幽门螺杆菌感染诱导microRNA-146a表达的机制研究

The Mechanism of MicroRNA-146a Expression in Helicobacter pylori Infection

目的:探讨幽门螺杆菌(H.pylori)感染引起人胃上皮细胞microRNA-146a(miR-146a)上调的分子机制。方法:分别用H.pylori重组蛋白、全菌蛋白、培养上清、感染相关炎性因子(IL-8、TNF-α、IL-1β)以及TLR配体刺激人胃上皮细胞,检测细胞miR-146a的表达;通过生物信息学软件预测和荧光素酶实验鉴定miR-146a启动子,分析诱导表达的相关信号通路。结果:除H.pylori感染相关炎性因子IL-8、TNF-α、IL-1β能够明显诱导miR-146a表达上调(P<0.01)外,其他刺激因素均不能诱导miR-146a的显著表达;当采用RNAi技术将IL-8、TNF-α、IL-1β分别沉默,检测H.pylori诱导miR-146a表达时,各沉默组与对照组均无显著差异。软件预测显示miR-146a启动子序列中含有多个NF-κB结合位点;H.pylori能够显著增加miR-146a启动子荧光素酶报告载体的相对荧光素酶值;当启动子序列中的NF-κB结合位点发生突变,其相对荧光素酶比值显著降低(P<0.05)。结论:H.pylori感染相关炎性因子IL-8、TNF-α、IL-1β能够诱导miR-146a表达明显上调;NF-κB信号通路在H.pylori感染诱导miR-146a的表达中发挥关键作用。

Objective： To investigate the mechanism of microRNA-146a（miR-146a） up-regulation in Helicobacter pylori（H.pylori,HP） infection.Methods： Human gastric epithelium cells（GES-1 cells） were stimulated respectively by H.pylori recombination proteins,whole HP protein,HP supernatant,infection-related cytokines and TLR ligands and the expressions of miR-146a of GES-1 cells were detected by Realtime quantitative PCR.The signal pathways of miR-146a induction were analyzed by bioinformatics soft and luciferase assay.Results： MiR-146a was induced by H.pylori infection-related cytokine IL-8,TNF-α,IL-1β（P0.01）.GES-1 cells transfected by siRNA for IL-8,TNF-α,IL-1β were stimulated by H.pylori and the miR-146a expression had no significant deviation compared with controls.Bioinformatics analysis showed the promoter subsequence of miR-146a contained several NF-κB combining sites and the luciferase assays demonstrated mutation of NF-kB sites significantly decreased miR-146a basal promoter activity upon H.pylori stimula-tion（P0.05）.Conclusions： H.pylori infection-related cytokine IL-8,TNF-α,IL-1β could induce the up-regulation of miR-146a and NF-kB pathway is required for the induction of miR-146a in H.pylori infection.