Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 0XZ, UK.
J Pathol. 2011 Aug;224(4):496-507. doi: 10.1002/path.2898. Epub 2011 May 18.
Although gain of chromosome 5p is one of the most frequent DNA copy-number imbalances in cervical squamous cell carcinoma (SCC), the genes that drive its selection remain poorly understood. In a previous cross-sectional clinical study, we showed that the microRNA processor Drosha (located on chromosome 5p) demonstrates frequent copy-number gain and overexpression in cervical SCC, associated with altered microRNA profiles. Here, we have conducted gene depletion/overexpression experiments to demonstrate the functional significance of up-regulated Drosha in cervical SCC cells. Drosha depletion by RNA interference (RNAi) produced significant, specific reductions in cell motility/invasiveness in vitro, with a silent RNAi-resistant Drosha mutation providing phenotype rescue. Unsupervised hierarchical clustering following global profiling of 319 microRNAs in 18 cervical SCC cell line specimens generated two groups according to Drosha expression levels. Altering Drosha levels in individual SCC lines changed the group into which the cells clustered, with gene depletion effects being rescued by the RNAi-resistant mutation. Forty-five microRNAs showed significant differential expression between the groups, including four of 14 that were differentially expressed in association with Drosha levels in clinical samples. miR-31 up-regulation in Drosha-overexpressing samples/cell lines was the highest-ranked change (by adjusted p value) in both analyses, an observation validated by northern blotting. These functional data support the role of Drosha as an oncogene in cervical SCC, by affecting expression of cancer-associated microRNAs that have the potential to regulate numerous protein-coding genes.
虽然染色体 5p 的获得是宫颈鳞状细胞癌 (SCC) 中最常见的 DNA 拷贝数失衡之一,但驱动其选择的基因仍知之甚少。在之前的横断面临床研究中,我们表明微处理器 Drosha(位于染色体 5p 上)在宫颈 SCC 中经常表现出拷贝数增益和过表达,与改变的 microRNA 谱相关。在这里,我们进行了基因耗竭/过表达实验,以证明上调的 Drosha 在宫颈 SCC 细胞中的功能意义。通过 RNA 干扰 (RNAi) 进行的 Drosha 耗竭在体外产生了显著的、特异性的细胞迁移/侵袭减少,而沉默的 RNAi 抗性 Drosha 突变提供了表型挽救。在 18 个宫颈 SCC 细胞系标本中对 319 个 microRNA 进行全局分析后,进行无监督层次聚类,根据 Drosha 表达水平将细胞分为两组。改变单个 SCC 系中的 Drosha 水平会改变细胞聚类的组,RNAi 抗性突变挽救了基因耗竭效应。45 个 microRNAs 在两组之间表现出显著的差异表达,包括在临床样本中与 Drosha 水平相关的 14 个中有 4 个差异表达。在 Drosha 过表达样本/细胞系中 miR-31 的上调是两种分析中排名最高的变化(通过调整后的 p 值),Northern blot 验证了这一观察结果。这些功能数据支持 Drosha 在宫颈 SCC 中作为癌基因的作用,通过影响与癌症相关的 microRNAs 的表达,这些 microRNAs 具有调节众多蛋白质编码基因的潜力。