乳腺癌MCF-7细胞p21^(WAF1/CIP1)启动子区HDAC1高功能结合位点的研究  

A study on high function binding site of HDAC1 in p21^(WAF1/CIP1) promoter region in breast cancer MCF-7 cells

作　　者：邹丹[1] 周伟强[2] 

机构地区：[1]沈阳医学院病理生理学教研室,辽宁沈阳110034 [2]沈阳医学院辽宁省环境污染与微生态重点实验室,辽宁沈阳110034

出　　处：《中国药理学通报》2017年第3期317-321,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81172509);辽宁省自然科学基金资助项目(No 201602735);沈阳市科技计划项目(No F15-199-1-28)

摘　　要：目的研究乳腺癌MCF-7细胞中组蛋白去乙酰化酶1(histone deacetylases 1,HDAC1)募集于p21^(WAF1/CIP1)启动子区调控其转录活性的特异性结合位点。方法将处于对数生长期的乳腺癌MCF-7细胞在无血清培养基中饥饿24 h后,分别用20μmol·L^(-1)0.88μL SAHA(S组)、0.625 nmol·L^(-1)10μL Leptin(L组)处理24 h,对照组(B组)细胞培养在完全型RPMI 1640培养基中。各组细胞裂解液与HDAC1抗体孵育,收集纯化结合HDAC1抗体的DNA片段,应用Realtime PCR法检测p21^(WAF1/CIP1)启动子区从TSS到其上游(+2^-4 000 bp)f1~f10片段的DNA相对表达量并用2-ΔΔCT法分析。结果 B组中,HDAC1抗体在p21^(WAF1/CIP1)启动子区f1、f8片段有高亲和力,f8片段达最高。S组中,HDAC1抗体与p21^(WAF1/CIP1)启动子区f1~f10片段结合量明显低于对照组,f8片段达最低,而在L组此片段与HDAC1抗体结合量达最大值。结论乳腺癌MCF-7细胞增殖过程中,HDAC1可被招募至p21^(WAF1/CIP1)启动子区,该启动子区上游-2 800 bp至-3 200 bp DNA片段是与HDAC1高度结合的靶功能区。Aim Toinvestigatethespecificbinding sites that HDAC1 can be recruited to regulate the tran-scriptional activity of p21 WAF1/CIP1 promoter in the breast cancer MCF-7 cells.Methods ThebreastcancerMCF-7 cells in logarithmic growth phase were starved with FBS free medium for 24 hours,and treated with 20 μmol·L-1 SAHA（S group）or 0. 625 nmol·L-1 Leptin（L group）for 24 hours,and the cells that were cultured in the complete RPMI 1640 medium without any treatment were assigned as control group （B group）.The DNA-ChIP was followed the manufactur-er′s protocol for the assay.The cell lysis was prepared and incubated with anti-HDAC1 antibody overnight at 4℃.DNA fragments binding anti-HDAC1 antibody were gathered and purified.The relative expression level of DNA fragments from TSS to the upstream of the p21 WAF1/CIP1 promoter region（＋2 ^-4000 bp）bind-ing with antibody was detected by Real-time PCR and analyzedby2-ΔΔCTmethod.Results InBgroup, HDAC1 had high affinity with the f1 and f 8 fragmentsof p21 WAF1/CIP1 promoter compared to the other fragemts,and showed the highest affinity with the f8 fragment.In S group,the binding ability of HDAC1 to the f1 ~f10 fragment of p21 WAF1/CIP1 promoter was sig-nificantly lower than that of the control.The binding activity of HDAC1 to f8 fragment was the lowest,while reversing to reach the peak after leptin treatment.Con-clusions HDAC1canberecruitedtop21WAF1/CIP1pro-moter by the cell proliferation signal during the prolifer-ation of breast cancer MCF-7 cells.The DNA fragment from -2800 to -3200 bp in the upstream of p21 WAF1/CIP1 promoter is the target functional region for the binding with HDAC1 .

关 键 词：乳腺癌 MCF-7细胞 p21(WAF1/CIP1) 组蛋白去乙酰化酶1 辛二酰苯胺异羟肟酸 瘦素 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R392.11[医药卫生—基础医学]