in implantation was investigated by intrauterine injection of OPN and ß3 integrin antibody. RESULTS: We found that PMSG+hCG injection significantly increased the number of blastocysts during implantation as well as the concentration of estradiol and progesterone in serum and endometrium tissues. OPN and ß3 integrin were co-expressed in luminal epithelium and their levels dynamically changed from day 4 to day 8 of pregnancy with peak expression on day 5. The percentages of OPN and ß3 integrin positive cells in the luminal epithelium were significantly higher in PMSG+hCG-stimulated mice on day 5 than in control mice. Functional blockade of OPN and ß3 integrin significantly inhibited implantation. CONCLUSIONS: This study suggests that co-expression of OPN and ß3 integrin is a biological marker for good endometrial receptivity and that both proteins play a crucial role in blastocyst implantation.

Reversal of paclitaxel resistance in epithelial ovarian carcinoma cells by a MUC1 aptamer-let-7i chimera.

The purpose of this study was to establish tumor tissue specific delivery of let-7i miRNA to reverse paclitaxel-induced chemoresistance. A chimera that combines MUC1 aptamer and let-7i miRNA was tested in OVCAR-3 ovarian cancer cells. Results demonstrated that the chimera can specifically be delivered into OVCAR-3 cells and the released let-7i significantly sensitized the role of paclitaxel in inhibiting cell proliferation, inducing cell apoptosis, and decreasing long-term cell survival. The chimera achieved reversal of chemoresistance through downregulation of cyclin D1, cyclin D2, Dicer 1, and PGRMC1 expressions. Our study indicated that this MUC1/let-7i chimera can specifically reverse chemoresistance to paclitaxel.

[Effects of calponin-1 gene silencing on the biological behavior of uterine smooth muscle cells].

OBJECTIVE: To study the effects of calponin-1 expression inhibition on the proliferation , invasiveness, apoptosis and cytoskeleton of uterine smooth muscle cells, and explore the molecular mechanism of calponin-1 in the uterine smooth muscle cells for labor onset. METHODS: siRNA-calponin-1 adenovirus plasmid was constructed and transfected into primarily cultured uterine smooth muscle cells. The proliferation, invasiveness and apoptosis of the cells were determined by MTT assay, matrigel invasion assays and flow cytometry, respectively. Rhodamine-Phalloidin was used for labeling filamentous actin (F-actin), and the morphology and the distribution of F-actin was observed under fluorescence microscopy and analyzed quantitatively. RESULTS: The motor ability of uterine smooth muscle cells decreased significantly after transfection with siRNA-calponin-1 adenovirus plasmid (P<0.05). The transfected cells showed thinner, loosened and irregular F-actin microfibers, and the cells in the empty vector and blank control groups showed thicker and longer F-actin microfibers. CONCLUSION: Inhibition of calponin-1 expression can inhibit uterine smooth muscle cell migration and cause the morphological change and rearrangement of F-actin without affecting its proliferation and apoptosis in vitro, suggesting that the morphological change and rearrangement of F-actin of uterine smooth muscle cell may be one of the important mechanisms in the labor onset.

[Effect of HPV16E6 on sensitivity of chemotherapy for cervical carcinoma in different p53 genotype cell lines].

