HDAC1在调控乳腺癌MCF-7细胞p21^(WAF1/CIP1)转录过程中与ERα的协同作用

Synergistic effect of ERα on HDAC1 regulating transcription of p21^(WAF1/CIP1) in breast cancer MCF-7 cells

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)、雌激素受体α(estrogen receptorα,ERα)共同募集于p21^(WAF1/CIP1)启动子特定区域,调控其转录活性的具体作用位点,同时明确辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid,SAHA)及瘦素(Leptin)在调节p21^(WAF1/CIP1)启动子功能中的作用机制。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(SAHA组)、0.625nmol·L^(-1)10μL Leptin(Leptin组)处理24 h,对照组(Basal组)细胞培养在完全型RPMI 1640培养基中。各组细胞裂解液先后与HDAC1抗体及ERα抗体进行染色质免疫共沉淀(chromatin-immunoprecipitation,Ch IP)孵育,收集纯化结合HDAC1及ERα抗体的DNA片段,应用Real-time PCR法检测p21^(WAF1/CIP1)启动子区从转录起始位点(transcription start site,TSS)到其上游(+2^-4 000 bp)f1~f10片段的DNA相对表达量,并用2^(-△△CT)法分析。结果Basal组中,HDAC1、ERα抗体在p21^(WAF1/CIP1)启动子区f1、f8片段有高亲和力。SAHA组中,HDAC1、ERα抗体与p21^(WAF1/CIP1)启动子区f1、f8片段结合量明显低于对照组,而在Leptin组两片段与HDAC1、ERα抗体结合量明显高于对照组。结论乳腺癌MCF-7细胞增殖过程中,细胞增殖信号可招募HDAC1、ERα至p21^(WAF1/CIP1)启动子区。该启动子区上游0^-400bp,-2 800^-3 200 bp DNA片段是与HDAC1、ERα共同作用的靶功能区。

Aim To investigate the specific binding sites that histone deacetylases 1（ HDAC1） and estrogen receptor α（ ERα） can be recruited to regulate the transcriptional activity of p21^WAF1/CIP1 promoter in the breast cancer MCF-7 cells,and to clarify the molecular mechanism of suberoylanilide hydroxamic acid（ SAHA） and leptin regulating p21^WAF1/CIP1 promoter function. Methods The breast cancer MCF-7 cells in logarithmic growth phase were starved with FBS free medium for 24 hours,and treated with 20 μmol · L^-1SAHA（ SAHA group） or 0. 625 nmol·L^-1leptin（ Leptin group） for24 hours. The cells that were cultured in complete RPMI 1640 medium without any treatment were assigned as control group（ Basal group）. The cell lysis was prepared and incubated respectively with anti-HDAC1 and anti-ERα antibody by chromatin-immunoprecipitation（ Ch IP） method overnight at 4℃. The DNA-Ch IP was followed the manufacturer＇ s protocol for the assay.DNA fragments binding anti-HDAC1 and anti-ERα 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 --4 000 bp）binding with antibody was detected by real-time PCR and analyzed by 2-（-△△CT）method. Results In basal group,HDAC1 and ERα had high affinity with the f1 and f8 fragments of p21^WAF1/CIP1 promoter compared to the f4 fragment. In SAHA group,the binding ability of HDAC1 and ERα to the f1 and f8 fragments of p21^WAF1/CIP1 promoter was significantly lower than that of the control,while reversing to reach the peak after leptin treatment. Conclusions HDAC1 and ERα can be recruited to p21^WAF1/CIP1 promoter by the cell prolif-eration signal during the proliferation of breast cancer MCF-7 cells. The DNA f1（ from 0 to-400 bp） and f8（ from-2 800 to-3 200 bp） fragment in the upstream of p21^WAF1/CIP1 promoter are the target functional region for the binding with HDAC1 and ERα.