Regulation of gene expression in MDAMB231 breast cancer cells by Parvin-beta in 2D vs 3D culture conditions. Homo sapiens

Parvin-beta is a focal adhesion protein downregulated in human breast cancer cells. Loss of Parvin-beta contributes to increased integrin-linked kinase activity, cell-matrix adhesion, and invasion through the extracellular matrix in vitro. The effect of ectopic Parvin-beta expression on the transcriptional profile of MDA-MB-231 breast cancer cells, which normally do not express Parvin-beta was evaluated. Particular emphasis was placed upon propagating MDA-MB-231 breast cancer cells in three-dimensional culture matrices. Gene expression profiles of vector control and Parvin-beta transfected MDA-MB-231 cells cultured on (A) monomeric type I collagen coated plastic, (B) embedded in a type I collagen gel, and (C) embedded in basement membrane (growth factor reduced Matrigel), were compared. Interestingly, Parvin-beta re-expression in MDA-MB-231 cells increased the mRNA expression, serine 82 phosphorylation (mediated by CDK9), and activity of the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARgamma) and a concomitant increase in lipogenic gene expression as a downstream effector of PPARgamma. Importantly, Parvin-beta suppressed breast cancer growth in vivo with associated decreased proliferation. These data suggest that Parvin-beta might influence breast cancer progression.. Keywords: Gene expression profiling in two dimensional vs three dimensional cell culture Overall design: The MDA-MB-0231 transfectants were cultured for 7 days in each growth condition and total RNA isolated using Trizol reagent (Invitrogen). Genes either upregulated or downregulated in Parvin-beta transfectants versus vector control cells were compared among the 3 different growth conditions. Venn diagrams were generated using GeneSpring software and used to produce gene lists that were unique to a particular growth condition, or shared between two or all three growth conditions. The focus was primarily on gene expression alterations observed in the 3D collagen type I and 3D Matrigel conditions.

Parvin-beta是一种黏着斑蛋白（focal adhesion protein），在人类乳腺癌细胞中表达下调。Parvin-beta的缺失会导致整合素连接激酶（integrin-linked kinase）活性、细胞-基质黏附能力以及体外经细胞外基质侵袭的能力增强。本研究评估了异位表达Parvin-beta对MDA-MB-231乳腺癌细胞转录组谱（transcriptional profile）的影响——该细胞系本身不表达Parvin-beta。研究特别着重于在三维培养基质中培养MDA-MB-231乳腺癌细胞。本研究比较了以下三种培养条件下，载体对照组与Parvin-beta转染组MDA-MB-231细胞的基因表达谱：(A) 包被有单体I型胶原蛋白的塑料培养板，(B) 嵌入I型胶原蛋白凝胶中，(C) 嵌入基底膜（生长因子缺陷型Matrigel）中。有趣的是，在MDA-MB-231细胞中重新表达Parvin-beta，可上调核激素受体过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor gamma, PPARγ）的mRNA表达、丝氨酸82位点的磷酸化（由CDK9介导）及其活性，并伴随作为PPARγ下游效应因子的生脂基因表达上调。重要的是，Parvin-beta在体内可抑制乳腺癌生长，并伴随增殖能力降低。上述数据表明，Parvin-beta或可影响乳腺癌的进展。
关键词：二维与三维细胞培养中的基因表达谱分析
实验设计概述：将MDA-MB-231转染细胞在每种培养条件下培养7天，使用Trizol试剂（Invitrogen）提取总RNA。比较了三种不同培养条件下，Parvin-beta转染组相较于载体对照组的差异表达基因（上调或下调）。使用GeneSpring软件绘制维恩图（Venn diagrams），并据此生成特定培养条件特有的、或在两种或全部三种培养条件下共有的基因列表。本研究的重点主要集中于三维I型胶原蛋白及三维Matrigel培养条件下观察到的基因表达变化。