Abstract: Objective To investigate the effect of -572C/G polymorphism on the binding activity of IL-6 gene promoter and nuclear extract, it provides a reference for predicting the occurrence and development of diseases. Method A sequence of the IL-6 promoter region, from -1029 to 52 obtained from a subject with C allelotype at position -572 was cloned into the pGL3-Basic vector. The pGL3/-572C vector was mutated to pGL3/-572G, using Site-DirectedMutagenesis Kit.The IL-6 promoter luciferase vectors were transfected into HeLa cells, and the effects of -572C/G gene polymorphism on promoter activity was determined using Dual-Glo luciferase assay system.The single-stranded complementary oligonucleotides covering the IL-6 promoter region -572 site were designed and synthesized, and biotin-labeled at the 3' end. Then annealed to form a double-stranded oligonucleotide, which is then incubated with HeLa cell nuclear extracts, and the effects of IL-6 allele-572C/G on the binding activity of nuclear extract was analyzed by electrophoretic mobility-shift assay (EMSA). Result The transcription activity of pGL3/-572C in HeLa cells was significantly higher than that of pGL3/-572G, and the difference was statistically significant (P<0.01); EMSA Resultshowed that one strong band (complex 1) and one super shift band was shifted when the nuclear extracts were incubated with biotin-labeled C probe, In contrast, there was much less complex 1 formation on the G probe. Furthermore, complex 1 formation was specifically blocked by unlabeled C or G competitor. These result indicated that -572 C alleles variant increased the binding activity of more nuclear protein to an IL-6 promoter region, to form the transcription initiation complex. Conclusion The -572C/G polymorphism can affect the transcriptional activity of the IL-6 promoter and regulate the binding activity of nuclear extracts of IL-6 promoters.

Key words: Interleukin-6, Gene polymorphism, Promoter, Binding activity of nuclear extract