Laboratory of Antineoplastic Pharmacology, Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Rome 00168, Italy.
BMC Cancer. 2013 Feb 8;13:72. doi: 10.1186/1471-2407-13-72.
MicroRNAs in solid malignancies can behave as predictors of either good or poor outcome. This is the case with members of the miR-200 family, which are the primary regulators of the epithelial to mesenchymal transition and have been reported to act as both oncogenes and tumor suppressors. This study assessed the role of miR-200c as regulator of class III β-tubulin (TUBB3), a factor associated with drug-resistance and poor prognosis in ovarian cancer.
Expression of miR-200c was assessed in a panel of ovarian cancer cell lines with inherent or acquired drug-resistance. Stable overexpression of miR-200c was obtained in A2780 and Hey cell lines. Crosslinking-coupled affinity purification method and ribonucleic-immunoprecipitation assay were used to characterise the complexes between miR-200c, HuR and 3'UTR region of TUBB3 mRNA. Nanofluidic technology and immunohistochemistry were used to analyze the expression of HuR, TUBB3 and miR-200c in 220 ovarian cancer patients.
In a panel of ovarian adenocarcinoma cell lines, we observed a direct correlation between miR-200c expression and chemoresistance. In A2780 cells miR-200c targeted TUBB3 3'UTR, while a positive correlation was observed between miR-200c and TUBB3 expression in most of the other cell lines. Through the analysis of 3'UTR-associated complexes, we found that the miR-200c can increase the association of the RNA binding protein HuR with TUBB3 mRNA, whereas HuR binding enhanced TUBB3 mRNA translation. Most importantly, in our analysis on 220 ovarian cancer patients we observed that overexpression of miR-200c correlated with poor or good outcome depending on the cellular localization of HuR.
This study suggests a model for the combined regulatory activity of miR-200c and HuR on TUBB3 expression in ovarian cancer. When HuR is nuclear, high expression of miR-200c inhibits TUBB3 expression and results in a good prognosis, whereas when HuR occurs in cytoplasm, the same miRNA enhances TUBB3 expression and produces a poor outcome. These findings reveal the usefulness of multidimensional analysis in the investigation of the prognostic role of miRNA expression.
实体恶性肿瘤中的 microRNAs 可以作为预后良好或不良的预测因子。miR-200 家族成员就是这种情况，它们是上皮间质转化的主要调节剂，已被报道具有癌基因和肿瘤抑制子的双重作用。本研究评估了 miR-200c 作为 III 类β-微管蛋白（TUBB3）调节剂的作用，TUBB3 是与卵巢癌药物耐药性和预后不良相关的因素。
在具有固有或获得性耐药性的卵巢癌细胞系中评估 miR-200c 的表达。在 A2780 和 Hey 细胞系中获得 miR-200c 的稳定过表达。采用交联偶联亲和纯化法和核糖核酸免疫沉淀法鉴定 miR-200c、HuR 和 TUBB3 mRNA 3'UTR 区之间的复合物。采用纳米流体技术和免疫组织化学方法分析 220 例卵巢癌患者中 HuR、TUBB3 和 miR-200c 的表达。
在一组卵巢腺癌细胞系中，我们观察到 miR-200c 表达与化疗耐药性之间存在直接相关性。在 A2780 细胞中，miR-200c 靶向 TUBB3 3'UTR，而在大多数其他细胞系中，miR-200c 与 TUBB3 表达之间存在正相关。通过对 3'UTR 相关复合物的分析，我们发现 miR-200c 可以增加 RNA 结合蛋白 HuR 与 TUBB3 mRNA 的结合，而 HuR 结合增强了 TUBB3 mRNA 的翻译。最重要的是，在对 220 例卵巢癌患者的分析中，我们观察到 miR-200c 的过表达与 HuR 的细胞内定位有关，与预后不良或良好相关。
本研究提出了 miR-200c 和 HuR 对卵巢癌 TUBB3 表达联合调控作用的模型。当 HuR 位于核内时，高表达 miR-200c 抑制 TUBB3 表达，导致预后良好，而当 HuR 位于细胞质中时，相同的 miRNA 增强 TUBB3 表达，导致预后不良。这些发现揭示了多维分析在 miRNA 表达预后作用研究中的有用性。
