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Objective To investigate the expression of Bcl-2 associated athanogene 3 (BAG3) in cervical cancer tissues and cells and its role in epithelial mesenchymal transition (EMT) of cervical cancer.Methods (1) Cervical cancer samples were collected from September 2015 to March 2017 in the Qilu Hospital of Shandong University and Shangdong Provincial Hospital.While,50 normal tissues were collected from August 2015 to March 2017 in the Dezhou Municiple Hospital,which were obtained from patients with uterine mnyoma underwent hysterectomy and patients with cervical biopsy.Reverse transcription (RT)-PCR and western blot were used to detect the expression of BAG3 mRNA and protein,and their clinical significances were analyzed.(2) The expression of BAG3 mRNA and protein was detected using RT-PCR and western blot method in HeLa and SiHa cell lines and normal cervical epithelial cells.The experiment was divided into two groups,BAG3 small interfering RNA transfected group (st-BAG3) and the control group transfected with small interfering RNA (siRNA).Cell counting kit 8 (CCK-8) analysis was used to detect cell proliferation of two groups.Wound-healing and transwell assay were used to detect the migration and invasion ability of HeLa and SiHa cells.The xenograft model of cervical cancer in nude mice was used to observe the effect of BAG3 on tumor xenografts and the tumor-related biomarkers were tested by western blot.Results (1) The expression levels of BAG3 mRNA and protein in cervical carcinoma tissues were 1.20±0.15 and 1.10±0.16,which were significantly higher than that in normal cervical tissue,0.23± 0.04 and 0.29 ± 0.03 (both P＜0.01).The results showed that the expression levels of BAG3 mRNA and protein were significantly correlated with cervical carcinoma staging and lymph node metastasis (P＜0.05).However,its expression was not conrelated with the patient's age,pathological grade,and diameter of tumor (all P＞0.05).(2) Compared with normal cervical epithelial cells,the expression of BAG3 mRNA and protein levels in HeLa and SiHa cells were significantly increased (P＜0.01),the expression levels of BAG3 mRNA and protein in HeLa and SiHa cells transfected with si-BAG3 were significantly lower than that in control group (all P＜0.01).After post-transfected 72 hours,A value of HeLa and SiHa with transfection were significantly lower than those in control group [(0.88±0.08) vs (1.22±0.13),(0.92±0.09) vs (1.35±0.12);both P＜0.01].After post-transfected 24 hours,the migration level of HeLa and SiHa cells with transfection were significantly lower than those in the control group [(20.1±2.1)％ vs (58.6±5.6)％,and (21.1±2.1)％ vs (61.7± 5.4)％;both P＜0.01].The transmembrane cell number in HeLa and SiHa cells with transfection were 76± 11 and 71±8,which were significantly less than those in control group (131± 12 and 129± 14;both P＜0.01).After the inoculation into nude mice,tumor formation time of HeLa and SiHa cells with transfection were (9.5±0.5) and (10.5 ± 1.3) days,respectively,which were significantly longer than those in control group [(4.5±0.5) and (5.2± 1.1) days;both P＜0.05].Compared with those in the control group,the expression level of Slug,N-cadherin and matrix metalloproteinase-2 (MMP-2) protein in HeLa and SiHa cells with transfected in tumor tissues were significantly decreased (all P＜0.01),while the expression level of E-cadberin protein was significantly increased (P＜0.01).Conclusion BAG3 could be involved in the proliferation,migration and invasion of cervical cancer cells by affecting cervical cancer EMT,and BAG3 may be an effective target for the treatment of cervical cancer.
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Retrieval of ancient DNA (aDNA) sequences from organism remains provide direct view of their evolutionary history. However, researches on aDNA have suffered from lots of technical problems. Specifically, discredited sequences were generated from damaged aDNA templates, and expensive and time-consuming methods were employed. Here, a method which could recover the endogenous aDNA as well as to reduce the cost and research period is described. This is achieved by improving the ancient DNA extraction method of isopropanol precipitation, and reevaluating the method of PCR after N-glycosylase (UNG) treatment, which could remove the damaged DNA from the aDNA extract. The efficiency of these methods were tested by comparing with traditional methods using ancient specimens of pig teeth aged between 4 300 years before present (BP) and 3 900 BP. The results showed that: firstly, the extraction efficiency of the improved method of isopropanol precipitation and current method with silica-based spin column were all 60%. Furthermore, the research period at least could be reduced by half with the application of the improved methods and the cost to 1/10 of the current method. Secondly, sequences obtained through the method of PCR after UNG treatment were 100% authentic. In contrast, 66%～ 88% sequences were authentic based on the results obtained with the method of multiple PCRs without UNG treatment. And the research cost and period needed by the method with UNG treatment were only half of the later one. These results demonstrate that the improved extraction method of isopropanol precipitation combined with the method of PCR after UNG treatment could increase the success rate of authentic DNA amplified and at least reduce the research cost and period by half. Therefore, this method can be applied in the large-scale detection of ancient specimens.
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0.05),while those of the cells treated with 6?10-5,6?10-6,6?10-7 mol/L of exemestane were significantly different from that of controls(P
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Objective To investigate the effect of mifepristone on the activity of proliferation and the apoptosis, the expression of estrogen receptor (ER),progesterone receptor (PR) protein and morphology changes of human ovarian carcinoma cell line 3AO and SKOV3 in vitro. Methods The proliferative activity of 3AO and SKOV3, which were cultured in vitro, was measured by tetrazolium-based colorimetric assay (MTT assay). Flow cytormetry (FCM) was used to measure the expressive rate of ER, PR , p53 protein, bcl-2 protein, cell apoptotic rate and cell proliferative cycle of 3AO cells, which were cultured with different concentration and duration of mifepristone. The morphologic and ultrastructure changes of apoptotic 3AO cells was observed by the light and electron microscopy. Results Mifepristone inhibited significantly the proliferation of 3AO cells in dose-time dependent manner in vitro. The inhibitory rate of 3AO cells growth, which were cultured with different concentration of mifepristone( 5,10,20,40,80 ?mol/L) and duration (24,48,72 h) was from 1.7% to 75.0%(P0.05). 3AO cells apoptosis activity appeared the positive correlation with the dose of mifepristone and cultured duration (P
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Background and purpose:Endometrial carcinoma is a common malignant tumour in women and expresses COX-2,CDK4;the expressions are associated with the development of the tumour. Mifepristone can inhibit the growth of the tumours, but its mechanisms are not very clear, We investigated the inhibitory effect of antiprogestins mifepristone n the growth of human endometrial carcinoma cell line HHUA xenografted in nude mice of and the expressions of COX-2,CDK4,the purpose was to find out whether or not mifepristone could influence the expressions of COX-2,CDK4. Methods:Human endometrial carcinoma HHUA cells were cultured in vitro.The models of xenografted tumor were established by the transplantation of human endometrial carcinoma HHUA cell on nude mice. The nude mice were randomly divided into two groups to receive either refined peanut oil (control group), or daily mifepristone for six weeks, respectively . The sizes of xenograft were measured in the pre-and post-treatment. The changes of morphology were observed by glass microscopy. The positive immunostaining for COX-2 and CDK4 were evaluated semiquantitatively by using an immunohistochemical scoring system (HSCORE) that it incorporated both the intensity and the distribution of specific staining.Results:After 6 weeks of treatment , the size of the xenografts in MIF group were 115.25?10.97 mm 3 ,compared with 313.25?43.92 mm 3 in the control group (P