MAF Amplification licenses Estrogen Receptor α to Drive Breast Cancer Metastasis [RNA-seq]

We performed mRNA profiling of control and MAF-overexpressing MCF7 cells to assess the transcriptional consequences of estrogen receptor (ER) activation upon metastatic MAF expression. To this end, we cultured MCF7 cells in hormone-deprived (HD) medium for 72 h and then estrogen (E2) or vehicle was added for 6h prior to RNA extraction. Samples were generated in triplicate. We report that MAF supports the expression of genes involved in metastatic functions and that E2 treatment correlates with E2 early and late gene responses and cell cycle regulation. Additionally, a set of MAF and E2-dependent gene responses was identified. Our results show that MAF expression causes an expansion of ER-mediated transcriptional events in MCF7 cells exposed to E2. Additionally, we silenced KDM1A expression in MAF-overexpressing MCF7 cells to test whether this histone demethylase mediates the transcriptional consequences of MAF overexpression. Samples with KDM1A knockdown were generated in duplicates. We report that KDM1A partially regulates MAF/E2-dependent gene responses. To assess gene expression changes governed by MAF and estrogen (E2), control or MAF-overexpressing MCF7 cells were maintained in hormone-deprived medium for 72 h. Then, E2 or vehicle was added for 6h prior to RNA extraction and mRNA profiling. To evaluate the effect of KDM1A expression on the regulation of MAF/E2-dependent gene responses, expression of the histone demethylase was silenced in MAF-overexpressing cells using shRNA followed by selection with 2µg/mL puromycin for 72 h. Then cells were cultured in hormone-deprived medium before E2 stimulation as previously explained.

本研究对对照组与过表达MAF的MCF7细胞开展mRNA表达谱分析，以探究转移性MAF表达时雌激素受体（ER）激活所引发的转录调控效应。为此，我们将MCF7细胞置于激素剥夺（HD medium）培养基中培养72小时，随后加入雌激素（E2）或溶剂对照，继续培养6小时后进行RNA提取，所有样本均设置三次生物学重复。研究结果表明，MAF可促进与转移功能相关基因的表达，而E2处理则与E2早期、晚期基因应答及细胞周期调控过程显著相关；此外，本研究还鉴定出一组同时依赖于MAF与E2的基因应答特征。我们的实验结果显示，在经E2处理的MCF7细胞中，MAF的表达会扩大ER介导的转录调控事件范围。此外，我们还通过短发夹RNA（shRNA）在过表达MAF的MCF7细胞中敲低KDM1A的表达，以验证该组蛋白去甲基化酶是否介导了MAF过表达所带来的转录调控变化，KDM1A敲低的样本设置两次生物学重复。研究发现，KDM1A可部分调控MAF/E2依赖的基因应答过程。为评估由MAF与雌激素（E2）介导的基因表达变化，我们将对照组或过表达MAF的MCF7细胞置于激素剥夺培养基中培养72小时，随后加入E2或溶剂对照，培养6小时后进行RNA提取与mRNA表达谱分析。为探究KDM1A表达对MAF/E2依赖基因应答调控的影响，我们先用shRNA在过表达MAF的细胞中沉默该组蛋白去甲基化酶的表达，随后以2µg/mL嘌呤霉素筛选72小时，后续实验步骤与此前一致：将细胞置于激素剥夺培养基中培养，再进行E2刺激。