Estrogen regulated microRNAs in mouse ovarian cancer cells.. Mus musculus

In a mouse model of ovarian cancer, we have established that prolonged exposure to 17β-estradiol (E2) accelerates tumour onset and increases the incidence of morphologically dysplastic ovarian surface epithelium (OSE). OSE cell proliferation and morphology are tightly regulated by the asymmetrical distribution of polarity proteins that provide positional cues for surface localization and growth inhibition. We hypothesized that E2 causes OSE dysplasia by inhibiting a tumour suppressor gene called Disabled-2 (Dab2). Dab2 is critical in mediating the polarized distribution of cell surface proteins and is highly expressed in normal OSE, but is absent in the majority of ovarian cancers. In this study, Dab2 is shown to be suppressed by E2 and we investigated the possibility that this occurs through E2 up-regulation of microRNAs. microRNA microarray analysis comparing control vs. E2 treated mouse ovarian cancer cells (MASE) was used to identify candidate miRNAs that have a seeding sequence capable of targeting the 3-prime untranslated region (3’UTR) of both human and mouse Dab2 transcript. Overall design: MASE ovarian cancer cells were isolated from ascites fluid of estrogen treated tgCAG-TAg mice. Once cells were established in culture, they were treated with 100nM E2 for 24H. An equivalent volume of 100% EtOH (vehicle) was added to control plates for a final concentration of 0.0002% EtOH.

本研究在卵巢癌小鼠模型中证实，长期暴露于17β-雌二醇（17β-estradiol, E2）可加速肿瘤发生，并提升形态异常卵巢表面上皮（OSE）的发生率。卵巢表面上皮细胞的增殖与形态，严格受极性蛋白的不对称分布调控——这类极性蛋白可为细胞表面定位及生长抑制提供位置信号。我们提出假说：E2通过抑制抑癌基因Disabled-2（Dab2）诱导卵巢表面上皮发育异常。Dab2在介导细胞表面蛋白的极性分布中发挥关键作用，正常卵巢表面上皮中Dab2呈高表达状态，但多数卵巢癌组织中无Dab2表达。本研究证实E2可抑制Dab2的表达，并探究了其通过E2上调微小RNA（microRNA, miRNA）实现该调控的可能性。本研究采用对比对照组与E2处理的小鼠卵巢癌细胞（MASE）的微小RNA芯片分析，筛选出具备种子序列、可靶向人及小鼠Dab2转录本3'非翻译区（3’UTR）的候选miRNA。实验整体设计如下：从经雌激素处理的tgCAG-TAg转基因小鼠的腹水中分离得到MASE卵巢癌细胞；待细胞成功建立体外培养体系后，用100nM的E2处理24小时；对照组则加入等体积的100%乙醇（溶剂对照），使最终乙醇浓度为0.0002%。