Cancer Science Institute of Singapore, Centre for Life Sciences #02-15, 28 Medical Drive, National University of Singapore, Singapore 117456, Singapore.
Eur J Cancer. 2010 May;46(8):1456-63. doi: 10.1016/j.ejca.2010.01.036. Epub 2010 Feb 20.
Accumulating evidence indicates that RUNX3 is an important tumour suppressor that is inactivated in many cancer types. This study aimed to assess the role of microRNA (miRNA) in the regulation of RUNX3.
Four bioinformatic algorithms were used to predict miRNA binding to RUNX3. The correlation between candidate miRNAs and RUNX3 expression in cell lines was determined by real-time reverse transcriptase quantitative PCR (RT-qPCR) and Western blot. Candidate miRNAs were tested for functional effects through transfection of miRNA precursors and inhibitors, and monitoring cell viability, apoptosis and Bim expression. miRNA and RUNX3 expression, RUNX3 methylation and RUNX3 protein levels were assessed in gastric tissue by RT-qPCR, Methylight analysis and immunohistochemistry, respectively.
Bioinformatics, gene and protein expression analysis in eight gastric cell lines identified miR-130b as the top candidate miRNA for RUNX3 binding. Overexpression of miR-130b increased cell viability, reduced cell death and decreased expression of Bim in TGF-beta mediated apoptosis, subsequent to the downregulation of RUNX3 protein expression. In 15 gastric tumours, miR-130b expression was significantly higher compared to matched normal tissue, and was inversely associated with RUNX3 hypermethylation.
Attenuation of RUNX3 protein levels by miRNA may reduce the growth suppressive potential of RUNX3 and contribute to tumourigenesis.
越来越多的证据表明 RUNX3 是一种重要的肿瘤抑制因子，在许多癌症类型中失活。本研究旨在评估 microRNA（miRNA）在 RUNX3 调控中的作用。
使用四种生物信息学算法预测 miRNA 与 RUNX3 的结合。通过实时逆转录定量 PCR（RT-qPCR）和 Western blot 确定候选 miRNA 与细胞系中 RUNX3 表达的相关性。通过转染 miRNA 前体和抑制剂，以及监测细胞活力、凋亡和 Bim 表达，测试候选 miRNA 的功能影响。通过 RT-qPCR、Methylight 分析和免疫组织化学分别评估 miRNA 和 RUNX3 表达、RUNX3 甲基化和 RUNX3 蛋白水平在胃组织中的变化。
八种胃细胞系的生物信息学、基因和蛋白表达分析确定 miR-130b 是 RUNX3 结合的首选候选 miRNA。miR-130b 的过表达增加了细胞活力，减少了 TGF-β介导的凋亡中的细胞死亡，并降低了 Bim 的表达，随后 RUNX3 蛋白表达下调。在 15 例胃癌组织中，miR-130b 的表达明显高于匹配的正常组织，与 RUNX3 高甲基化呈负相关。
miRNA 对 RUNX3 蛋白水平的下调可能会降低 RUNX3 的生长抑制潜力，并促进肿瘤发生。
