Rizzo Milena, Berti Gabriele, Russo Francesco, Fazio Sofia, Evangelista Monica, D'Aurizio Romina, Pellegrini Marco, Rainaldi Giuseppe
Non-coding RNA Laboratory, Institute of Clinical Physiology (IFC), CNR, Pisa, Italy.
Tuscan Tumor Institute (ITT), Firenze, Italy.
J Cancer. 2017 Aug 22;8(14):2729-2739. doi: 10.7150/jca.18396. eCollection 2017.
miR-26a-5p is a tumor suppressor (TS) miRNA often downregulated in several tumor tissues and tumor cell lines. In this work, we performed the re-expression of the miR-26a-5p in DU-145 prostate cancer cells to collect genes interacting with miR-26a-5p and analyzed their integration in the tumorigenesis related pathways. The transfection of DU-145 prostate cancer cells with miR-26a-5p was done using nucleofection. The biological effects induced by miR-26a-5p re-expression were detected with routine assays for cell proliferation, cell cycle, survival, apoptosis and cell migration. The miRNA pull out technique was used to collect and next generation sequencing to identify the complete repertoire of the miR-26a-5p targets (miR-26a-5p/targetome). TargetScan 7, PITA and RNA22 were used to find the predicted miR-26a-5p targets in the miR-26a-5p/targetome. Gene set enrichment analysis were used to integrate target genes in KEGG pathways and Protein-Protein Interaction networks (PPINs) and modules were built. miR-26a-5p exerted an anti-proliferative effect acting at several levels, by decreasing survival and migration and inducing both cell cycle block and apoptosis The analysis of the miR-26a-5p/targetome showed that 1423 (1352 coding and 71 non-coding) transcripts interacted with miR-26a-5p. Filtering the miR-26a-5p/targetome with prediction algorithms, 628 out of 1353 transcripts were miR-26a-5p predicted targets and 73 of them were already validated miR-26a-5p targets. Finally, miR-26a-5p targets were involved in 22 KEGG pathways and 20 significant protein-protein interaction modules The TS-miR-26a-5p/targetome is a platform that shows both unknown and known miRNA/target interactions thus offering the possibility to validate genes and discover pathways in which these genes could be involved.
miR-26a-5p是一种肿瘤抑制（TS）微小RNA，在多种肿瘤组织和肿瘤细胞系中常呈下调状态。在本研究中，我们在DU-145前列腺癌细胞中重新表达miR-26a-5p，以收集与miR-26a-5p相互作用的基因，并分析它们在肿瘤发生相关途径中的整合情况。使用核转染法将miR-26a-5p转染到DU-145前列腺癌细胞中。通过细胞增殖、细胞周期、存活、凋亡和细胞迁移的常规检测来检测miR-26a-5p重新表达所诱导的生物学效应。使用miRNA pull out技术收集并通过下一代测序来鉴定miR-26a-5p靶标的完整库（miR-26a-5p/靶标组）。使用TargetScan 7、PITA和RNA22在miR-26a-5p/靶标组中寻找预测的miR-26a-5p靶标。使用基因集富集分析将靶基因整合到KEGG途径和蛋白质-蛋白质相互作用网络（PPINs）中并构建模块。miR-26a-5p通过降低存活和迁移并诱导细胞周期阻滞和凋亡，在多个水平发挥抗增殖作用。对miR-26a-5p/靶标组的分析表明，1423个（1352个编码和71个非编码）转录本与miR-26a-5p相互作用。用预测算法筛选miR-26a-5p/靶标组，1353个转录本中有628个是miR-26a-5p预测靶标，其中73个是已验证的miR-26a-5p靶标。最后，miR-26a-5p靶标参与22条KEGG途径和20个重要的蛋白质-蛋白质相互作用模块。TS-miR-26a-5p/靶标组是一个展示未知和已知miRNA/靶标相互作用的平台，从而为验证基因和发现这些基因可能参与的途径提供了可能性。