OBJECTIVE: To investigate the effect of human papillomavirus types 16E6 on the sensitivity of chemotherapy for cervical carcinoma in different p53 genotype cell lines. METHODS: The apoptosis rates of each group were detected by AO/EB, immunofluorescence and Annexin V/PI stained methods. The expressions of protein HPV16E6 and p53(mt) after the treatments of different concentration of DDP were detected by Western blot. HPV16E6 mRNA in C33A, C33A-E6, C33A-P, and CaSki cell lines under different DDP treatments were detected by RT-PCR. RESULTS: AO/EB and Annexin V/PI stained tests showed that the apoptosis rates of C33A, C33A-E6, C33A-P, and CaSki cells were significantly increased when DDP concentration increased. Western blot showed that the HPV16E6 protein could be detected only in C33A-E6 and CaSki cell lines. The expression of HPV16E6 protein in C33A-E6 and CaSki cell lines gradually decreased and was hardly detected with increased dosage of DDP and the prolonged treatment time (P<0.01), and slightly increased in C33A-E6 and Caski cell lines without the treatment, but there was no significant difference between them (P>0.05). Protein p53(mt) persistently expressed in C33A-E6, C33A, and C33A-P cell lines following the increased dosage of DDP and the prolonged treatment time(P>0.05), while it couldn't be found in CaSki cell line. RT-PCR showed that without DDP intervention, there was no significant difference of HPV16E6 mRNA in C33A-E6 and CaSki cell lines within 24 h.The HPV16E6 mRNA in C33A-E6 cell line expressed much higher than that in CaSki (P<0.05), and HPV16E6 mRNA of 2 cell lines expressed much higher at 48 h than at 24 h (P<0.05).The expression of HPV16E6 mRNA in C33A-E6 and CaSki cell lines gradually decreased with the increased DDP and prolonged treatment time (P<0.01), while there was no significant difference between C33A-E6 and CaSki cell lines under the same DDP concentration (P>0.05). CONCLUSION: Effect of HPV16E6 on the sensitivity of chemotherapy for cervical carcinoma cell lines is not markedly related with the different p53 genotype forms(p53(mt)/p53wt ). HPV16E6 may affect the proliferation and sensitivity of chemotherapy in C33A cell line through other mechanism.

[Expression of aspartyl-(asparaginyl) beta-hydroxylase in villi in patients with missed abortion].

OBJECTIVE: To determine the difference in aspartyl-(asparaginyl) beta-hydroxylase (AAH) expression level in villi between patients with missed abortion and normal women with early pregnancy, and to confirm the expression loci of AAH in villi. METHODS: A total of 50 patients of missed abortion were collected and categorized into a test group, which was subdivided into Group 1 and Group 2. Patients in Group 1 (n=20) were of confirmed etiological disorders while those in Group 2 (n=30) showed no obviously etiological clues. In addition, 20 women of early pregnancy with artificial abortion were categorized into a control group, whose embryos were sonographically confirmed alive before surgery. The 50 patients of missed abortion were also subdivided into a group within 4 weeks and a group over 4 weeks according to the time that the embryo stayed in utrine after death. Immunohistochemical technique and computer image analysis were used to detect the expression loci and the level of AAH in villi. RESULTS: AAH was expressed in the endochylema and nucleus of trephocyte both in missed abortion and normal early pregnancy. The expression level of AAH in villi of missed abortion was much lower than that of in villi of normal early pregnancy (P<0.05). The expression level had no difference between different groups of patients with missed abortion(P>0.05). CONCLUSION: Low expression of AAH in the endochylema and nucleus of trephocyte may play a role in patients with missed abortion.

[Construction of adenoviral vector encoding Calponin-1 siRNA and its effect on human myometrium cells in vitro].

OBJECTIVE: To investigate the effect of Calponin-1 suppression on human myometrium cells through adenovirus mediated siRNA. METHODS: Human uterine smooth muscle tissues were digested with enzymes, cultured and confirmed with immunocytochemistry. Adenovirus siRNA-Calponin-1 plasmid was transfected into primary cultured uterine smooth muscle cells in vitro. The expressions of Calponin-1 mRNA and protein were analyzed by RT-PCR and Western blot, respectively. RESULTS: The pAdEasy-pShuttle-U6-Calponin-1 siRNA plasmid was successfully constructed, and Calponin-1 siRNA mediated by recombinant adenovirus resulted in markedly reduced expression of Calponin-1 mRNA and protein in human myometrium cells. The gray values of Calponin-1 mRNA in the uterine smooth muscle cells in the experimental, blank control, and empty vector groups were 316.3+/-39.2, 1048.5+/-126.4 and 1027.2+/-127.5, respectively. The gray values of Calponin-1 protein were 323.3+/-43.2, 1021.5+/-143.4, and 1019.2+/-144.5, respectively. The difference between the experimental group and the blank control group as well as the empty vector group was significant (P< 0.05). There was no significant difference between the empty vector group and the blank control group (P>0.05). CONCLUSION: The pAdEasy-pShuttle-U6-Calponin-1 siRNA plasmid can inhibit the expression of Calponin-1 in human myometrium cells in vitro, which may be a useful approach to determine the role of Calponin-1 in delivery.

[Study of the role of nuclear factor-kappa B in preterm birth with subclinical chorioamnionitis].

