Substrate stiffness-dependent gene expression profile for cortical neurons

We undertook mRNA microarray and gene ontology analyses to screen out substrate stiffness-dependent genes. Total mRNA were extracted from E17 cortical neurons grown on soft or stiff substrates at 5 or 16 hr time points. We identified 114 differentially-expressed mRNA transcripts in cells grown on 0.1 kPa and 20 kPa gels at the 5 hr time-point. Among them, 66 were upregulated in 0.1 kPa gel cultures and the remainder were downregulated (compared to cells grown on stiffer substrates). The expressions of three endocytic genes (Cltc, Dab2, and Myo6) and four adhesion genes(Vcl, Robo2, Nrcam, and Cad11) were confirmed by QGP and smRNA FISH.本研究采用mRNA微阵列（mRNA microarray）与基因本体分析（gene ontology），筛选出依赖底物刚度的基因。我们从培养于柔软或坚硬底物、处于5小时或16小时时间点的E17皮层神经元中提取总mRNA。在5小时时间点下，于培养于0.1 kPa与20 kPa水凝胶的细胞中鉴定出114个差异表达mRNA转录本；其中66个在0.1 kPa水凝胶培养体系中呈上调表达，剩余则相较于培养于更坚硬底物的细胞呈下调表达。本研究通过QGP与单分子RNA荧光原位杂交（smRNA FISH），验证了3个内吞基因（endocytic genes：Cltc、Dab2及Myo6）与4个黏附基因（adhesion genes：Vcl、Robo2、Nrcam及Cad11）的表达水平。