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@# Objective To explore the relationship between the expression of decoy receptor 3 (DcR3) and high-risk human papilloma virus(HR-HPV)infection in cervical carcinoma. Methods Immunohistochemistry and hybird capture Ⅱ assay were used to detect the expression of DcR3 and HR-HPV in 35 cases of normal cervical tissues(NCE), 39 cases of cervical intraepithelial neoplasm(CIN)and 44 cases of cervical squamous epithelial carcinoma(CSES). Specific HR-HPV16 E7 siRNA and nonspecific HR-HPV16 E7 siRNA were synthesized and transfected to SiHa cells by Lipofectamine. The expression of DcR3 at mRNA and protein levels was examined by real-time polymerase chain reaction and western blot. The growth inhibition was examined by MTT assay. Results In NCE, CIN and CSES, the positive expression rates of DcR3 were 8.6%(3/35), 48.7%(19/39)and 77.3%(34/44), respectively, and the expression intensity was increasing(2=36.942, P<0.001). In NCE, CIN and CSES, the infection rates of HR-HPV were 5.7%(2/35), 56.4%(22/39)and 93.2%(41/44), respectively(2=60.322, P<0.001) . The protein expression of DcR3 was positively correlated with the infection of HR-HPV in CSES(r=0.893, P=0.004). Conclusions DcR3 was highly expressed in cervical cancer. Its expression was positively correlated with HR-HPV infection, which may contribute to the occurrence and development of cervical cancer. HR-HPV silencing inhibited cellular growth and proliferation by down-regulating the expression of DcR3.
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Objective:To construct the human DcR3 expression vector and verify its expression in vitro. Methods: 915 bp human DcR3 gene CDS was amplified from porcine lung tissues,and was cloned into eukaryotic expression vector pEF1a-IRES-DsRed-Express2 which show red fluorescence. And then pEF1a-IRES-DsRed-Express2-DcR3 was transfected into LX-2 cells by FuGene HD. Expression of mRNA and protein lever of Human DcR3 were detected by RT-PCR and Western blot. Results:The levels of DcR3 gene transcription and translation in the hepatic stellate cells were significantly increased after transfection with pEF1a-IRES-DsRed-Ex-press2-DcR3 by RT-PCR and Western blot analysis. Conclusion: DcR3 expression vector was successfully constructed and highly expressed in LX-2 cells.
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Objective:To study the expression of DcR3 of myocardial tissue in diabetic mouse and normal rats and the impact of DcR3 recombinant protein to the expression of related molecules and myocardial cell apoptosis to discuss the action of DcR3 to myocardial cell apoptosis in Diabetic rats.Methods:Intraperitoneally injected streptozotocin one time to establish the model of Diabetic rats.Injected different doses of DcR3 recombinant protein to tail vein[1.2 mg/(rat? d),0.8 mg/(rat? d),0.4 mg/(rat? d)] 40 d. The expression of DcR3 mRNA, Fas mRNA and FasL mRNA of myocardial tissue was detected with RT-PCR;the expression of apoptosis related molecules Bcl-2 and Caspase-8 was analyzed with Western blot;the IL-1β, TNF-αand LFN-γof the blood was detected with double antibody sandwich ELISA;the percentage of myocardial cell apoptosis was observed with HE dyeing.Results:To compare the DcR3 treatment group with diabetic group,the expression DcR3 of myocardial tissue was high,the expression of Fas mRNA and FasL mRNA was descended.The Caspase-8 protein was ascended and the Bcl-2 protein was descended.The middle dose group was the most obvious.the IL-1β,TNF-αand IFN-γin the blood was descended differently in each DcR3 treatment group(P<0.05,P<0.01).The percentage of myocardial cell apoptosis was declined(P<0.05).Conclusion:DcR3 recombinant protein have the action of inhibiting the rats′myocardial cell apoptosis,the mechanism is related to competing with Fas,blocking-up FasL of inducing apoptosis, expressing DcR3 of myocardial cell,the descending of apoptosis related factors Caspase-8,the ascending of Bcl-2 and the reduction of cytokine levels.
