Analysis Of SNPs In MD-2 Promoter And Their Relationship With LPS Response

Clinical epidemiology study has shown that sepsis is an important cause of death of severe patients, and LPS plays a central role in the pathogenesis of sepsis. We carried out a great quantity of exploratory study on LPS receptors in the past ten years, reseach results demonstrate that TLR4/CD14/MD-2 complex is the crucial pattern recognition receptor of LPS, and CD14, TLR4 gene polymorphisms are associated with susceptivity and prognosis of sepsis. MD-2 is also a key molecule of pattern recognition receptor in LPS passthway, but the role of its polymorphisms in sepsis susceptivity, and its distribution in China population is still unclear.In this study, we selected the potential SNPs in MD-2 gene promoter, which maybe influence MD-2 gene expression through bioinformatics analysis. Then, we genotyped selected SNPs in Chongqing Han population by method of restriction fragment length polymorphism. To study the function of the SNPs, we constructed different allele promoter plasmids and measured MD-2 promoter activity by Dual-Luciferase Reporter assay system. We also examined different genotype carriers' MD-2 mRNA expression levels and the significance of their whole-blood leukocytes response to LPS stimulation. The main results and conclusions were summarized as follows:1. Bioinformatics analysis shows that there are eight SNPs in the upstream 3000bp of MD-2 promoter. Among them, -1625C→G, -1064A→G and -475A→T can change the type or numbers of binding transcription factors, which indicats that the three SNPs may affect the transcription activities of MD-2 promoter.2. Genotyping 711 cases of Chongqing Han healthy population shows that -1625C→G and -1064A→G both exist in Chinese population, with the frequencies 19.84% and 34.71% respectively. While, no polymorphisms are found in -475 site.3. MD-2 gene promoter (-1704 ~ +61), -1625G and -1064G base variation promoter are constructed through PCR and single-site mutation technique. Luciferase Reporter assay shows that -1625G promoter has a higher activity than -1625C promoter, it implys that -1625 C→G base variation can enhance the activity of MD-2 promoter. There are no...