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Objective To explore the effects of cisplatin on the proliferation and autophagy of endometrial carcinoma Ishikawa cells.Methods Ishikawa cell proliferation was detected by MTS assay after the cells were treated with CDDP.To assess the level of autophagy,transmission electron microscope and Western blot were used to detect LC3 and Beclin1 expression;fluorescence microscopy was used to observe the fluorescence aggregation of green fluorescent protein and microtubule associated protein 1 light chain 3 fusionprotein (GFP-LC3).Results Cisplatin of 10 g/mL inhibited the proliferation of Ishikawa cells,with an increase of time and concentration,the inhibition of cell proliferation was significantly elevated (P ＜ 0.01).Transmission electron microscopy showed that under a condition of cisplatin on Ishikawa endometrial cancer cell,autophagy occurred.With an increase of concentration and dosage,Western blot showed that autophagy related protein LC3 expression was up-regulated,but becline-1 had no obvious change.LC3 expression level was higher in 12h-treatment with 20 μg/mL cisplatin group than in the control group,and was higher in 24h-treatment group than in 12h-treatment group.Conclusions Cisplatin inhibits proliferation of Ishikawa cells and induces autophagy of the cells in a time-and dose-dependent manner.Autophagy related MAP-LC3 is involved in the molecular mechanisms of autophagy induced by cisplatin in endometrial carciHom.
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Objective To evaluate the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) in ovarian cancer cell lines, and to investigate the biological effects of down-regulated MALAT-1 on OVCAR3 cells.Methods qRT-PCR analysis was used to examine the expression level of MALAT-1 gene in ovarian cancer cells, including ES-2, A2780, SKOV3 and OVCAR3 cell lines.For functional research, four shRNA oligos specially targeting MALAT-1 and a empty vector were designed and constructed into pGPU6/GFP/Neo, then transfected into OVCAR3 cells.qRT-PCR was used to confirm the effective suppression of MALAT-1.Changes of proliferation and adhesion of cells were analyzed by CCK-8 and adhesion assays.Wound-healing, transwell migration and invasion assays were used to examine migration and invasion of MALAT-l-silencing cells in vitro.Results The expression of MALAT-1 gene in OVCAR3 cells was high, and qRT-PCR results confirmed successfully the knockdown of MALAT-1 after transient transfection.After successful suppression of MALAT-1, the proliferation, wound-healing and adhesion ability in vitro were inhibited to some degree.In transwell migration assay, the number of migration cells in MALAT-1-silencing group was 52.17±4.48, which is much less than that in the negative and control groups (286.50± 12.23 and 295.67±6.96, respectively).In invasion assay, the number of invasion cells passing the transwell membrane in MALAT-1-silencing group (37.33±2.40) was also decreased significantly, compared to that in the negative and control groups (239.00±15.72 and 222.67±20.85, P ＜ 0.05).Conclusions shRNA-mediated silence of MALAT-1 can effectively inhibit the proliferation, adhesion, migration and invasion abilities of ovarian cancer cell line OVCAR3 in vitro, indicating MALAT-1 is expected to be a target gene for the treatment of ovarian cancer.
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<p><b>OBJECTIVE</b>To investigate the reversal effects of different concentrations of DNA methylation inhibitor, 5-aza-2-deoxycytidine, on the hypermethylation of maternally expressed gene 3 (MEG3) gene promoter, and then the inhibitory effect of restoration of MEG3 expression on the proliferation of ovarian cancer cells.</p><p><b>METHODS</b>Human ovarian cancer OVCAR3 cells were treated with various concentration of 5-aza-2-deoxycytidine (0, 1, 5, 10, 20 µmol/L, respectively) for 6 days. Then the methylation status of MEG3 promoter was detected by methylation specific PCR (MSP). The alteration of MEG3 gene expression was detected by RT-PCR. Cell proliferation was determined by MTT assay and EdU incorporation assay.