乳腺癌MCF-7细胞p21^(WAF1/CIP1)启动子区雌激素受体α的高功能结合位点

High-affinity Binding Sites for Estrogen Receptor α in the p21^(WAF1/CIP1) Promoter Region in Breast Cancer MCF-7 Cells

目的研究雌激素受体(ER)α募集于p21^(WAF1/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p21^(WAF1/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 0.88μL(SAHA组)、0.625 nmol/L的leptin 10μL(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p21^(WAF1/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-ΔΔCt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p21^(WAF1/CIP1)启动子区,且p21^(WAF1/CIP1)启动子区-2 800 bp^-3 200 bp区域存在与ERα高度结合的靶功能区。

Objective To investigate the specific sites that estrogen receptor （ER）α could be recruited to in the p21^WAF1/CIP1 promoter region to regulate its transcriptional activity in M CF-7 ceils, and to clarify the molecular mechanism of suberoylanilide hydroxamic acid （SAHA） and leptin in the regulation of p21^WAF1/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 p, mol/L of 0.88 μL SAHA （SAHA group） or 0.625 nmol/L of 10 μL leptin （leptin group） for 24 hours,or cul- tured in complete RPMI-1640 medium （control group）. Cell lysates were incubated with anti-ERct antibody for ChIP analysis. The relative expres- sion levels of DNA fragments, ranging from the TSS to upstream of the p21^WAF1/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 anti- ERet antibody were two-fold higher than the relative expression of the f9 fragment （P 〈 0.01 ）. In the SAHA and leptin groups, the relative expres- sion of fl to f10 DNA fragment that bound anti-ERα antibody was significantly lower than that of the control. The binding affinity of ERa 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 p21^WAF1/CIP1 promoter via signaling pathways activated during the proliferation of breast cancer MCF-7 cells. More- over,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 bind- ing with ERα.