Lettlova Sandra, Brynychova Veronika, Blecha Jan, Vrana David, Vondrusova Magdalena, Soucek Pavel, Truksa Jaroslav
Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV Research Center, Prague, Czech Republic.
Faculty of Science, Charles University, Prague, Czech Republic.
Cell Physiol Biochem. 2018;46(6):2601-2615. doi: 10.1159/000489687. Epub 2018 May 7.
BACKGROUND/AIMS: MiRNA-301a-3p is an oncogenic miRNA whose expression is associated with tumor development, metastases and overall poor prognosis. Estrogen receptor α (ERα) is one of the estrogen hormone-activated transcription factors, which regulates a large number of genes and is involved in the mammary gland development. Expression of ERα is considered to be a good indicator for endocrine therapy and breast cancer survival. Loss of ERα in breast cancer patients indicates invasiveness and poor prognosis. In this study, we focus on the regulation of ERα by miR-301a and its role in transition from estrogen-dependent to estrogen-independent breast cancer.
Expression of miR-301a-3p was measured by qRT-PCR in tumor tissue samples from 111 patients with primary breast carcinoma and in mammospheres representing in vitro model of cancer stem-like cells. Dual reporter luciferase assay and complementary experiments were performed to validate ESR1 as a direct target of miR-301a-3p. The effect of miR-301a-3p on estrogen signaling was evaluated on the level of gene and protein expression and growth response to estrogens. Finally, the effect of miR-301a-3p expression on tumor growth was studied in nude mice.
We identified ESR1 as a direct target of miR-301a-3p. Ectopic miR-301a-3p causes a decrease in ESR1 mRNA and protein level and modulates the expression of ERα target genes in ERα positive breast cancer cells. Consistently, miR-301a-3p causes a decrease in sensitivity of MCF7 cells to 17β-estradiol and inhibits the growth of estrogen dependent tumor in nude mice. Yet, the mice tumors have significantly increased expression of genes related to cancer stem-like cells and epithelial to mesenchymal transition suggesting enrichment of the population of cells with more invasive properties, in line with our observation that miR-301a-3p expression is highly increased in mammospheres which show a decrease in estrogenic signaling. Importantly, miR-301a-3P level is also increased in primary breast cancer samples exhibiting an ER/PR negative phenotype.
Our results confirm ESR1 as a direct target of miR-301a-3p and suggest that miR-301a-3p likely contributes to development of estrogen independence, which leads to a more invasive phenotype of breast cancer.
背景/目的：微小RNA-301a-3p是一种致癌性微小RNA，其表达与肿瘤发展、转移及总体预后不良相关。雌激素受体α（ERα）是雌激素激活的转录因子之一，可调节大量基因并参与乳腺发育。ERα的表达被认为是内分泌治疗及乳腺癌生存的良好指标。乳腺癌患者中ERα的缺失表明肿瘤具有侵袭性且预后不良。在本研究中，我们聚焦于miR-301a对ERα的调控及其在乳腺癌从雌激素依赖向雌激素非依赖转变过程中的作用。
通过qRT-PCR检测了111例原发性乳腺癌患者肿瘤组织样本及代表癌干细胞体外模型的乳腺球中miR-301a-3p的表达。进行了双荧光素酶报告基因检测及补充实验以验证ESR1是miR-301a-3p的直接靶点。在基因和蛋白表达水平以及对雌激素的生长反应方面评估了miR-301a-3p对雌激素信号传导的影响。最后，在裸鼠中研究了miR-301a-3p表达对肿瘤生长的影响。
我们确定ESR1是miR-301a-3p的直接靶点。异位表达miR-301a-3p会导致ERα阳性乳腺癌细胞中ESR1 mRNA和蛋白水平降低，并调节ERα靶基因的表达。同样，miR-301a-3p会降低MCF7细胞对17β-雌二醇的敏感性，并抑制裸鼠中雌激素依赖型肿瘤的生长。然而，小鼠肿瘤中与癌干细胞及上皮-间质转化相关的基因表达显著增加，这表明具有更强侵袭性的细胞群体增多，这与我们在雌激素信号传导降低的乳腺球中观察到miR-301a-3p表达高度增加一致。重要的是，在表现出ER/PR阴性表型的原发性乳腺癌样本中，miR-301a-3P水平也升高。
我们的结果证实ESR1是miR-301a-3p的直接靶点，并表明miR-301a-3p可能促成雌激素非依赖状态的发展，进而导致乳腺癌更具侵袭性的表型。
