HTLV-1 Tax蛋白对T细胞中HMGB1调控的影响  

The regulatory effects of HTLV-1 Tax protein on HMGB1 gene in T cells

作　　者：张晨光[1,2] 牛志国[1] 王辉[1] 尹明梅[1] 李月[1] 朱琳琳[1] 赵庆伟[1] 丁肖华[1] 化瑞芳[1] 蒲亚陆[1] 胡丽华[2] 

机构地区：[1]新乡医学院医学检验系,453003 [2]华中科技大学附属协和医院检验科,武汉430022

出　　处：《中华微生物学和免疫学杂志》2013年第7期501-506,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81273241）;济南省科技厅项目（132102310163）;河南省教育厅重点项目（12A310006）;新乡医学院重点研究领域招标课题（ZD2011-13,ZD2011-15）

摘　　要：目的探讨人T淋巴细胞白血病1型病毒（humanT—cellleukemiavirus1，HTLV-1）Tax蛋白对人高迁移率族蛋白1（highmobilitygroupbox1，HMGB1）基因转录调控的影响。方法提取TaxN和TaxP细胞总RNA和蛋白质，通过real—timePCR和Westernblot分析HMGB1mRNA和蛋白质的表达情况；利用脂质体介导方法，将6个含有不同长度HMGB1调控序列的pGL3-HMGB1-luc瞬时转染至TaxN和TaxP细胞，观察HMGB1基因在不同T细胞中的转录活性；pCMV—Tax与pGL3-HMGB1-luc瞬时共转染至Jurkat细胞，观察Tax蛋白对HMGB1基因的转录调控影响；染色体免疫共沉淀（ChIP）找寻Tax蛋白影响HMGB1基因转录调控的区段。结果TaxP细胞中HMGB1mRNA和蛋白质表达水平高于TaxN细胞。TaxN和TaxP细胞中HMGB1调控趋势基本相似，均表现出3号质粒（pHLuc3，含有-504～＋83HMGB1区段）的相对荧光素酶活性（HMGB1／neo）最高，但是6号质粒（含有-1163-＋83HMGB1区段）却表现出TaxP细胞HMGB1的转录活性明显高于TaxN细胞。pCMV．Tax与pGL3．HMGB1．Luc报告基因共转染到Jurkat细胞也显示，6号质粒（pHLuc6）中Tax促进HMGB1基因的转录。ChIP分析证实了Tax蛋白可能富集在HMGB1的-1163—-1043区段。结论-504—-383可能是HMGB1基因转录激活的关键启动子区，Tax蛋白可能富集在HMGB1的-1163～-1043区段促进HMGB1基因转录。Obiective To explore the regulatory effects of HTLV-1 （human T-cell leukemia virus type 1 ） Tax protein on the expression of HMGB 1 （ high mobility group box 1 ） gene in T cells. Methods Total RNA and protein were extracted from Tax＋-T cells （TaxP）, Tax--T ceils （TaxN） and Jurkat cells which were stably transfected with pCMV-Tax and pCMV-Neo, respectively. Then, the expression levels of HMGB1 mRNA and protein in different CIM＋T cells were analyzed by real-time PCR and Western blot （WB）. By using liposome-mediated method, pGL3-HMGB1-luc reporter genes and pGL3-neo-luc were tran- siently transfected into TaxP and TaxN cells and the basal transcriptional activity was observed in different T cells. Additionally, pCMV-Tax and pGL3-HMGBI-luc reporter genes were also co-transfected into Jurkat cells and the regulatory effects of Tax protein on HMGB1 gene was detected. The chromatin immunoprecipi- tation （CHIP） assay was used to identify HMGB.1 genomic sites directly targeted by Tax. Results The ex- pression levels of HMGB1 mRNA and protein in Tax＋-T cells （TaxP） were higher than those in Tax--T cells （TaxN）. The transcription regulation trends for HMGB1 gene in TaxN and TaxP cells were similar but not identical in diverse T ceils, pHLuc3 （ containing -504-＋83 HMGB1 ） showed the highest transcriptional ac- tivity of HMGB1 gene in both TaxP and TaxN cells, but HMGB1 transcriptional activity of pHLuc6 in TaxPcells was significantly stronger than that in TaxN cells. Luciferase assays also showed that Tax protein promo- ted the transcription of HMGB1 gene in a dose-dependent manner. The ChIP assay further confirmed that Tax protein enriched at the HMGB1 region of -1163--1043. Conclusion The region of nt -504--383 is essen- tial for the basal promoter activity of -1163-＋83 HMGB1 gene originated from pHLuc6 reporter plasmid, and Tax protein enriched probably at the HMGB1 site of -1163--1043 enhances HMGB1 transcription.

关 键 词：成人T细胞白血病病毒1型 TAX蛋白 高迁移率族蛋白1 T淋巴细胞 基因调控 

分 类 号：R733[医药卫生—肿瘤]