共查询到20条相似文献，搜索用时 31 毫秒
1.
AIM: To construct a prostate-specific expression vector of promoter and enhancer of human prostate-specific membrane antigen (PSMA).METHODS: The promoter and enhancer of PSMA were amplified by PCR separately. The two segments were cloned into the expression vector pGL3-Basic, a prostate-specific expression vector pGL3-PSMP-PSME was constructed. The vector was transfected into prostatic carcinoma cell PC-3M and four kinds of non-prostatic carcinoma cells by lipofectamine. The activity of luciferase of transfected cells and tissue specificity of vector was examined at 48 hours after transfection. RESULTS: With DNA sequence of the vector pGL3-PSMP-PSME, the segment of clone was proved correct. The activity of luciferase of the pGL3-PSMP-PSME was expressed distinctly in PC-3M and was not expressed in other nonprostate cell lines. CONCLUSION: The prostate-specific expression vector was constructed successfully. It lays foundation for studying target gene therapy of the prostate cancer. 相似文献
2.
AIM: To investigate the effect of rs35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 (IPO8) gene on its mRNA expression. METHODS: A 342-bp fragment of IPO8 gene promoter containing the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced. The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples. The PCR products were sequenced and inserted into the luciferase reporter vector pGL3-Basic. Recombinant vectors were transfected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system. The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells. RESULTS：The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT,CCT/- and -/-, and the gene frequencies were 1837%, 5510% and 2653%, respectively. The recombinant expression vectors pGL3-3N Insertion and pGL3-3N Deletion were successfully constructed. The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL3-3N Deletion. Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote. CONCLUSION：The CCT 3-nucleotide insertion variant decreases the promoter activity of IPO8, thus affecting the gene expression. 相似文献
3.
4.
5.
6.
AIM: To investigate the effect of G/A mutation in rs5418 site of solute carrier family 2,facilitated glucose transporter,member 4 protein(SLC2A4) promoter region on gene expression. METHODS: The core promoter region of SLC2A4 gene was amplified by PCR. Mutant and wild-type recombinant expression vectors containing promoter of SLC2A4 gene were constructed by recombinant gene technique and the strategy of site-directed mutagenesis. Recombinant vectors were transfected into HEK293T cells by lipofectamine and the expression activity of the reporter gene in the recombinant expression vectors with different alleles was detected by a dual-luciferase reporter assay system. RESULTS: The 716-bp SLC2A4 promoter was amplified and the recombinant expression vectors pGL3-SLC2A4-prom(A) and pGL3-SLC2A4-prom(G) were successfully constructed. The luciferase reporter vector containing SLC2A4 promoter with rs5418-A alleles produced significantly higher relative luciferase activity (19.49±4.41) than that with rs5418-G allele (13.04±4.45; P<0.05). CONCLUSION: The G→A variation of rs5418 site in SLC2A4 promoter region increases the expression of SLC2A4 gene,thereby affecting the gene function. 相似文献
7.
DONG Pei JIANG Li-juan GUO Sheng-jie LIU Zhuo-wei LI Yong-hong YAO Kai QIN Zi-ke HAN Hui ZHOU Fang-jian 《园艺学报》2013,29(10):1821-1825
AIM: To verify the cholesterol-sensitive sites in the promoter of prohibitin (PHB) gene. METHODS: A series of successive PHB promoter truncated segments were amplified by PCR and cloned into pGL3-Basic luciferase reporter vector. Human prostate cancer PC-3 cells were transfected with the recombinant plasmids and then cultured in normal medium (NM) or cholesterol-depleted medium (CDM). The activity of PHB promoter was detected by luciferase activity analysis to find the cholesterol-sensitive region of approximately 200 bp in PHB promoter. Point mutations in sterol regulatory element (SRE) homologous loci of this 200 bp region were made to confirm the cholesterol-sensitive role of this SRE site. RESULTS: Plasmids with PHB promoter segments were successfully constructed. Luciferase activity in PC-3 cells transfected with PHB promoter segment pPHB-179 (-35/+138) plasmid was significantly decreased compared with the cells transfected with the whole PHB promoter pPHB-1192 plasmid (P<0.05). After point mutation was made in the SRE homologous locus located at -117 to -108 bp and this plasmid was transfected into PC-3 cells, the luciferase activity in these cells was significant decreased compared with the cells transfected with pPHB-1192 plasmid (P<0.05). CONCLUSION: Cholesterol regulates the activity of PHB promoter in human prostate cancer PC-3 cells. The cholesterol-sensitive site in PHB promoter is located at -117 to -108 bp of SRE homologous locus. PHB may become a new target of gene therapy for prostate cancer. 相似文献
8.
CHEN Lin-lin XIE Qing SHAN Xiao-liang QUAN Hui-qin CHEN Qing-mei ZHOU Fan NIE Dan TANG Ni 《园艺学报》2013,29(2):200-204
AIM: To investigate the molecular mechanism that hepatitis B virus X protein (HBx) inhibits the promoter activity of secreted frizzled-related protein 5 (SFRP5). METHODS: Serial truncated fragments of SFRP5 promoter were amplified and cloned into luciferase reporter vector pGL3-Basic. LO2 cells were transfected with recombinants, and then infected with the adenoviral vector expressing HBx protein (Ad-HBx) or the analogous adenovirus expressing green fluorescent protein (Ad-GFP) for control. The infection efficiency was examined under a fluorescence microscope and the activity of SFRP5 promoter was measured by luciferase assay. RESULTS: Truncated fragments of SFRP5 promoter were successfully constructed. Compared with the pGL3-Basic control, the luciferase activity was 6.32±0.04 of the -478 bp~+47 bp fragment, 5.79±0.32 of the -811 bp~+47 bp fragment, 3.59±0.34 of the -1 235 bp~+47 bp fragment, 3.86±0.39 of the -1 677 bp~+47 bp fragment and 3.26±0.42 of the -2 072 bp~+47 bp fragment, respectively. Overexpression of HBx inhibited the activity of SFRP5 promoter. The average inhibitory rates were 44% for -478 bp~+47b, 46% for -811 bp~+47 bp, 28% for -1 235 bp~+47 bp, 24% for -1 677 bp~+47 bp and 40% for -2 072 bp~+47 bp, respectively. CONCLUSION: HBx suppresses the activity of SFRP5 promoter, leading to down-regulation of SFRP5 expression. 相似文献
9.