</p><p><b>RESULTS</b>After treated with 5-aza-2-deoxycytidine, the methylation status of MEG3 in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 0.79 ± 0.00, 0.67 ± 0.00, 0.65 ± 0.03 and 0.61 ± 0.01 folds, respectively (P < 0.05 for all). The relative expressions of MEG3 mRNA in the 0, 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine groups were 1.00 ± 0.00, 2.04 ± 0.16, 2.44 ± 0.17, 3.19 ± 0.34 and 5.34 ± 0.39, respectively (P < 0.05 for all). In contrast to the negative control, the inhibition rates of the OVCAR3 cell growth were increased significantly when treated with 1, 5, 10, 20 µmol/L 5-aza-2-deoxycytidine in 2, 4 and 6 days. There were (40.78 ± 0.80)%, (35.65 ± 0.33)%, (31.81 ± 0.66)%, (27.33 ± 1.27)% and (17.75 ± 1.85)% of EdU-positive cells in the 0, 1, 5, 10 and 20 µmol/L 5-aza-2-deoxycytidine groups (P < 0.01 for all).</p><p><b>CONCLUSIONS</b>Maternally expressed gene 3 promoter hypermethylation is reversed by 5-aza-2-deoxycytidine in ovarian cancer cells. The downregulation of MEG3 gene might be resulted from the methylation, and the re-expression of MEG3 partly contribute to the growth inhibition of epithelial ovarian cancer cells.</p>
Subject(s)
Female , Humans , Antimetabolites, Antineoplastic , Pharmacology , Azacitidine , Pharmacology , Cell Cycle , Cell Line, Tumor , Cell Proliferation , DNA Methylation , Neoplasms, Glandular and Epithelial , Ovarian Neoplasms , Promoter Regions, Genetic , RNA, MessengerABSTRACT
Objective To explore the relationship between pregnancy associated plasma protein A (PAPP-A) and threatened abortion,whether low level of PAPP-A in early pregnancy can be used as indicators to predict the pregnancy outcomes.Methods The level of serum PAPP-A was measured by double antibody sandwich enzyme-linked immunosorbent assay in 110 cases with threatened abortion (threatened abortion group) and 131 cases with normal intrauterine pregnancy (control group),and track to 20 weeks pregnant.Results The serum PAPP-A multiple of median (MOM) value was 1.02 ± 0.15 in control group,0.98 ± 0.17 in threatened abortion group,and there was no significant difference(P ＞ 0.05).In threatened abortion group,90 cases of spuc success,the serum PAPP-A MOM value was 1.03 ± 0.11,20 patients of spuc failure,the serum PAPP-A MOM value was 0.73±0.21,and there was significant difference (P ＜ 0.01).There was no significant difference in the serum PAPP-A MOM value between control group and the spuc success of threatened abortion group (P ＞ 0.05).Conclusion Pregnant women with PAPP-A levels embryonic development is closely related to the good,as one can predict miscarriage in pregnant women with threatened abortion outcome evaluation.
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Objective To examine the expression of metastasis-associated in colon cancer 1 (MACC1) gene in ovarian cancer cell lines and investigate its effect on biological behaviors of ovarian cancer cells.Methods The expression of MACC1 was examined by qRT-PCR and Western blot analysis in four ovarian cancer cell lines inculding OVCAR3,ES-2,SKOV3 and HO-8910.When the MACC1 was transfected to OVCAR3 cells,fluorogenic quantitative PCR was used to filter and identify MACC1 gene after the efficient silencing.Changes of adhesion in the cells were analyzed by an adhesion assay.Transwell migration and invasion assay and in vitro vascular mimicry assay were used to detect migration,invasion and angiogenesis of OVCAR3 cells in vitro.Results The expression of MACC1 gene was higher in OVCAR3 compared to other cell lines.qRT-PCR confirmed that the expression of MACC1 was silenced successfully after transient transfected MACC1-siRNA into OVCAR3 cells.After successful silencing the MACC1 expression,the adhesion ability was inhibited to some degree.In transwell migration assay,the numbers of cells in upper chamber passing through the membrane in transfected group were less than control groups (245.5 ±12.8,500.3±16.5 and 496.3±13.1 respectively),while in transwell invasion assay,the numbers of cells in upper chamber passing through the membrane in transfected group were less than the negative group and control group (185.3±14.1,405.7±9.1 and 416.3±11.5 respectively),both with markedly differences among the three groups.In tube formation assay,the distrubition of HUVECs was diffused with less junctions,and the average number of complete tubular structure was decreased in transfected group compared to the corresponding controls.Conclusion RNA interference inhibits the expression of MACC1 and effectively inhibits the metastasis and invasion abilities of ovarian cancer cells in vitro,and MACC1 is expected to become the target gene of ovarian cancer treatment.
