Negative Regulation Role Of SLC22A3Gene Intron7in Gene Transcription

Objective: To explore the regulatory effect of the human SLC22A3gene intron7and SNPrs2048327on gene transcription.Methods: The hSLC22A3intron7, amplified from the bacterialartificial chromosome (BAC) include whole human SLC22A3gene, wascloned into pGL3-Basic and pGL3-Promoter respectively to construct thewide-type recombinants pGL3-Basic-SLCi7wt andpGL3-Promoter-SLCi7wt. The mutant recombinant plasmidpGL3-Promoter-SLCi7mut was constructed through the site-directedmutagenesis based on pGL3-Promoter-SLCi7wt. The recombinants withinternal reference plasmid pRL-SV40were co-transfected into HEK293Tcells, then the luciferase activity was detected. The luciferase activity wasanalyzed by statistical software SPSS17.0.Results: The recombinant plasmid pGL3-Basic-SLCi7wt,pGL3-Promoter-SLCi7wt and pGL3-Promoter-SLCi7mut were constructedsuccessfully. The luciferase activity of pGL3-Basic-SLCi7wt has nosignificant difference with pGL3-Basic (P=0.986). The luciferase activity ofpGL3-Promoter-SLCi7wt was reduced compared with pGL3-Promoter ((3.514±0.09609) vs (6.286±0.3699), P=0.000), and that ofpGL3-Promoter-SLCi7mut (2.089±0.3315) was decreased as well, nomatter compared with pGL3-Promoter (P=0.000) orpGL3-Promoter-SLCi7wt (P=0.000).Conclusion: The human SLC22A3gene intron7has negativeregulatory effect on gene transcription, which could significantlydown-regulate the expression of luciferase reporter gene. The mutation(A→G) of SNPrs2048327located in the hSLC22A3intron7may enhancethe negative regulatory activity of this intron.