Spl对肠三叶因子启动子基础转录活性的影响

Effects of Spl on the basic transcriptional activity of intestinal trefoil factor promoter

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-basle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500ng及15ng海肾荧光素酶报告质粒pRL—TK，培养48h，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、pCL3—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500ng及15ngpRL—TK质粒。培养48h，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、50μmol／L光神霉素组，每组3孔，转染500ngpGL3—300质粒及15ngpRL·TK质粒后，空白对照组不加，后2组分别加10、50μmol／L光神霉素，培养24h，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、0．3μgpeDNA3．1-Spl组，每组3孔，转染500ngpGL3-300质粒及15ngpRL—TK质粒后，空白对照组不转染，后3组分别转染0．1、0．2、0．3μgpcDNA3．1-Spl质粒。培养48h，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、pCL3-240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O．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、50Ixmol／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、0．3IxgpeDNA3．1-Spl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～-293bp区域存在-个Spl反应元件，是维持ITF启动子基础转录活性的核心元件。

Objective To explore response element that maintains basic transcriptional activity of intestinal trefoil factor （[TF） promoter. Methods Truncated and mutant 5＇ flanking sequences of ITF gone were cloned from ITF promoter sequences by PCR, and then they were inserted into the pGL3-hasic vector to construct truncated and mutant luciferase vectors to conduct the following experiments. （ 1 ） Human embryonic kidney 293 （ HEK293 ） cells were divided into nGL3-hasie , pGL3-300 group, pGL3-280group, 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 transfeeted 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 sys- tem. （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-basie, pGL3-300, mutant 1 , 2, 3, and 4 plasmids and 15 ng pRL-TK plasmids. After being cultured for 48 hours, the relative lueiferase 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 transfeeted with mithramycin, while cells in the latter two groups were respectively transfectcd with 10 and 50 μmol/L mithramycin. After being cultured for 24 hours, the relative lueiferase activity of cells was measured as in （1）. （4） Another batch of HEK293 cells were divided into blank con- trol group and 0.1, 0.2, and 0.3 μg pcDNA3.1-Spl groups, with 3 wells in each group. After being trans- fected with 500 ng pGL3-300 plasmids and 15 ng pRL-TK plasmids, cells in blank control group were not transfected with pcDNA3.1-Spl plasmids, while cells in the latter three groups were respectively transfected with 0.1 , 0.2, and 0.3 μg pcDNA3.1-Spl plasmids. After being cultured for 48 hours, the relative lueifer- ase 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 rela- tive 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 lueiferase 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 con- trol 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 g pcDNA3.1-Spl 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 Spl 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.