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 (with
P 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 (with
P 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, with
P 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, with
P 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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