OBJECTIVE: To investigate the role of nuclear factor-kappa B (NF-kappaB) in preterm birth with subclinical chorioamnionitis. METHODS: From October 2005 to October 2006, 111 cases including 36 cases of preterm birth in labor, 37 cases of full term gravida with spontaneous labor and 38 cases of full term gravida without threatened labor in the Hunan Province People's Hospital, third Xiangya Hospital of Central South University and Changsha Maternal and Child Care Service Center were enrolled in the study. After delivery, by pathology results of fetal membrane they were divided into two groups: subclinical chorioamnionitis group (subclinical infectious group) and non-infectious group. Immunohistochemical staining and RT-PCR were used to observe the change of the p65 subunit of NF-kappaB family in maternal blood and fetal membrane in subclinical infectious group and non-infectious group. RESULTS: (1) The incidence of subclinical chorioamnionitis: there were 24 cases of subclinical chorioamnionitis in the 36 cases of preterm birth in labor (67%), 7 cases in the 37 cases of full term gravida with spontaneous labor group (19%) and 3 cases in the 38 cases of full term gravida without threatened labor group (8%). There was a significant difference among the three groups (P < 0.01). In the totally 111 cases, 34 cases were classified as subclinical infectious group and 77 cases as non-infectious group. (2) In fetal membrane, the median of the average staining intensity of NF-kappaB p65 protein was higher in the subclinical chorioamnionitis group (8.0) than those in non-infectious group (4.0). Similarly, the average staining intensity of NF-kappaB p65 mRNA was higher in the subclinical infectious group (47.5 +/- 17.2) than those in non-infectious group (31.3 +/- 13.6). There was a significant difference between the two groups (P < 0.01). (3) In maternal blood, the expression of NF-kappaB p65 protein and mRNA was higher in subclinical chorioamnionitis group(8.0 and 42.6)than those in non-infectious group(4.0 and 23.6).There was a significant difference between the two groups (P < 0.01). (4) The concentration of NF-kappaB p65 protein in fetal membrane was positively correlated with that of maternal blood (r = 0.581, P < 0.01) and the concentration of NF-kappaB p65 mRNA in fetal membrane was positively correlated with that of maternal blood (r = 0.571, P < 0.01). CONCLUSION: The expression of NF-kappaB in maternal blood and fetal membrane in preterm birth with subclinical chorioamnionitis is higher and the two are correlated with each other. NF-kappaB p65 could be an accurate biochemical marker in predicting subclinical chorioamnionitis in preterm birth. NF-kappaB p65 plays an important role in the pathogenesis of subclinical chorioamnionitis in preterm birth.

[Killing activity of cytotoxic T lymphocytes stimulated by dendritic cell vaccine loaded with autologous cervical cancer antigen].

BACKGROUND & OBJECTIVE: Dendritic cells (DCs), the strongest antigen-presenting cells (APCs), can present antigens to T lymphocytes in vivo and in vitro, and induce cytotoxic T lymphocyte (CTL) reaction. This study was designed to investigate the killing activity of CTLs stimulated by Dcs loaded with autologous cervical cancer antigen in vitro. METHODS: Tumor antigens were made from frozen-thawed cervical cancer cells from patients after operation. DCs were isolated from peripheral blood mononuclear cells of patients with cervical cancer, cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), loaded with tumor antigen to prepare DC vaccine, and used to stimulate autologous T lymphocytes to prepare antigen-specific CTLs. The killing activities of CTLs on autologous cervical cancer cells and HeLa, HepG2, MCF7, A549, and MGC803 cells were observed. RESULTS: CTLs stimulated by the DC vaccine had high killing activity on autologous cervical cancer cells, with killing rates of 79.32%-89.27% which were obviously higher than that of lymphokine-activated killing cells (t> or =2.89, P<0.05). The killing activity of CTLs was significantly weaker on HeLa cells (40.35%-58.09%) than on autologous cervical cancer cells (t> or =2.97, P<0.05). The specific CTLs had no obvious killing activity on HepG2, MCF7, A549, and MGC803 cells. CONCLUSIONS: CTLs stimulated by autologous cervical cancer antigen-loaded DCs have highly efficient and specific immune activity on autologous cervical cancer cells. It may be used in biotherapy for cervical cancer.