Objective To investigate the specific sites that estrogen receptor (ER)α could be recruited to in the p21WAF1/CIP1 promoter region to regulate its transcriptional activity in MCF-7 cells,and to clarify the molecular mechanism of suberoylanilide hydroxamic acid (SAHA) and leptin in the regulation of p21WAFI/CIP1 promoter function.Methods MCF-7 cells were starved by culturing them in fetal calf serum-free medium for 24 hours,and then treated with 20 μmol/L of 0.88 μL SAHA (SAHA group) or 0.625 nmol/L of 10 μL leptin (leptin group) for 24 hours,or cultured in complete RPMI-1640 medium (control group).Cell lysates were incubated with anti-ERα antibody for ChIP analysis.The relative expression levels of DNA fragments,ranging from the TSS to upstream of the p21WAF1/CIP1 promoter region (+2 to-4 000 bp),that bound the antibody were detected by real-time PCR.Results In the control group,the relative expression levels of f1,f2,and f8 DNA fragments that bound the antiERα antibody were two-fold higher than the relative expression of the f9 fragment (P ＜ 0.01).In the SAHA and leptin groups,the relative expression of f1 to f10 DNA fragment that bound anti-ERα antibody was significantly lower than that of the control.The binding affinity of ERα for the f8 fragment was the lowest (P ＜ 0.01) in the SAHA group,and it was significantly lower than that in the leptin group (P ＜ 0.01).Conclusion ERα could be recruited to the p21WAFI/CIP1 promoter via signaling pathways activated during the proliferation of breast cancer MCF-7 cells.Moreover,the DNA fragment ranging from-2 800 to-3 200 bp upstream of the p21 WAF1/CIP1 promoter is the target functional region for high-affinity binding with ERα.%目的 研究雌激素受体(ER)α募集于p21WAF1/CIP1启动子区调控其转录活性的具体作用位点,明确辛二酰苯胺异羟肟酸(SAHA)及瘦素(leptin)在调节p21WAF1/CIP1启动子功能中的分子机制.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的SAHA 0.88 μL(SAHA组)、0.625 nmol/L的leptin 10 μL(leptin组)处理24 h,对照组在完全型RPMI-1640培养基中培养细胞.应用染色质免疫共沉淀技术将各组细胞裂解液与ERα抗体孵育,收集纯化结合ERα抗体的DNA片段,应用实时PCR法检测p21WAF1/CIP1启动子区从转录起始点到其上游(+ 2～-4 000 bp) f1 ～f10片段的DNA相对表达量并用2-△△Ct法分析.结果 对照组中,与ERα抗体结合的f1、f2、f8片段DNA相对表达量较f9片段高出2倍以上(P＜0.01).与对照组比较,SAHA及leptin组f1～f10片段与ERα抗体结合能力均降低,其中SAHA组f8片段DNA相对表达量达最低值(P＜0.01),且明显低于leptin组(P＜0.01).SAHA组中以f8片段为对照,其他片段与ERα抗体结合能力均较其升高(P＜ 0.05或0.01).leptin组中以f8片段为对照,其他片段与ERα抗体结合能力均较其降低,除f1外均有统计学差异(P＜0.01).结论 乳腺癌细胞增殖过程中细胞增殖信号招募ERα至p21WAF1/CIP1启动子区,且p21WAF1/CIP1启动子区-2 800bp～-3 200 bp区域存在与ERα高度结合的靶功能区.
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