LPS对牛子宫内膜细胞IL-6和IL-8启动子区甲基化状态的影响

摘要

为了解细菌脂多糖(Lipopolysaccharide,LPS)诱导牛子宫内膜细胞白介素6(Interleukin 6,IL-6)和白介素8(Interleukin 8,IL-8) mRNA表达上调的分子机制,利用亚硫酸氢测序(Bisulfite sequencing PCR,BSP)技术分析LPS对牛子宫内膜细胞IL-6和IL-8基因启动子区甲基化状态的影响,明确DNA甲基化对LPS诱导的牛子宫内膜细胞IL-6和IL-8基因表达的调控作用.用1μg/mL的LPS处理牛子宫内膜细胞24h,用BSP技术检测IL-6和IL-8表达量及其启动子区甲基化水平.结果表明:LPS可显著提高IL-6和IL-8表达量,伴随着IL-6和IL-8启动子区DNA甲基化水平降低,尤其在IL-6启动子区-660及IL-8启动子区-120和-48位点降低最明显.结果提示,LPS可降低牛子宫内膜细胞IL-6和IL-8启动子特定位点甲基化水平.%LPS could increase mRNA expression of interleukin 6 (IL-6) and interleukin 8 (IL-8)in bovine endomnetrial cells,but its underlying mechanisms remain unclear.In the present study,in order to confirm the role of DNA methylation in LPS induced expression of IL-6 and IL-8 in bovine endometrial cells,bisulfite sequencing PCR (BSP) was used to investigate the effect of LPS on methylation levels of IL-6 and IL-8 promoter in bovine endometrial cells.Bovine endometrial cells were treated with 1.0 μg/mL of LPS for 24 h to evaluate mRNA expression of IL-6 and IL-8 and methylation levels of their promoter using real time PCR and BSP.The results showed that LPS stimulation increased mRNA expression of IL-6 and IL-8,accompanying with the decreased methylation levels in the IL-6 and IL-8 promoter regions,which is robust at specific CpG sites in the IL-6 promoter (at-660) and the IL-8 promoter (at-120 and-48).These results indicated that LPS could decrease methylation levels at specific CpG sites in IL-6 and IL-8 promoter regions.