CAJ | 학술논문

目的探讨人T淋巴细胞白血病1型病毒（human T-cell leukemia virus 1，HTLV-1）Tax蛋白对人高迁移率族蛋白1（ high mobility group box 1，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-time PCR和Western blot分析HMGB1 mRNA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HMGB1 mRNA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～＋83 HMGB1区段）的相对荧光素酶活性（HMGB1/neo）最高，但是6号质粒（含有-1163～＋83 HMGB1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。
목적탐토인T림파세포백혈병1형병독（human T-cell leukemia virus 1，HTLV-1）Tax단백대인고천이솔족단백1（ high mobility group box 1，HMGB1）기인전록조공적영향。방법제취TaxN화TaxP세포총RNA화단백질，통과real-time PCR화Western blot분석HMGB1 mRNA화단백질적표체정황；이용지질체개도방법，장6개함유불동장도HMGB1조공서렬적pGL3-HMGB1-luc순시전염지TaxN 화 TaxP 세포，관찰 HMGB1기인재불동 T 세포중적전록활성；pCMV-Tax 여 pGL3-HMGB1-luc순시공전염지Jurkat세포，관찰Tax단백대HMGB1기인적전록조공영향；염색체면역공침정（ ChIP）조심Tax단백영향HMGB1기인전록조공적구단。결과 TaxP세포중HMGB1 mRNA화단백질표체수평고우TaxN세포。 TaxN화TaxP세포중HMGB1조공추세기본상사，균표현출3호질립（pHLuc3，함유-504～＋83 HMGB1구단）적상대형광소매활성（HMGB1/neo）최고，단시6호질립（함유-1163～＋83 HMGB1구단）각표현출 TaxP 세포 HMGB1적전록활성명현고우 TaxN 세포。pCMV-Tax여pGL3-HMGB1-Luc보고기인공전염도Jurkat세포야현시，6호질립（pHLuc6）중Tax촉진HMGB1기인적전록。 ChIP분석증실료Tax단백가능부집재HMGB1적-1163～-1043구단。결론-504～-383가능시HMGB1기인전록격활적관건계동자구，Tax단백가능부집재HMGB1적-1163～-1043구단촉진HMGB1기인전록。
Objective 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 cells ( 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 CD 4+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-HMGB1-luc reporter genes were also co-transfected into Jurkat cells and the regulatory effects of Tax protein on HMGB 1 gene was detected .The chromatin immunoprecipi-tation (ChIP) assay was used to identify HMGB1 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 cells.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 pHLuc 6 in TaxP cells 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 pHLuc 6 reporter plasmid , and Tax protein enriched probably at the HMGB 1 site of -1163--1043 enhances HMGB1 transcription.