10.
11.
12.
13.
ZHANG Peng-ju JIANG An-li HE Mei-lan YUAN Hui-qing CHEN Wei-wen GUO Qiang ZHANG Jian-ye△ 《园艺学报》2006,22(7):1324-1329
AIM:To observe the effect of exogenous androgen responsive element decoy on the promoter of prostate specific antigen (PSA) and the growth of LNCaP cells for searching the possibility of gene therapy for prostate cancer. METHODS:Firstly, pGL3-PSA luciferase expression vector containing 640bp-promoter fragment of PSA gene was constructed. Then, a 23-mer phosphorothioated ARE decoy based on the deduced ARE sequence at the promoter region of PSA gene was synthesized. pGL3-PSA and ARE decoy DNA were cotransfected into PC3-M cell by lipofectamineTM 2000. Through detecting the activity of luciferase, the effect of ARE decoy on the promoter of PSA was studied. Then the ARE decoy DNA was transfected into LNCaP cells. The effect of decoy DNA on the proliferation of LNCaP cells was examined by using MTT assay. The effects of apoptosis were detected by phase contrast microscopy, DNA agrose gel electrophoresis and flow cytometry. Meanwhile, the nuclear extract was prepared from LNCaP cells and DNA-protein interactions were examined by electrophoretic mobility shift assay (EMSA). RESULTS:The reporter assay showed that the activity of luciferase was significantly reduced in the ARE decoy-transfected cells, bnt not in the cells transfected with the control decoy. EMSA demonstrated specific binding of the ARE decoy to androgen receptor. The growth of LNCaP was remarkably inhibited and apoptotic morphological changes as well as DNA fragmentation were observed in the ARE decoy-transfected cells. The rate of apoptosis was 22.4% detected by FCM. CONCLUSION:The ARE decoy is capable of inhibiting the promoter of PSA gene and inducing the apoptosis in prostate cancer cells. It may become a potential therapeutic tool for prostate cancers. 相似文献
14.
AIM:To study the mechanisms of nicotine-induced expression of intercellular adhesion molecule-1(ICAM-1).METHODS:Related luciferase reporter gene plasmids were constructed with molecular cloning techniques;above plasmids and intracontrol plasmid pSV-β-gal were co-transfected into human umbilical vein endothelial cells(HUVECs) with eukaryotic gene transfection techniques; the relative luciferase activities were detected in the transfected HUVECs.RESULTS:Series of luciferase reporter gene containing different sequences of human ICAM-1 promotor and site-directed mutants of NF-κB and Sp-1 in promotor were successfully constructed; Nicotine could increase the expression of luciferase reporter gene plasmid containing-579 bp(pGL3E-579/+36),-230 bp(pGL3E-230/+36) and mutated Sp-1 version(pGL3E-Sp-1-MU)(P＜0.05 vs control) of ICAM-1 promotor in the transfected HUVECs, whereas deletion derivative (pGL3E-134/+36) and mutation (pGL3E- NF-κB -MU) of downstream NF-κB site of ICAM-1 promotor prevent nicotine-induced increase in expression of luciferase reporter gene plasmid.CONCLUSION:NF-κB site of promotor mediates nicotine-induced ICAM-1 expression in human umbilical vein endothelial cells. 相似文献
15.
今年是《园艺学报》创刊发行50周年。50年来,《园艺学报》坚持为学术交流服务,为促进学科发展作贡献的办刊原则,以"科学性;创新性;对生产和科研有参考启迪作用"的标准,收录和发表了大量高水平的论文,记载了几代科技工作者呕心沥血创新之作,反映了中国园艺科学技术和园艺产业的发展历程。 相似文献
16.
17.
AIM: To explore the regulatory effect of microRNA-3666 (miR-3666) on the expression of its target gene phosphatase and tensin homologue deleted on chromosome ten (PTEN) in leukemic cells. METHODS: miR-3666 expression levels in normal peripheral blood mononuclear cells and leukemic cells were determined by quantitative real-time PCR. miR-3666 targeting PTEN 3-untranslated region (3UTR) was predicted by TargetScan software. 3UTR of PTEN was inserted in the dual luciferase reporter vector psiCHECK2. The reporter activity was evaluated by the Dual-Luciferase Reporter Assay System after the luciferase promoter vector and miRNA were co-transfected into HEK293T cell line. K562 cells were transfected with synthetic miR-3666 inhibitor (anti-miR-3666) or a synthetic control miRNA (anti-miR-C). The expression of PTEN protein in the above transfected K562 cells was determined by Western blotting. RESULTS: miR-3666 was up-regulated in the human leukemic cell lines and primary leukemic cells compared to normal peripheral blood mononuclear cells. The results of dual luciferase assays validated PTEN as a specific target gene of miR-3666. Inhibition of miR-3666 resulted in an up-regulation of PTEN protein expression in the K562 cells. CONCLUSION: miR-3666 is over-expressed in leukemic cells. The abnormal over-expression of miR-3666 may play a key role in leukemia due to the down-regulation of PTEN. 相似文献
18.
19.