DC-SIGN启动子活化信号通路对HIV-1 5'LTR活性的影响

摘要

目的 研究诱导DC-SIGN启动子活化的信号通路对HIV-1 5'LTR活性的影响.方法 PCR法扩增DC-SIGN启动子及HIV-1 5'LTR序列,通过双酶切重组到荧光素酶报告质粒中,构建DCSIGN启动子和HIV-1 5'LTR荧光素酶报告质粒.以PMA分化的THP-1细胞作为细胞模型,分别转染DC-SIGN启动子和HIV-1 5 'LTR荧光素酶报告质粒,并以相应的信号通路阻断剂处理,以1000IU/ml的IL-4诱导24 h后检测荧光素酶活性.结果 HIV-1 5'LTR荧光素酶报告质粒的活性明显弱于DC-SIGN启动子,IL-4诱导可以使DC-SIGN启动子和HIV-1 5'LTR荧光素酶报告质粒的活性升高至2倍以上.ERK、JAK-STAT和NF-κB信号通路阻断剂均能够降低DC-SIGN启动子荧光素酶报告质粒的活性,其中ERK1/2抑制剂抑制活性最强,几乎完全阻断了IL-4的诱导效应.对于HIV-1 5 'LTR荧光素酶报告质粒,NF-κB信号通路阻断剂阻断效果最明显,阻断效率为52.32％,ERK信号通路阻断剂阻断效率为43.31％.结论 IL-4诱导的信号通路在活化DC-SIGN启动子的同时,对HIV-15 'LTR也具有一定的活化作用,并主要通过NF-κB和ERK信号通路实现.%Objective To explore the effects of signaling pathways inducing activation of DC-SIGN promoter on the activity of HIV-1 5'LTR.Methods The sequences of DC-SIGN promoter and HIV-1 5'LTR were amplified by PCR and then cloned into pGL-3/Basic plasmid to constructluciferase reporter plasmids for DC-SIGN promoter and HIV-1 5'LTR.Differentiated THP-1 cells stimulated by PMA (phorbol myristate acetate) were used as the in vitro model of DCs.The activaties of DC-SIGN promoter and HIV-1 5'LTR induced by IL-4 in differentiated THP-1 cells were studied using luciferase reporter plasmids.The signaling pathways were identified by using specific inhibitors.Results IL-4 induced signaling pathways could increase the activities of HIV-1 5'LTR and DC-SIGN promoter for more than two times in THP-1 cells transfected with luciferase reporter plasmids.However,the activity of HIV-1 5'LTR was weaker than that of DCSIGN promoter.ERK/JAK-STAT/NF-κB signal pathway blockers could inhibit the luciferase activity driven by DC-SIGN promoter,of which ERKI/2 blocker showed the strongest inhibitory effect that almost completely blocked IL-4 induction.NF-κB blocker had a significant inhibitory effect on HIV-1 5'LTR activity at a rate of 52.32％,followed by the ERK blocker at a rate of 43.31％.Conclusion This study suggested that IL-4-induced signaling pathways mediate the activation of DC-SIGN promoter and HIV-1 5'LTR through NFκB and ERK.