Effects of rs35100176 polymorphism in IPO8 promoter on gene expression
10.3969/j.issn.1000-4718.2014.09.016
- VernacularTitle:IPO8基因启动子区rs35100176位点变异对基因表达的影响
- Author:
Jianjun XIONG
;
He JIANG
;
Xiaoyuan XU
;
Xiaoou ZHOU
;
Ting WANG
;
Weidong LI
- Publication Type:Journal Article
- Keywords:
Importin 8;
Genetic polymorphism;
Gene expression
- From:
Chinese Journal of Pathophysiology
2014;(9):1640-1644
- CountryChina
- Language:Chinese
-
Abstract:
AIM:To investigate the effect of rs 35100176 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 con-taining 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 pGL 3-Basic.Recombinant vectors were trans-fected 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 re-combinant expression vectors pGL 3-3N Insertion and pGL3-3N Deletion were successfully constructed .The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL 3-3N Deletion.Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO 8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote .CONCLUSION: The CCT 3-nucleotide insertion variant decreases the pro-moter activity of IPO8, thus affecting the gene expression .
