Objective To investigate the effects of allele-31 C>T on the binding activity to IL-1βpromoter of the nuclear transcription factor C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection.Methods The electrophoretic mobility shift assay ( EMSA) was performed to explore whether the nuclear transcription factor C/EBPβand PU.1 could bind to -31 region in IL-1βpromoter.The C/EBPβ-and PU.1-expressing vectors were constructed and co-transfected into HeLa cells with IL-1βpromoter luciferase vector.The expression of C/EBPβand PU.1 was confirmed using Western blotting assay, and the promoter activity was determined using Dual-Glo Luciferase system under various transfection conditions. Lentivirus-mediated RNA interference was used to explore the effects of C/EBPβand PU.1 on IL-1βexpression.GraphPad Prism 5.0 was used for data analysis.Results EMSA results showed that both C/EBPβand PU.1 could bind to -31 region in IL-1βpromoter.Both C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection could increase IL-1βpromoter activity, especially for the -31 T allele (t=22.33 and 7.98,P<0.01), and there was a synergy on the promoter activity between C/EBPβand PU.1.The promoter activity decreased significantly when C/EBPβand/or PU.1 were silenced by lentivirus-mediated RNA interference (q=5.79, 6.23 and 11.66,P<0.01).Conclusion The allele-31 C>T can induce IL-1βpromoter activity and gene transcription through regulation of binding activity to C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection.%目的：探讨IL-1β等位基因-31 C＞T对结核分枝杆菌诱导的核转录因子C／EBPβ和PU．1与启动子结合活性的影响。方法首先，采用凝胶电泳迁移率电泳实验（ EMSA）证明C／EBPβ和PU．1可以结合在IL-1β启动子-31位置区域。然后，构建表达PU．1和C／EBPβ的真核载体，协同不同等位基因型IL-1β启动子荧光素载体共同转染至HeLa细胞，采用蛋白免疫印迹实验验证PU．1和C／EBPβ是否成功表达，利用双荧光素酶系统分析在不同转染条件下IL-1β等位基因-31 C＞T对启动子活性的影响。最后，利用慢病毒介导的RNA干扰技术（ siRNA）反向验证结核分枝杆菌诱导的C／EBPβ和PU．1对IL-1β基因的转录调控。所有统计分析使用GraphPad Prism 5．0软件完成。结果EMSA证实核转录因子C／EBPβ和PU．1均可以结合在IL-1β启动子-31位置区域；过表达C／EBPβ或者PU．1均可以增加IL-1β启动子活性，尤其以增强-31 T等位基因启动子活性最为明显（ t＝22．33和7．98，P＜0．01），在共表达C／EBPβ和PU．1时两者具有显著的协同效应；利用siRNA 单一或者同时沉默C／EBPβ和PU．1基因后，IL-1β启动子活性显著降低（q＝5．79、6．23和11．66，P＜0．01）。结论IL-1β等位基因-31 C＞T通过影响结核分枝杆菌诱导的核转录因子C／EBPβ和PU．1与启动子的结合活性，进而调控IL-1β基因转录水平。
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