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High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
Published on: June 1, 2014
Distribution of PAI-1 promoter 4G/5G polymorphism in healthy Chinese and functional characterization using a
1Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, 1 Shuaifuyuan, Beijing 100730, China. zding@temple.edu
Abstract:
The aim of this study was to analyze the distribution of plasminogen activator inhibitor-1 (PAI-1) promoter 4G/5G polymorphism in healthy Chinese and compare the transcription activity of the two PAI-1 promoters using luciferase reporter vector pGL(3). PAI-1 promoter 4G/5G polymorphism was analyzed in 77 healthy Chinese using allele-specific polymerase chain reaction (PCR). The PAI-1 promoters from individuals homozygous for the insertion (5G) and deletion (4G) were cloned into luciferase reporter vector pGL(3), respectively. The transcription activities of the two promoters (PAI-1-4G and PAI-1-5G) were then compared using the luciferase reporter vectors expressed in ECV304 and HepG(2) cell lines. Among the 77 healthy Chinese the genotype frequency for 4G/4G, 4G/5G, and 5G/5G was 49.1, 44.1, and 6.5%, respectively. Both of the two PAI-1 promoters cloned into luciferase reporter vector pGL(3), expressed very well both in ECV304 and HepG(2), and have similar basic transcription activity. Thrombin (10-20 U/ml) has no effects on the transcription activity of the two promoters expressed in ECV304, while in the HepG(2) cell line interleukin-1 (IL-1) 158 U/ml increased the transcription activity of PAI-1-4G by 67% (p<0.05, n=5) without effects on PAI-1-5G transcription activity. The distribution of PAI-1 promoter 4G/5G polymorphism in Chinese is obviously different from that in Caucasians with the 4G allele more frequently found in Chinese. PAI-1 promoter 4G/5G polymorphism does not alter basic transcription activity and 4G promoter has increased response to IL-1.
