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

摘要:

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pRL-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、pGL3-con-SLCi7-447S、pGL3-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。

Abstract:

Objective：To investigate the properties and function of two negative regulatory DNA sequences in intron 7 of the coronary heart disease-related SLC22A3 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 HEK293T cells. pGL3-Control was used as a blank control. The luciferase activity was deter－mined 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-SLCi7-447S, pGL3-con-SLCi7-460X，and pGL3-con-SLCi7-460S, 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 iden－tified 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.