mRNA sequencing of MDA-MB-231 engineered for doxycycline-inducible overexpression of wild-type or mutant myocardin related transcription factor A

Myocardin Related Transcription Factor A (MRTF-A), a co-factor or serum response factor (SRF), has been implicated in breast cancer cell migration and metastatic potential. The goal of this sequencing effort was to determine which mRNA were regulated by MRTF-A overexpression and which mRNA were differentially regulated when function-inhibited (SAP-domain deleted or SRF-binding deficient) mutant MRTF-A were overexpressed. GFP and luciferase expressing MDA-MB-231 cells engineered with doxycycline inducible overexpression of wild-type or mutant MRTF-A were treated with doxycycline for 72 hours prior to RNA collection. Two mutant versions of MRTF-A were used 1) a SAP-domain deleted mutant (deleted base-pairs 343-378) and 2) an SRF binding deficient mutant (mutated K237A, Y238A, H239A, Y241A). The cells were plated in a tissue-treated culture dish or on top of matrigel during doxycycline induction. RNA was extracted using the Qiagen RNeasy Plus Mini Kit according the manufacturer's instructions. Libraries were obtained starting from total RNA following the Illumina Stranded Total RNA preparation Ligation with Ribo-Zero Plus protocol. mRNA was then sequenced on the Illumina NextSeq2000.

心肌素相关转录因子A（Myocardin Related Transcription Factor A, MRTF-A）作为血清反应因子（serum response factor, SRF）的共辅因子，已被证实与乳腺癌细胞迁移及转移潜能密切相关。本测序研究的目标为明确两类受调控的mRNA：一类是受MRTF-A过表达调控的mRNA，另一类是当功能抑制型（SAP结构域缺失或SRF结合缺陷）突变型MRTF-A过表达时存在差异调控的mRNA。我们构建了可经多西环素（doxycycline）诱导过表达野生型或突变型MRTF-A、且表达绿色荧光蛋白（green fluorescent protein, GFP）与荧光素酶（luciferase）的MDA-MB-231细胞，经多西环素处理72小时后收集RNA。本次实验使用两种MRTF-A突变体：1）SAP结构域缺失突变体（缺失343至378位碱基对）；2）SRF结合缺陷突变体（K237A、Y238A、H239A、Y241A位点发生突变）。在多西环素诱导阶段，细胞分别接种于经细胞培养处理的培养皿及基质胶（matrigel）表面。RNA提取采用Qiagen RNeasy Plus Mini试剂盒，严格遵循制造商提供的操作说明书。测序文库以总RNA为起始材料，按照Illumina Stranded Total RNA制备（搭配Ribo-Zero Plus）的连接建库方案构建。最终在Illumina NextSeq2000测序平台上完成mRNA测序。