Effects of Sp1 on the basic transcriptional activity of intestinal trefoil factor promoter
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摘要: 目的 寻找维持肠三叶因子(ITF)启动子基础转录活性的反应元件。 方法 以ITF启动子序列为模板,采用PCR方法获取不同长度及突变的ITF基因5'侧翼序列,插入pGL3-basic质粒,构建截短及突变荧光报告载体,进行以下实验。(1)按照随机数字表法(分组方法下同)将人胚胎肾293(HEK293)细胞分为pGL3-basic组、pGL3-300组、pGL3-280组、pGL3-260组、pGL3-240组、pGL3-220组、pGL3-200组,每组3孔,分别转染对应的质粒500 ng及15 ng海肾荧光素酶报告质粒pRL-TK,培养48 h,单管型多功能检测仪检测细胞荧光素酶相对活性。(2)另取HEK293细胞,分为pGL3-basic组、pGL3-300组及突变体1、2、3、4组,每组3孔,分别转染pGL3-basic、pGL3-300及突变体1、2、3、4质粒500 ng及15 ng pRL-TK质粒。培养48 h,同(1)检测荧光素酶相对活性。(3)另取HEK293细胞,分为空白对照组及10、50 μmol/L光神霉素组,每组3孔,转染500 ng pGL3-300质粒及15 ng pRL-TK质粒后,空白对照组不加,后2组分别加10、50 μmol/L光神霉素,培养24 h,同(1)检测荧光素酶相对活性。(4)另取HEK293细胞,分为空白对照组及0.1、0.2、0.3 μg pcDNA3.1-Sp1组,每组3孔,转染500 ng pGL3-300质粒及15 ng pRL-TK质粒后,空白对照组不转染,后3组分别转染0.1、0.2、0.3 μg pcDNA3.1-Sp1质粒。培养48 h,同(1)检测荧光素酶相对活性。对数据行单因素方差分析、LSD检验。 结果 (1)pGL3-basic组、pGL3-300组、pGL3-280组、pGL3-260组、pGL3-240组、pGL3-220组、pGL3-200组细胞荧光素酶相对活性分别为1.00、7.99±0.51、2.03±0.55、2.50±0.40、2.50±0.15、1.72±0.19、2.10±0.21,pGL3-280组、pGL3-260组、pGL3-240组、pGL3-220组、pGL3-200组细胞荧光素酶相对活性较pGL3-300组显著下降(
P 值均小于0.01)。(2)pGL3-basic组、pGL3-300组及突变体1、2、3、4组细胞荧光素酶相对活性分别为1.00、7.99±0.51、2.10±0.56、7.03±1.05、5.09±1.40、8.15±1.48,其中突变体1组细胞荧光素酶相对活性较pGL3-300组显著下降(P <0.01),突变体2、3、4组细胞荧光素酶相对活性与pGL3-300组相近(P 值均大于0.05)。(3)10、50 μmol/L光神霉素组细胞荧光素酶相对活性分别为3.07±0.60、2.93±0.55,均显著低于空白对照组的8.05±0.83(P 值均小于0.01)。(4)0.1、0.2、0.3 μg pcDNA3.1-Sp1组细胞荧光素酶相对活性分别为12.74±1.12、14.52±1.25、15.66±1.82,均显著高于空白对照组的8.13±0.71(P 值均小于0.05)。 结论 ITF启动子的-301~-293 bp区域存在一个Sp1反应元件,是维持ITF启动子基础转录活性的核心元件。Abstract: Objective To explore response element that maintains basic transcriptional activity of intestinal trefoil factor (ITF) promoter. Methods Truncated and mutant 5' flanking sequences of ITF gene were cloned from ITF promoter sequences by PCR, and then they were inserted into the pGL3-basic vector to construct truncated and mutant luciferase vectors to conduct the following experiments. (1) Human embryonic kidney 293 (HEK293) cells were divided into pGL3-basic group, pGL3-300 group, pGL3-280 group, pGL3-260 group, pGL3-240 group, pGL3-220 group, and pGL3-200 group according to the random number table (the same grouping method below), with 3 wells in each group, and they were respectively transfected with 500 ng corresponding plasmids and 15 ng renilla luciferase reporter plasmids pRL-TK. After being cultured for 48 hours, the relative luciferase activity of cells was measured by single tube detection system. (2) Another batch of HEK293 cells were divided into pGL3-basic group, pGL3-300 group, mutant 1, 2, 3, and 4 groups, with 3 wells in each group, and they were respectively transfected with 500 ng pGL3-basic, pGL3-300, mutant 1, 2, 3, and 4 plasmids and 15 ng pRL-TK plasmids. After being cultured for 48 hours, the relative luciferase activity of cells was measured as in (1). (3) Another batch of HEK293 cells were divided into blank control group and 10, 50 μmol/L mithramycin groups, with 3 wells in each group. After being transfected with 500 ng pGL3-300 plasmids and 15 ng pRL-TK plasmids, cells in blank control group were not transfected with mithramycin, while cells in the latter two groups were respectively transfected with 10 and 50 μmol/L mithramycin. After being cultured for 24 hours, the relative luciferase activity of cells was measured as in (1). (4) Another batch of HEK293 cells were divided into blank control group and 0.1, 0.2, and 0.3 μg pcDNA3.1-Sp1 groups, with 3 wells in each group. After being transfected with 500 ng pGL3-300 plasmids and 15 ng pRL-TK plasmids, cells in blank control group were not transfected with pcDNA3.1-Sp1 plasmids, while cells in the latter three groups were respectively transfected with 0.1, 0.2, and 0.3 μg pcDNA3.1-Sp1 plasmids. After being cultured for 48 hours, the relative luciferase activity of cells was measured as in (1). Data were processed with one-way analysis of variance and LSD test. Results (1) The relative luciferase activity of cells in pGL3-basic group, pGL3-300 group, pGL3-280 group, pGL3-260 group, pGL3-240 group, pGL3-220 group, and pGL3-200 group was 1.00, 7.99±0.51, 2.03±0.55, 2.50±0.40, 2.50±0.15, 1.72±0.19 and 2.10±0.21, respectively. The relative luciferase activity of cells in pGL3-280 group, pGL3-260 group, pGL3-240 group, pGL3-220 group, and pGL3-200 group was significantly lower than that in pGL3-300 group (withP values below 0.01). (2) The relative luciferase activity of cells in pGL3-basic group, pGL3-300 group, mutant 1, 2, 3, and 4 groups was 1.00, 7.99±0.51, 2.10±0.56, 7.03±1.05, 5.09±1.40 and 8.15±1.48, respectively. The relative luciferase activity of cells in mutant 1 group was significantly lower than that in pGL3-300 group (P <0.01). The relative luciferase activity of cells in pGL3-300 group, mutant 2, 3, and 4 groups was similar (withP values above 0.05). (3) The relative luciferase activity of cells in 10 and 50 μmol/L mithramycin groups was respectively 3.07±0.60 and 2.93±0.55, which was significantly lower than that in blank control group (8.05±0.83, withP values below 0.01). (4) The relative luciferase activity of cells in 0.1, 0.2, and 0.3 μg pcDNA3.1-Sp1 groups was respectively 12.74±1.12, 14.52±1.25, and 15.66±1.82, which was significantly higher than that in blank control group (8.13±0.71, withP values below 0.05). Conclusions One Sp1 binding site, locating in the region from -301 to -293 bp of ITF promoter, is the core element for regulating the basic transcriptional activity of ITF.
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