机构地区:[1]中山大学附属第一医院病理科,广州510080
出 处:《中华实验和临床病毒学杂志》2022年第1期8-14,共7页Chinese Journal of Experimental and Clinical Virology
基 金:广东省基础与应用基础研究基金(2019A1515011336);广东省自然科学基金(2018A030313054)。
摘 要:目的探索依托泊苷(VP-16)对人类免疫缺陷病毒(human immunodeficiency virus-1,HIV-1)潜伏感染的激活作用并研究其潜在的分子机制。方法将HIV-1潜伏感染细胞系J-Lat分为二甲基亚砜(DMSO)处理组、VP-16处理组及伏立诺他(SAHA)处理组,利用流式细胞技术检测细胞绿色荧光蛋白(green fluorescent protein,GFP)阳性比例,评估高浓度VP-16对HIV-1潜伏感染的激活效率;通过流式细胞技术检测VP-16在不同作用浓度及不同作用时间下对HIV-1潜伏感染的激活效果;通过流式细胞技术检测在VP-16作用下J-Lat细胞中CD25、CD69及白介素-6(interleukin-6,IL-6)的表达情况;运用双荧光素酶报告系统检测VP-16对HIV-1长末端重复序列(long terminal repeat,LTR)转录水平的影响;使用蛋白质印迹技术(western blot,WB)检测VP-16作用下蛋白沉默信息调节因子1(silent information regulator 1,SIRT1)及核因子κB(nuclear factorκB,NF-κB)乙酰化p65的表达水平;利用慢病毒介导的靶向SIRT1短发夹RNA(SIRT1 short-hairpin RNA,SIRT1-shRNA)敲低J-Lat细胞的SIRT1表达并检测相应的激活效果。结果流式细胞技术分析结果显示,VP-16能够特异性地激活HIV-1潜伏感染细胞系J-Lat,其激活效果存在浓度依赖和时间依赖;VP-16激活HIV-1潜伏感染的同时会一定程度上调J-Lat细胞的CD25及CD69,但IL-6的表达水平无明显变化;双荧光素酶报告实验提示VP-16通过NF-κB信号通路正向调控HIV-1 LTR的转录水平;WB结果表明VP-16能够抑制SIRT1的蛋白表达并促进NF-κB p65的乙酰化水平;慢病毒SIRT1-shRNA成功敲低J-Lat细胞中SIRT1的mRNA和蛋白表达,能够有效激活HIV-1潜伏感染。结论VP-16通过抑制SIRT1表达从而上调NF-κB p65的乙酰化水平,继而促进HIV-1 LTR的转录并最终激活HIV-1的潜伏感染。Objective To explore the effect of the etoposide(VP-16)on the reactivation of human immunodeficiency virus(HIV-1)from latent infection and study its potential molecular mechanism.Methods The HIV-1 latently infected cell line J-Lat was treated with DMSO,VP-16 and vorinostat(SAHA),flow cytometry was used to detect the positive ratio of green fluorescent protein(GFP)in J-Lat,which can reflect the efficacy of high concentration VP-16 on reactivation.Then the reactivation efficiency of VP-16 on HIV-1 latently infected cells at different concentration and treatment time was measured by flow cytometry.The expression of CD25,CD69 and interleukin-6(IL-6)of the VP-16-treated J-Lat cells were also detected in the same way.The effect of VP-16 on the transcription of long terminal repeat(LTR)was tested using a dual-luciferase reporter assay.The protein expression of the silent information regulator 1(SIRT1)and the acetylated nuclear factorκB(NF-κB)p65 under the effect of VP-16 was checked by western blot(WB).Lentivirus-mediated SIRT1 short-hairpin RNA(SIRT1-shRNA)was employed to knock down SIRT1,and then tested for efficiency of reactivation.Results The results of flow cytometry showed that VP-16 specifically reactivated HIV-1 latently infected J-Lat cells in a concentration and time-dependent manner.Meanwhile,the expression of CD25 and CD69 was upregulated to a certain extent,but the expression of IL-6 was not affected.Dual-luciferase reporter assay suggested that VP-16 positively regulated the transcription of HIV-1 LTR through NF-κB signaling pathway.WB results indicated that VP-16 can inhibit the protein expression of SIRT1 and promote the acetylation of NF-κB p65.Lentivirus-mediated SIRT1-shRNA successfully knocked down the mRNA and protein expression of SIRT1,and reactivated the HIV-1 latency effectively.Conclusions VP-16 upregulates the acetylation of NF-κB p65 by inhibiting the expression of SIRT1,which subsequently promotes the transcription of HIV-1 LTR and reactivates the latent HIV-1 infection.
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