Effect of fibulin-3 knockdown on malignant mesothelioma cells

Whole transcriptome comparison of mesothelioma cells transfected with control vs Fibulin-3 (EFEMP1) siRNAs. Mesothelioma cells H-226 (ATCC) and H-2595 (Harvey Pass, New York University) were transfected with two fibulin-3 or control siRNAs and collected 48h after transfection. Gene expression was quantified by RNAseq using standard procedures. The results suggest that downregulation of fibulin-3 negatively affects a PI3K/AKT-dependent gene signature involved in cell growth, adhesion, and association with the extracellular matrix. Overall design: Three independent cultures from each cell line were transfected with two previously validated fibulin-3 siRNAs or control siRNA (Hu et al., Cancer Res (2012) 72: 3873-3885). Total RNA was extracted using column purification and analyzed by RNAseq using the Illumina NSQ 500/550 Hi Output KT v2.5 (75 CYS, #20024906). Analysis of gene expression was used to determine a gene signature correlated with fibulin-3 downregulation. Design: 2 cell lines x 2 conditions per cell line x 3 independent replicates

本研究针对转染对照小干扰RNA（siRNA）与纤连蛋白-3（Fibulin-3, EFEMP1）小干扰RNA的间皮瘤细胞开展全转录组比较分析。所用间皮瘤细胞株H-226（购自美国典型培养物保藏中心（ATCC））与H-2595（由纽约大学Harvey Pass惠赠），分别以2条靶向纤连蛋白-3的小干扰RNA或对照小干扰RNA进行转染，并于转染后48小时收集细胞。采用标准流程通过RNA测序（RNAseq）对基因表达水平进行定量。结果显示，纤连蛋白-3的下调会对PI3K/AKT依赖的基因特征产生负面影响，该特征参与细胞生长、黏附及细胞外基质关联过程。实验整体设计：每个细胞株设置3份独立培养样本，分别使用2条已验证的靶向纤连蛋白-3的小干扰RNA或对照小干扰RNA进行转染（Hu等人，"Cancer Res"，2012年，72卷：3873-3885）。采用柱纯化法提取总RNA，使用Illumina NSQ 500/550 Hi Output KT v2.5（75 CYS, #20024906）进行RNA测序分析。通过基因表达分析筛选得到与纤连蛋白-3下调相关的基因特征。实验设计：2种细胞株 × 每种细胞株2种处理条件 × 3次独立生物学重复