Time-course effect of estradiol and ERa17p on Early Gene expression in MDA-MB-231 cells. Homo sapiens

ERα17p is a synthetic peptide corresponding to the sequence P295LMIKRSKKNSLALSLT311 of the estrogen receptor alpha (ERα) and initially synthesized to mimic its calmodulin binding site. ERα17p was subsequently found to elicit estrogenic responses in E2-deprived ERα-positive breast cancer cells, increasing proliferation and E2-dependent gene transcription. Surprisingly, in E2-supplemented media, ERα17p induced apoptosis and modified the actin network, influencing thereby cell motility. Here, we report that ERα17p induces a massive early (3h) transcriptional activity in breast cancer cell line MDA-MB-231. Overall design: Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with or without E2 (10-6M) or ERa17p in RPMI 1640 supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturer’s instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.ERα17p是对应雌激素受体α（estrogen receptor alpha, ERα）序列P295LMIKRSKKNSLALSLT311的合成多肽，最初为模拟其钙调蛋白结合位点而合成。后续研究发现，ERα17p可在雌激素剥夺（E2-deprived）的ERα阳性乳腺癌细胞中触发雌激素样应答，促进细胞增殖并增强E2依赖的基因转录。令人意外的是，在添加了E2的培养基中，ERα17p反而会诱导细胞凋亡并重塑肌动蛋白网络，进而影响细胞运动能力。 本研究报道了ERα17p可在乳腺癌细胞系MDA-MB-231中诱导大规模的早期（3小时）转录激活活性。 总体实验设计：将细胞在含10%活性炭吸附胎牛血清的培养基中孵育4小时后，置于添加10%活性炭吸附胎牛血清的RPMI 1640培养基中，分别用或不用10^-6 M E2或ERα17p处理3小时。依照制造商说明书，采用Nucleospin II柱式试剂盒（Macherey-Nagel，德国迪伦）提取总RNA。按照Affymetrix基因芯片表达分析技术手册的标准流程完成RNA标记与杂交，使用HGU133A plus 2芯片，共分析54675个转录本。信号通过Affymetrix基因芯片扫描仪进行检测。