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Objective To study the relationship between the methylation status of CPG islands in MEG3 gene promoter region of epithelial ovarian cancer and its clinical and pathological features. Methods The promoter methylation status was evaluated by MSP (methylation-specific polymerase chain reaction ) in 47 cases of ovarian cancer tissue and 15 cases of normal control. Results The methylation ratio (42.6%) of the MEG3 genes in the ovarian cancer was statistically significantly higher (P = 0.035 ) than that (13.3%) in the normal control. The methyation rate of the group with an age > 60 years old was slightly higher than that of the group with an age≤60 years old, without statistically significant (P > 0.05), so was observed in ovarian cancers of stage Ⅰ andⅡ than that in stage Ⅲ and Ⅳ. There were also no significant differences in MEG3 gene methylation positive rate neither in different pathological grading nor in various ovarian cancer tissues (P > 0.05). Conclusion Abnormal methylation in MEG3 gene may be associated with epithelial ovarian cancer , but no relation to its clinical pathology.
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Minichromosome maintenance proteins (MCMs) are the primary control factors for eukaryotes DNA replication.MCMs play important roles in the starting place and the extending process of DNA replication.MCM2,one member of MCMs,expresses little in stationary phase while highly in proliferative and transformational phase.MCM2 can accurately reflect the cell proliferation activity and is considered as a specific maker for carcinoma and precancerous lesions.The overexpression of MCM2 is closely correlated with the genesis and development of tumors,and it maybe a good maker for early screening and prognosis assessment in many cancers and used in clinical.
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Metastasis-associated in colon cancer-1 (MACC1),a newly discovered gene which controls tumor growth and metastasis,abnormally expresses in a variety of malignant tumors.As a key regulator of HGF-MET signal pathway,its coding protein can obviously promote invasion and metastasis of tumor cells.
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Objective To investigate expression of VEGF and the in vitro angiogenesis ability induced by ovarian cancer cells after RNA interference on expression of matrix metalloproteinase-2 (MMP-2)gene.Methods One specific target sequence of MMP-2 and one non-specific sequence (NC group) were chosen,the DMEM as blank group.After transfection of ovarian cancer OVCAR-3 cells,mRNA and protein expression of MMP-2 and VEGF genes were examined by RT-PCR and Western blot analysis,and the angiogenesis ability was detected by in vitro angiogenic assay.Results When compared with the NC group,the mRNA expression of MMP-2 and VEGF were decreased by 78.8 ％ and 75.5 ％ (P ＜ 0.05) in 48 h after transfected,respectively,and protein expression was decreased by 81.2 ％ and 78.3 ％ (P ＜ 0.05) at the same time point.In vitro angiogenic assay suggested that the ability of angiogenesis was inhibited when down-regulated of MMP-2 gene (P ＜ 0.05).Conclusion Down-regulation of MMP-2 gene in ovarian cancer cells by RNA interference could inhibit its VEGF expression and in vitro angiogenesis induced by ovarian cancer cells,which suggestes that the inhibition of MMP-2 gene has an anti-angiogenesis effect,and MMP-2 gene could be a potential target for ovarian cancer gene-therapy.
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Objective To investigate the inhibitory effects of RNA interference (RNAi) on the expression of matrix metalloproteinase-2 (MMP-2) gene and growth, adhesion,invasiveness and migration of ovarian cancer cells. Methods One specific target sequence of MMP-2 gone and one non-specific sequence (NC group) were chosen,the medium DMEM as blank group.After transfection of ovarian cancer OVCAR-3 cells, the RT-PCR and Western blot were used to detect mRNA and protein expression of MMP-2 gene, the growth ability was detected by MTT assay, the abilities of adhesion was detected by cell adhesion assay, the invasion and migration were detected by Matrigel invasion assay and wound healing assay. Results By contrast to the NC group,the mRNA expression was decreased by 73.8 ％,78.8 ％ and 78.4 ％(P＜ 0.05) in 24 h,48 h and 72 h after transfection and protein expression was decreased by 72.6 ％,81.2 ％ and 76.4 ％(P＜ 0.05) respectively at the same time. The 48 h group had the most efficient inhibitory effect. Cell growth curve revealed that cell growth was not significantly inhibited (P＞ 0.05). Adhesion was significantly reduced,the inhibitory rate was 55.0 ％ at 60 min and 44.8 ％ at 90 min (P＜ 0.05),respectively. Invasion and migration were significantly reduced as well,the inhibitory rate on invasion and migration were 29.7 ％ and 35.8 ％(P＜0.05), respectively. Conclusion siRNA mediated MMP-2 down-regulation in ovarian OVCAR-3 cells can inhibits its adhesion,invasion and migration,but do not significantly affect its growth,suggesting a important target to ovarian cancer gene-therapies.
