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 .%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p21WAF1/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-10.88μL SAHA(S组)、0.625 nmol·L-110μL 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Real-time PCR法检测p21 WAF1/CIP1启动子区从TSS到其上游(+2~-4000 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p21WAF1/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-2800 bp至-3200 bp DNA片段是与HDAC1高度结合的靶功能区.
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