Identification of miR-145 targets involved in colon cancer. Homo sapiens

MicroRNAs (miRNAs) have emerged as important gene regulators and are recognized as key players in tumorigenesis. miR-145 is reported to be down-regulated in several cancers, but knowledge of its targets in colon cancer remains limited. To investigate the role of miR-145 in colon cancer, we have employed a microarray based approach to identify miR-145 targets. Based on seed site enrichment analyses and unbiased word analyses, we found a significant enrichment of miRNA binding sites in the 3’-untranslated regions (UTRs) of transcripts down-regulated upon miRNA overexpression, which represent potential miR-145 targets. Gene Ontology analysis showed an overrepresentation of genes involved in cell death, gene expression, cancer, cell cycle, DNA replication, recombination and repair. A number of the identified miRNA targets have previously been implicated in cancer, including YES, FSCN1, ADAM17, BIRC2, VANGL1 as well as the transcription factor STAT1. Both YES and STAT1 were verified as direct miR-145 targets based on 3’UTR luciferase assays and western blots for endogenous proteins. Overall design: DLD-1 cells were transfected with 50 nM miR-145 duplex or mock transfected. Total RNA was harvested 24 hours post-transfection and analyzed on Affymetrix HG-U133 Plus 2.0 human arrays.

微小RNA（miRNAs）已被证实为重要的基因调控因子，且被认为是肿瘤发生进程中的关键参与者。已有研究表明，miR-145在多种癌症中表达下调，但目前对其在结肠癌中的靶基因的认知仍较为有限。为探究miR-145在结肠癌中的作用，本研究采用基于微阵列的方法鉴定miR-145的靶基因。基于种子位点富集分析与无偏倚单词分析，我们发现，在miRNA过表达后下调的转录本的3'非翻译区（UTRs）中，miRNA结合位点显著富集，这类转录本即为潜在的miR-145靶基因。基因本体（Gene Ontology, GO）分析显示，参与细胞死亡、基因表达、癌症、细胞周期、DNA复制、重组与修复的基因显著富集。本研究鉴定出的多个miRNA靶基因此前已被证实与癌症相关，包括YES、FSCN1、ADAM17、BIRC2、VANGL1以及转录因子STAT1。基于3'UTR荧光素酶报告基因实验与免疫印迹（Western blot）实验，YES与STAT1均被验证为miR-145的直接靶基因。实验整体设计：将DLD-1细胞用50 nM的miR-145双链体转染，同时设置模拟转染对照组。转染24小时后收集总RNA，通过Affymetrix HG-U133 Plus 2.0人类基因表达芯片进行分析。