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Objective To detect the expressions of decoy receptor 3 (DcR3) in pancreatic cancer tissues and to analyze the significance of DcR3 in the diagnosis, treatment and prognosis of patients with pancreatic cancer.Methods The expressions of DcR3 in pancreatic cancer tissues (n =100), paracancer tissues (n =15) and normal tissues (n =15) were detected with immunohistochemical method (Envision method).Results The positive rate of DcR3 in pancreatic cancer tissues was significantly higher than that in adjacent-tumor pancreatic cancer tissues (86.0％ vs.46.6％, P ＜ 0.05).The positive rate of DcR3 in adjacent-tumor pancreatic cancer tissues was significantly higher than that in normal tissues (46.6％ vs.13.3％, P ＜ 0.05).In clinical stage Ⅲ, the positive rate of DcR3 was significantly higher than that in stage Ⅱ and stage Ⅰ (100％ vs.87.0％, P＜0.05;100％ vs.62.5％, P＜0.05).There were significant differences among the three groups (P ＜ 0.05).With lymph node metastasis, the DcR3 positive rate was significantly higher than that in the group without lymph node metastasis (93.4％ vs.79.6％, P ＜ 0.05).In poorly differentiated pancreatic cancer, the positive rate of DcR3 was significantly higher than that in the highly differentiated group (100％ vs.64.0％, P ＜0.05), the positive rate of DcR3 was significantly higher in the moderately differentiated group than that in the highly differentiated group (88.6％ vs.64.0％, P ＜ 0.05) , There were significant differences among the three groups (P ＜ 0.05).There was no significant difference in the positive rate of DcR3 between the different age groups or the different gender groups (P ＞ 0.05).Conclusions The expression levels of DcR3 in patients with pancreatic cancer gradually increased from normal tissues to paracancer tissues, to pancreas tissues.The expression level of DcR3 protein was closely related to clinical stage, degree of tissue differentiation and presence of lymph node metastasis, but not associated with age, sex, and tumor diameter size.
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Objective To explore the effect of HTLV-1 (human T-cell leukemia virus type 1 ) Tax protein on the DcR3 gene expression in T cells.Methods The construction of DcR3 (-1010 bp to +114 bp) luciferase reporter gene; MT2,TaxP,and Jurkat E6-1 cells were transfected with DcR3 luciferase reporter gene (pGL3-DcR3-1uc) using liposomes according to the manufacturer's instructions.For the control group,pGL3-basic replaced it respectively.At 48 h after incubation,luciferase activity was measured with a luciferase assay system; Jurkat cells were transfected with pCMV-Tax-Bam using liposomes,and total RNA was extracted from the cells at 48 h after incubation.Reverse transcription was performed using standard protocols.Then real time PCR with primers DcR3 and 3-actin was conducted;The expression of DcR3 protein was detected by flow cytometry in MT2,TaxP,and Jurkat cells.Results The construction of DcR3 luciferase reporter gene is identified correctly; The detection of luciferase activity showed that the luciferase activity of experimental group in MT2 cells was increased by (32.07±12.43)-fold,of which in TaxP cells was increased by ( 13.27±4.04)-fold,and the luciferase activity of experimental group in Jurkat cells was increased by ( 1.26±0.49 ) -fold.And there is statistic significance about the relative luciferase activity of experimental group in MT2 cells and TaxP cells compared to the relative lueiferase activity of experimental group in Jurkat cells respectively ( P＜0.01 ) ; The result of real-time PCR showed that the level of DcR3 mRNA in the experimental group was higher compared with the control group(P＜0.05) ; The result of FCM shows the expression of DcR3 protein in MT2,TaxP cells is higher than that in Jurkat cells( P＜0.05 ).Conclusion Tax protein can promote the expression of DcR3 gene in T cells.
