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

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.