冠心病相关SLC22A3基因中新发现负性调控序列的性质与功能鉴定

Properties and function of newly discovered negative regulatory sequences in coronary heart disease-related SLC22A3 gene

目的:拟对课题组前期发现的冠心病相关SLC22A3基因intron7区域中的2段DNA负性调控序列的性质及功能进行研究,以明确其为沉默子还是绝缘子。方法:利用生物信息学分析2段负性调控序列在所培养细胞系中的脱氧核糖核酸酶I(DNaseⅠ)的敏感性,并以包含人SLC22A3基因全长序列的细菌人工染色体(bacterial artificial chromosome,BAC)为模板,PCR扩增2段负性调控序列,以pGL3-Control作为工具载体,将2段序列克隆入报告基因Luciferase(LUC)的启动子与增强子之间以及增强子的下游,构建4组重组质粒,并以pGL3-Control质粒作为空白对照,与内参质粒p RL-SV40共转染人胚肾细胞HEK293T,24 h后检测荧光素酶活性。结果:2段负性调控序列均位于DNaseⅠ超敏位点,明确了其为顺式调控元件的性质。4组重组质粒pGL3-con-SLCi7-447X、p GL3-con-SLCi7-447S、p GL3-con-SLCi7-460X及pGL3-con-SLCi7-460S荧光素酶活性分别为2.353±0.323、3.046±0.415、3.016±0.119、3.833±0.282,相较于空白对照pGL3-Control(7.423±0.230),荧光素酶活性均明显降低(P<0.05)。结论:SLC22A3基因的intron7区域中存在2个负性调控元件,其负性调控作用不受位置的影响发挥沉默子调控功能,为进一步对冠心病相关SLC22A3基因的表达调控研究提供了理论依据。

Objective:To investigate the properties and function of two negative regulatory DNA sequences in intron 7 of the coronary heart disease-related SLC22 A3 gene,and to determine if they are silencers or insulators. Methods:The DNase Ⅰ sensitivity of the two negative regulatory sequences in the cultured cell lines was analyzed using bioinformatics. A bacterial artificial chromosome li-brary containing the full-length human SLC22A3 gene was used as a template to amplify the two negative regulatory sequences by PCR. Using pGL3-Control as a vector,the two sequences were inserted between the promoter and the enhancer and the downstream of the enhancer of luciferase reporter gene to construct four recombinant plasmids. These plasmids were co-transfected together with the internal control plasmid pRL-SV40 into HEK293 T cells. pGL3-Control was used as a blank control. The luciferase activity was determined in 24 hours. Results:The two negative regulatory sequences were both located in the DNase Ⅰhypersensitive sites,suggesting that they were both cis-regulatory elements. The four recombinant plasmids,pGL3-con-SLCi7-447X,pGL3-con-SLCi7-447 S,pGL3-con-SLCi7-460 X,and pGL3-con-SLCi7-460 S, had significantly lower luciferase activities than the blank control(2.353 ±0.323,3.046±0.415,3.016±0.119,3.833±0.282 vs. 7.423±0.230,P<0.05). Conclusion:Two negative regulatory elements are identified in intron 7 of the SLC22A3 gene. They act as silencers and their negative regulatory function is independent of their positions.This study provides a theoretical basis for further research on regulation of expression of the coronary heart disease-related SLC22A3 gene.