中华皮肤科杂志 ›› 2019, Vol. 52 ›› Issue (9): 624-630.doi: 10.3760/cma.j.issn.0412-4030.2019.09.007

• 论著 • 上一篇    下一篇

病毒巨噬细胞炎症蛋白Ⅱ对293T细胞APOBEC3G表达的影响

郑国霞    刘如锦    齐燕    王小波    闫玉涛    谭晓华    杨磊   

  1. 杭州师范大学医学院  310036
  • 收稿日期:2018-11-07 修回日期:2019-06-10 出版日期:2019-09-15 发布日期:2019-08-30
  • 通讯作者: 杨磊；谭晓华 E-mail:yanglei62@hznu.edu.cn; xiaohuatan@hznu.edu.cn
  • 基金资助:
    国家自然科学基金（81772168）；浙江省自然科学基金（LY12H16028）；浙江省科技计划项目（2009C14014）

Effect of viral macrophage inflammatory protein Ⅱ on the expression of APOBEC3G in 293T cells

Zheng Guoxia, Liu Rujin, Qi Yan, Wang Xiaobo, Yan Yutao, Tan Xiaohua, Yang Lei   

  1. Division of Preventive Medicine, School of Medicine, Hangzhou Normal University, Hangzhou 310036, China
  • Received:2018-11-07 Revised:2019-06-10 Online:2019-09-15 Published:2019-08-30
  • Contact: Yang Lei; Tan Xiaohua E-mail:yanglei62@hznu.edu.cn; xiaohuatan@hznu.edu.cn
  • Supported by:
    National Natural Science Foundation of China （81772168）; Natural Science Foundation of Zhejiang Province of China（LY12H16028）; Science and Technology Planning Project of Zhejiang Province （2009C14014）

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摘要： 【摘要】 目的 探讨病毒巨噬细胞炎症蛋白Ⅱ（vMIP-Ⅱ）对载脂蛋白B mRNA编辑酶催化多肽样蛋白3G（APOBEC3G）表达的影响及其机制。方法 转染vMIP-Ⅱ质粒（vMIP-Ⅱ质粒组）、空质粒（空质粒组）至293T细胞。定量PCR和Western 印迹法分析转染vMIP-Ⅱ基因对293T细胞APOBEC3G表达的影响。空质粒组、vMIP-Ⅱ质粒组细胞分别加入1 000 IU/ml干扰素α（IFN-α），培养36 h后，Western印迹法检测空质粒组、vMIP-Ⅱ质粒组、空质粒 + IFN-α组、vMIP-Ⅱ质粒 + IFN-α组APOBEC3G的蛋白水平。对转染vMIP-Ⅱ质粒的293T细胞，分别用75 μmol/L JAK/STAT信号通路抑制剂AG490和20 μmol/LERK信号通路抑制剂U0126处理，24 h后收集细胞总蛋白，Western印迹法检测APOBEC3G蛋白水平。构建包含APOBEC3G启动子的荧光素酶报告基因重组质粒，启动子片段包括APOBEC3G全长启动子序列（POS）与长度1 560、960、720、480、420、360、330、240 bp序列及APOBEC3G启动子序列中不含调控元件的区域（NEG），将荧光素酶报告基因重组质粒和vMIP-Ⅱ质粒共转染293T细胞为实验组，以与空质粒共转染的293T细胞为对照，检测APOBEC3G启动子活性，分析vMIP-Ⅱ调控APOBEC3G转录活性的关键启动子作用区域。统计学比较采用t检验、单因素方差分析、LSD-t检验。结果 vMIP-Ⅱ转染组APOBEC3G mRNA、蛋白水平（2.500 ± 0.013、1.472 ± 0.013）均高于对照组（1、0.364 ± 0.030，t值分别为 6.22、6.54，均P < 0.05）。空质粒组、vMIP-Ⅱ质粒组、空质粒 + IFN-α组、vMIP-Ⅱ质粒 + IFN-α组APOBEC3G水平（分别为1、2.030 ± 0.108、2.700 ± 0.081、2.600 ± 0.099）差异有统计学意义（F = 67.026，P < 0.001），vMIP-Ⅱ质粒组与空质粒 + IFN-α组差异无统计学意义（t = 3.46，P > 0.05）。vMIP-Ⅱ质粒组、vMIP-Ⅱ质粒 + AG490组、vMIP-Ⅱ质粒 + U0126组APOBEC3G水平（0.617 ± 0.025、0.179 ± 0.061、0.359 ± 0.012）差异有统计学意义（F = 70.019，P < 0.001），后两组均低于vMIP-Ⅱ质粒组（t值分别为9.66、11.836，P < 0.01）。荧光素酶活性检测显示，转染了POS、1 560、960、720、480、420、360、330、240 bp及NEG序列的vMIP-Ⅱ质粒组启动子活性差异有统计学意义（F = 81.092，P < 0.001），从720 bp片段至480 bp片段，APOBEC3G启动子活性降低幅度巨大。结论 vMIP-Ⅱ上调APOBEC3G的表达，可能是通过JAK/STAT信号通路或作用于APOBEC3G转录活性的关键启动子区域。

