Functional Profiling of Precursor MicroRNAs Identifies MicroRNAs Essential for Glioma Proliferation

Cancer initiation and progression involve microRNAs that can function like tumor suppressors and oncogenes. The functional significance of most miRNAs is currently unknown. To determine systematically which microRNAs are essential for glioma growth, we screened a precursor microRNA library in three human glioblastoma and one astroglial cell line model systems. The most prominent and consistent cell proliferation–reducing hits were validated in secondary screening with an additional apoptosis endpoint. The functional screening data were integrated in the miRNA expression data to find underexpressed true functional tumor suppressor miRNAs. In addition, we used miRNA-target gene predictions and combined siRNA functional screening data to find the most probable miRNA-target gene pairs with a similar functional effect on proliferation. Nine novel functional miRNAs (hsa-miR-129, -136, -145, -155, -181b, -342-5p, -342-3p, -376a/b) in GBM cell lines were validated for their importance in glioma cell growth, and similar effects for six target genes (ROCK1, RHOA, MET, CSF1R, EIF2AK1, FGF7) of these miRNAs were shown functionally. The clinical significance of the functional hits was validated in miRNA expression data from the TCGA glioblastoma multiforme (GBM) tumor cohort. Five tumor suppressor miRNAs (hsa-miR-136, -145, -342, -129, -376a) showed significant underexpression in clinical GBM tumor samples from the TCGA GBM cohort further supporting the role of these miRNAs in vivo. Most importantly, higher hsa-miR-145 expression in GBM tumors yielded significantly better survival (p<0.005) in a subset of patients thus validating it as a genuine tumor suppressor miRNA. This systematic functional profiling provides important new knowledge about functionally relevant miRNAs in GBM biology and may offer new targets for treating glioma.

肿瘤的发生与进展涉及可兼具肿瘤抑制因子与致癌基因功能的微小RNA（microRNA，简称miRNA）。目前绝大多数miRNA的功能意义仍未明确。为系统性明确哪些miRNA对胶质瘤生长至关重要，本研究在3株人胶质母细胞瘤（glioblastoma multiforme，简称GBM）细胞系及1株星形胶质细胞系模型中，对前体微小RNA（precursor microRNA）文库开展了筛选。本研究通过附加细胞凋亡作为检测终点的二次筛选，对其中效果最显著且稳定性最佳的细胞增殖抑制命中靶点进行了验证。将功能筛选数据与miRNA表达数据整合，以筛选出表达下调的真正功能性肿瘤抑制性miRNA。此外，本研究结合miRNA靶基因预测结果与小干扰RNA（small interfering RNA，简称siRNA）功能筛选数据，筛选出对细胞增殖具有相似功能效应的潜在miRNA-靶基因配对。本研究验证了9种在GBM细胞系中发挥功能的新型miRNA（hsa-miR-129、-136、-145、-155、-181b、-342-5p、-342-3p、-376a/b）在胶质瘤细胞生长中的关键作用，并通过功能实验证实了这些miRNA的6个靶基因（ROCK1、RHOA、MET、CSF1R、EIF2AK1、FGF7）具有相似的功能效应。本研究通过癌症基因组图谱（The Cancer Genome Atlas，简称TCGA）GBM队列的miRNA表达数据，验证了上述功能性命中靶点的临床意义。在TCGA GBM队列的临床GBM样本中，5种肿瘤抑制性miRNA（hsa-miR-136、-145、-342、-129、-376a）呈现显著表达下调，进一步证实了这些miRNA在体内的功能作用。最为关键的是，在部分患者亚群中，GBM肿瘤组织内hsa-miR-145的高表达与更优的生存预后显著相关（p<0.005），从而证实其为真正的肿瘤抑制性miRNA。本系统性功能表征分析为GBM生物学中与功能相关的miRNA研究提供了重要的新认知，并可为胶质瘤治疗提供全新的潜在靶点。