SLC22A3基因intron7对基因转录的负性调控作用研究  

Negative regulation role of SLC22A3 gene intron7 in gene transcription

作　　者：封雪[1] 柳青[1] 雷寒[2] 丁艳辉[1] 

机构地区：[1]重庆医科大学附属第一医院临床研究中心,重庆400016 [2]重庆医科大学附属第一医院心血管内科,重庆400016

出　　处：《重庆医科大学学报》2014年第5期651-656,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81170189);高等学校博士学科点专项科研基金资助项目(编号:20115503110006);重庆市卫生局医学科学技术研究资助项目(编号:2010-2-118);重庆市渝中区科研计划资助项目(编号:20110302)

摘　　要：目的:探讨人SLC22A3基因intron7序列及其SNPrs2048327(A/G)位点对基因转录的调控作用。方法: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hSLC22A3 intron7,克隆入荧光素酶报告基因载体pGL3-Basic和pGL3-Promoter,得到重组质粒pGL3-Basic-SLCi7wt、pGL3-Promoter-SLCi7wt和pGL3-Promoter-SLCi7mut;重组质粒与内参质粒pRL-SV40共转染入人胚肾细胞HEK293T,24 h后检测荧光素酶活性。结果:野生型重组质粒pGL3-Basic-SLCi7wt荧光素酶活性与pGL3-Basic无统计学差异(P=0.986);野生型重组质粒pGL3-Promoter-SLCi7wt荧光素酶活性(3.514±0.096)相较于pGL3-Promoter(6.286±0.370)降低(P=0.000);突变型重组质粒pGL3-Promoter-SLCi7mut荧光素酶活性(2.089±0.332)相对于pGL3-Promoter亦有所下降(P=0.000),且较野生型重组质粒pGL3-Promoter-SLCi7wt下调(P=0.000)。结论:人SLC22A3基因intron7序列具有负性调节活性,能下调荧光素酶报告基因的表达水平;定位于该序列上的SNP rs2048327(A/G)位点A→G的改变,可能增强该内含子所具有的负性调节作用。Objective：To explore the regulatory effect of the human SLC22A3 gene intron7 and SNPrs2048327（A/G）on gene transcription. Methods：The hSLC22A3 intron7,amplified from the bacterial artificial chromosome（BAC）including whole human SLC22A3 gene,was cloned into pGL3-Basic and pGL3-Promoter respectively to construct the recombinant plasmid pGL3-Basic-SLCi7wt, pGL3-Promoter-SLCi7wt and pGL3-Promoter-SLCi7mut. The recombinants with internal reference plasmid pRL-SV40 were co-transfected into HEK293T cells,then the luciferase activity was detected. Results：There was no significant difference in luciferase activity between pGL3-Basic-SLCi7wt and pGL3-Basic（P=0.986）. Luciferase activity of pGL3-Basic-SLCi7wt was lower than that of pGL3-Pro moter（（3.514 ± 0.096）vs.（6.286 ± 0.370）;P=0.000）. Luciferase activity of pGL3-Promoter-SLCi7mut was lower than that of pGL3-Promoter and pGL3-Promoter-SLCi7wt（P=0.000）. Conclusions：The human SLC22A3 gene intron7 has negative regulatory activity,which could significantly down-regulate the expression of luciferase reporter gene. The mutation（A→G）of SNPrs2048327（A/G） located in the hSLC22A3 intron7 may enhance the negative regulatory activity of this intron.

关 键 词：冠心病 SLC22A3基因 内含子 启动子活性 负性调节活性 

分 类 号：R541.4[医药卫生—心血管疾病]