关键词: HIV-1； 肉瘤, 卡波西； 萤光素酶类； 病毒巨噬细胞炎症蛋白Ⅱ； 抗HIV-1基因； 载脂蛋白B mRNA编辑酶催化多肽3G； JAK-STAT信号通路 

Abstract: 【Abstract】 Objective To assess the effect of viral macrophage inflammatory protein （vMIP）-Ⅱ on the expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G （APOBEC3G）, and to explore the mechanisms. Methods A recombinant plasmid pEGFP-N3-K4 （vMIP-Ⅱ plasmid group） and an empty plasmid pEGFP-N3 （empty plasmid group） were separately transfected into 293T cells, and quantitative PCR and Western blot analysis were performed to evaluate the effect of transfection with vMIP-Ⅱ gene on the APOBEC3G expression in 293T cells. Some 293T cells in the empty plasmid group and vMIP-Ⅱ plasmid group were treated with 1 000 IU/ml interferon （IFN）-α for 36 hours, and then Western blot analysis was conducted to determine the APOBEC3G expression in the empty plasmid group and vMIP-Ⅱ plasmid group with or without IFN-α treatment. Some 293T cells transfected with vMIP-Ⅱ plasmids were treated with 75 μmol/L AG490 （a JAK/STAT signaling pathway inhibitor） and 20 μmol/L U0126 （an ERK signaling pathway inhibitor） separately; after 24 hours, total protein was extracted from 293T cells, and Western blot analysis was conducted to determine the expression of APOBEC3G. A recombinant plasmid containing APOBEC3G promoter was constructed by using a luciferase reporter gene, and the promoter fragment included the full-length promoter sequence （POS） of APOBEC3G, sequences with the lengths of 1 560, 960, 720, 480, 420, 360, 330 and 240 bp, and the regulatory element-free region （NEG） of APOBEC3G, separately. Some 293T cells were co-transfected with the recombinant plasmid carrying luciferase reporter gene and vMIP-Ⅱ plasmid (experimental group), or the recombinant plasmid and empty plasmid (control group). Subsequently, the activity of the APOBEC3G promoter was evaluated, and the key promoter region through which the transcriptional activity of APOBEC3G was regulated by vMIP-Ⅱ was analyzed. Statistical analysis was carried out by using t test, one-way analysis of variance and least significant difference （LSD）-t test. Results The mRNA and protein expression of APOBEC3G was significantly higher in the vMIP-Ⅱ plasmid group （2.500 ± 0.013, 1.472 ± 0.013 respectively） than in the control group （1, 0.364 ± 0.030 respectively; t = 6.22, 6.54 respectively, both P < 0.05）. The APOBEC3G expression significantly differed among the empty plasmid group, vMIP-Ⅱ plasmid group, empty plasmid + IFN-α group and vMIP-Ⅱ plasmid + IFN-α group （1, 2.030 ± 0.108, 2.700 ± 0.081 and 2.600 ± 0.099 respectively; F = 67.026, P < 0.001）, but there was no significant difference between the vMIP-Ⅱ plasmid group and empty plasmid + IFN-α group （t = 3.46, P > 0.05）. The APOBEC3G expression also significantly differed among the vMIP-Ⅱ plasmid group, vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group （0.617 ± 0.025, 0.179 ± 0.061, 0.359 ± 0.012 respectively; F = 70.019, P < 0.001）, and was significantly lower in the vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group than in the vMIP-Ⅱ plasmid group （t = 9.66, 11.836 respectively, both P < 0.01）. Luciferase activity assay showed that the promoter activity significantly differed among the vMIP-Ⅱ plasmid groups transfected with POS, 1 560-, 960-, 720-, 480-, 420-, 360-, 330-, 240-bp or NEG sequences （F = 81.092, P < 0.001）, and the APOBEC3G promoter activity decreased greatly from the 720-bp group to 480-bp group. Conclusion vMIP-Ⅱ upregulates the expression of APOBEC3G, likely through the JAK/STAT signaling pathway or the key promoter region regulating the transcriptional activity of APOBEC3G.

Key words: HIV-1, Sarcoma, Kaposi, Luciferases, Viral macrophage inflammatory protein-Ⅱ, Anti-HIV genes, Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G, JAK-STAT signaling pathway