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Objective: To study the expression of decoy receptor 3 (DcR3) in gastrointestinal tumors and elucidate the relationship between DcR3 and gastrointestinal tumors. Methods: The human colon carcinoma cells (SW480), gastric cancer cells (SGC7901), hepatoma carcinoma cell (HepG2) and human fibroblasts (3T3) were cultured in vitro. The expression level of DcR3 mRNA was determined by RT-PCR. The level of DcR3 protein expression was detected by Western blotting. Results: The mRNA and protein expression levels of DcR3 were higher in the SW480 cells than those in SGC7901, HepG2, and 3T3 cells. The difference was significant (P <0.05). Conclusion: Over-expression of DcR3 gene in the colon carcinoma cells may be related with the initiation and development of colon cancer.
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Objective To investigate the expression of DcR3 mRNA in cells of acute leukemia(AL) and its clinical significance.Methods The DcR3 mRNA of bone marrow mononuclear cells(BMNC) in the patients with AL was detected by RTPCR.There were 28 AML cases,and 18 ALL cases.27 cases of non-malignant hematopathy were used as control group.The expression level of DcR3 mRNA in BMNC was detected with RT-PCR.Results The positive rate of DcR3 mRNA was 67.4% and gene expression level was 0.56?0.09 in AL group,while they were 40.7% and 0.39?0.19 in control group,there were significant differences(P0.05).The positive expression level of DcR3 mRNA increased when the white blood cell count was ≥30?109?L-1.Conclusion DcR3 gene might play a role by suppressing apoptosis in leukemogenesis.
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Objective To clone ORF of DcR3 gene and insert it into eukaryotic expression vector and express it in COS-7 cells. Methods Encoding sequence of human DcR3 gene was cloned by PCR and sequenced. The sequenced ORF was cloned into eukaryotic expression vector pAAV-IRES-hrGFP to construct recombinant plasmid. COS-7 cells were transfected with recombinant plasmid by lipofectamine2000. Expression of recombinant DcR3 gene was verified by Western blotting and confocal microscopy. Results A 1 000-bp gene segment was obtained by PCR and inserted into pAAV-IRES-hrGFP to construct recombinant plasmid. The gene segment was proved to be encoding sequence of human DcR3 gene by sequencing. DcR3 expression in COS-7 cells was verified by Western blotting and confocal microscopy. Conclusion DcR3 gene was successfully cloned and expressed in COS-7 cells.
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Fas transduces apoptotic signals upon cross-linking with the Fas ligand (FasL), which is experimentally replaced by agonistic anti-Fas monoclonal antibodies (mAb). Of eight human malignant hematopoietic cell lines (HL-60, KG-1, THP-1, K562, U937, Jurkat, IM-9, RPMI-8226) examined by flow cytometric analysis, all, except K562, were found to be positive for surface Fas antigen. However, despite surface Fas expression, the agonistic anti-Fas mAb (7C11) induced apoptosis in only three of seven Fas-expressing cell lines (KG-1, Jurkat and IM-9). This Fas-resistance did not correlated with high levels of mRNA either for DcR3, a decoy receptor for FasL, or for FAP-1, a Fas-associated phosphatase that can block the apoptotic function of Fas. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis did not show consistent differences in the expression of Bcl-2 and Bax between Fas-sensitive and Fas-resistant cell lines examined. These findings indicated that the presence or absence of mRNA expression of DcR3, FAP-1, Bcl-2 and Bax did not always correlate with relative sensitivity to Fas-mediated apoptosis. Treatment of cells with cycloheximide converted the phenotype of resistant cell lines from Fas-resistant to Fas-sensitive, and enhanced the sensitivity of Fas-sensitive cell lines. These results suggest that the Fas-resistance is dependent on the presence of labile proteins that determine resistance to Fas-mediated apoptosis and the apoptotic machinery is already in place in Fas-resistant cell lines.