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 .