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Objective To explore the lowest effective dosage of mifepristone combined with misoprostol in terminating ultra-early pregnancy.Methods All the cases of ultra-early pregnancy classified by amenorrhea days,β-hCG and vaginal B-ultrasonic were randomly divided into two groups.One hundred cases in G1 group (minimized dosage) were orally administered 25 mg mifepristone once a day for 2 days and combined with 200 μg misoprostol 48 hours later,while 150 mg mifepristone combined with 600 μg misoprostol 48 hours later were given to 100 cases in G2 group (normal dosage).All cases were observed for 6 hours after taking misoprostol and returned for assessment three days later.Results None missing.Expulsion of conceptus:G1 and G2 group were 22 (22.0％,22/100) and 25 (25.0％,25/100;P ＞ 0.05).Failure rate:cases with incomplete abortion were 1 (1.0％,1/100) and 2 (2.0％,2/100) in G1 and G2 group,hospitalization for suspected ectopic pregnancies both was 1 (1.0％).Bleeding:bleeding cases during the administration of mifepristone in G1 and G2 group were 71 (71.0％,71/100) and 78 (78.0％,78/100; P＞0.05); the mcan bleeding time were (5.3 ± 1.4) days and (6.0± 1.5) days (P ＜0.01).Other side effects:in G1 group,majority showed light nausea (7.0％,7/100) and light abdominal pain (20.0％,20/100).Menses recovery:99 (99.0％,99/100) for G1 group and 98 (98.0％,98/100) for G2 group to recovery on scheduled time.Satisfactions:both were 99 (99.0％,99/100).Except mean bleeding days and side-effects,the differences above showed no significance (P ＞ 0.05).Conclusion It is safe and effective treatment with the lowest dosages of mifepristone and misoprostol to terminate ultra-early pregnancies.
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Breast cancer metastasis suppressor 1(BRMS1)can suppress the tumor cells metastasis. and significantly reduces the metastasis without affecting tumor growth. The relevant mechanism(s)may be related to intercellular communication, phosphoinositide signal transduction and interaction with NF-KB, and so on. Therefore, exploring the mechanisms of inhibition of tumor metastasis might be helpful for use of BRMS1 as tumor gene therapy.
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Objective To study the effect of BRMS1 on the invasion and metastasis of ovarian cancer cells. Methods BRMS1 small interfering RNA (BRMS1-siRNA) was transfected into human ovarian cancer cell-line OVCAR3 by liposome transfection method. The cells were divided into 3 groups: experimental group with BRMSl-siRNA, negative control group with siRNA that did not impact any gene, blank control group without any transfection. Changes of invasion and migration in the cells were per-formed using transwell invasion and migration assay. Results BRMS1 gene was silenced in OVCAR3 cell line successfully detecting by quantitative real-time RT-PCR and immunoblotting.In transwell invasion assay,the numbers of cells in lower chamber passing through the membrane in transfected group were more than negative control group and blank control group (190±8.5, 144±7.8 and 146±6.8 respectively) (t=8.747, t=8.869, P=0.000), while in transwell migration assay, the numbers of cells in lower chamber passing through the membrane in transfected group were more than negative control group and blank control group were (231 ±8.9, 177±9.7 and 182±7.9 respectively) (t=9.314, t=9.224, P=0.000), both with significant differences among the 3 groups. Conclusion BRMS1 gene could suppress the invasion and metastasis of ovarian cancer cells.
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Objective To screen the Doc 1R gene recombinant plasmid by use of colony PCR. Method The recombinant colonies were transfered into the PCR reaction mixture. The PCR primers were used for constructing mouse Doc 1R genomic sequence. Result Among the 5, 3 positive strips in the size of 1 500 bp were visible, which were the same as the Doc 1R gene fractions in terms of their sizes were screened as positive clones. The positive colony were further confirmed by double digestion and DNA sequencing. Conclusion Colony PCR is a simple, efficient and reliable technique for screening the recombinant.
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Objective To study the express of matrix metalloproteinase(MMP)-2 and tissue inhibitor of metalloproteinase(TIMP)-2 protein in human endometrial carcinoma. Methods The Immunohistochemistry was used to measure the MMP-2, TIMP-2 in endometrial carcinoma tissue of 40 patients, the atypical hyperplasia of 12 samples and the normal endometrium of 12 samples. Results The positive express of MMP-2 and TIMP-2 in the normal endometrium , atypical hyperplasia and endometrial carcinoma was increased gradually. In endometrial carcinoma, with progression of clinical stage and decrease of histological grade as well as deepening of cancer invasion, the ratio of MMP-2/TIMP-2 was increased. Conclusions The expression of MMP-2 and TIMP-2 were connected with endometrial carcinoma. The imbalance of MMP-2/TIMP-2 in the endometrial cancer might be one of the pathogenesis and development.