Medical Molecular Biology Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.
Breast Cancer Res. 2011 Jan 17;13(1):R5. doi: 10.1186/bcr2809.
In cancer cells, elevated transcription factor-related Brn-3a regulator isolated from brain cDNA (Brn-3b) transcription factor enhances proliferation in vitro and increases tumour growth in vivo whilst conferring drug resistance and migratory potential, whereas reducing Brn-3b slows growth both in vitro and in vivo. Brn-3b regulates distinct groups of key target genes that control cell growth and behaviour. Brn-3b is elevated in >65% of breast cancer biopsies, but mechanisms controlling its expression in these cells are not known.
Bioinformatics analysis was used to identify the regulatory promoter region and map transcription start site as well as transcription factor binding sites. Polymerase chain reaction (PCR) cloning was used to generate promoter constructs for reporter assays. Chromatin immunoprecipitation and site-directed mutagenesis were used to confirm the transcription start site and autoregulation. MCF-7 and Cos-7 breast cancer cells were used. Cells grown in culture were transfected with Brn-3b promoter and treated with growth factors or estradiol to test for effects on promoter activity. Quantitative reverse transcriptase PCR assays and immunoblotting were used to confirm changes in gene and protein expression.
We cloned the Brn-3b promoter, mapped the transcription start site and showed stimulation by estradiol and growth factors, nerve growth factor and epidermal growth factor, which are implicated in breast cancer initiation and/or progression. The effects of growth factors are mediated through the mitogen-activated protein kinase pathway, whereas hormone effects act via oestrogen receptor α (ERα). Brn-3b also autoregulates its expression and cooperates with ERα to further enhance levels.
Key regulators of growth in cancer cells, for example, oestrogens and growth factors, can stimulate Brn-3b expression, and autoregulation also contributes to increasing Brn-3b in breast cancers. Since increasing Brn-3b profoundly enhances growth in these cells, understanding how Brn-3b is increased in breast cancers will help to identify strategies for reducing its expression and thus its effects on target genes, thereby reversing its effects in breast cancer cells.
在癌细胞中，从脑 cDNA（Brn-3b）转录因子中分离出的转录因子相关 Brn-3a 调节剂升高，体外增强增殖，并在体内增加肿瘤生长，同时赋予耐药性和迁移潜能，而降低 Brn-3b 则会在体外和体内均减缓生长。Brn-3b 调节控制细胞生长和行为的关键靶基因的不同组。Brn-3b 在>65%的乳腺癌活检中升高，但控制其在这些细胞中表达的机制尚不清楚。
使用生物信息学分析来识别调节启动子区域并绘制转录起始位点以及转录因子结合位点。聚合酶链反应（PCR）克隆用于生成报告基因检测的启动子构建体。使用染色质免疫沉淀和定点突变来确认转录起始位点和自我调节。使用 MCF-7 和 Cos-7 乳腺癌细胞。在培养中生长的细胞用 Brn-3b 启动子转染，并用生长因子或雌二醇处理，以检测对启动子活性的影响。使用定量逆转录 PCR 检测和免疫印迹来确认基因和蛋白质表达的变化。
我们克隆了 Brn-3b 启动子，绘制了转录起始位点，并显示雌激素和生长因子（神经生长因子和表皮生长因子）刺激，这些因子与乳腺癌的发生和/或进展有关。生长因子的作用是通过丝裂原激活的蛋白激酶途径介导的，而激素作用则通过雌激素受体 α（ERα）起作用。Brn-3b 还自我调节其表达，并与 ERα 合作进一步增强水平。
癌细胞中生长的关键调节剂，例如雌激素和生长因子，可以刺激 Brn-3b 的表达，自我调节也有助于增加乳腺癌中的 Brn-3b。由于增加 Brn-3b 会极大地增强这些细胞的生长，因此了解 Brn-3b 在乳腺癌中如何增加将有助于确定减少其表达的策略，从而减少其对靶基因的影响，从而逆转其在乳腺癌细胞中的作用。
