IPO8基因启动子区rs35100176位点变异对基因表达的影响  

Effects of rs35100176 polymorphism in IPO8 promoter on gene expression

作　　者：熊建军[1,2] 江和[1,2] 许晓源[1,2] 周小鸥[1,2] 王庭[1,2] 李卫东[2] 

机构地区：[1]九江学院基础医学院,江西九江332000 [2]九江学院江西省系统生物医学重点实验室,江西九江332000

出　　处：《中国病理生理杂志》2014年第9期1640-1644,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81060075;No.81260140);江西省教育厅科学研究项目(No.GJJ12683)

摘　　要：目的:研究人importin 8(IPO8)基因启动子区rs35100176多态性位点CCT插入/缺失对基因表达的影响。方法:PCR扩增IPO8基因启动子区包含rs35100176多态位点的342 bp序列,通过测序获得了49例DNA样本的基因多态性分布。以CCT/CCT插入纯合子或-/-缺失纯合子DNA样本为模板,扩增含有不同插入/缺失序列的IPO8基因启动子片段,插入萤光素酶报告质粒pGL3-Basic,构建pGL3-3N Insertion和pGL3-3N Deletion重组表达载体。重组质粒经Fugene 6.0转染入细胞,采用双萤光素酶报告系统,检测携带不同多态性序列的重组质粒中报告基因的表达活性;real-time PCR检测各不同基因型细胞中IPO8 mRNA表达。结果:通过测序发现rs35100176多态位点存在CCT/CCT、CCT/-和-/-3种表型,其基因分布频率分别为18.37%、55.10%和26.53%。成功构建pGL3-3N Insertion和pGL3-3N Deletion重组表达载体。双萤光素酶报告基因活性检测结果显示,pGL3-3N Insertion启动下游报告基因表达的相对活性与pGL3-3N Deletion相比显著减弱,两者存在显著差异(P<0.05);real-time PCR结果显示,CCT纯合插入的HEK293细胞中IPO8 mRNA的表达显著低于CCT纯合缺失的Saos-2细胞。结论:IPO8基因启动子区rs35100176位点的CCT碱基插入变异能显著抑制启动子活性,从而影响IPO8基因的转录。AIM： To investigate the effect of rs35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8（ IPO8） gene on its mRNA expression. METHODS： A 342-bp fragment of IPO8 gene promoter containing the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced. The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples. The PCR products were sequenced and inserted into the luciferase reporter vector pGL3-Basic. Recombinant vectors were transfected into the cells by Fugene 6. 0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system. The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells. RESULTS： The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism,CCT /CCT,CCT /- and- /-,and the gene frequencies were 18. 37%,55. 10% and 26. 53%,respectively. The recombinant expression vectors pGL3-3N Insertion and pGL3-3N Deletion were successfully constructed. The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL3-3N Deletion. Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO8 mRNA than Saos-2cells with 3-nucleotide deletion homozygote. CONCLUSION： The CCT 3-nucleotide insertion variant decreases the promoter activity of IPO8,thus affecting the gene expression.

关 键 词：IMPORTIN 8 基因多态性 基因表达 

分 类 号：R363[医药卫生—病理学]