Gene expression profiling of MDA231 cells depleted of MRCKalpha (CDC42BPA) and MRCKbeta (CDC42BPB). Gene expression profiling of MDA231 cells depleted of MRCKalpha (CDC42BPA) and MRCKbeta (CDC42BPB)

Aim: The MRCK family of kinases (namely MRCKalpha and MRCKbeta) are downstream effectors of the Rho GTPase signaling pathways with roles in mediating cell migration via regulating the actin cytoskeleon. As such, it is thought that the MRCKs might also be involved in promoting cancer cell invasion or metastasis. However, crucial in vivo data and a general view of the signaling pathways regulated by the MRCKs are still lacking in order to substantiate the hypothesis. Therefore, the goal of this study is to perform transcriptome profiling (RNA-seq) of MDA-MB-231 cells depleted of either CDC42BPA, CDC42BPB, or both, in order to obtain insight into the differential genes expressed and whether CDC42BPA and CDC42BPB contribute as a causative factor of metastatic breast cancer. Method: We performed CRIPSR-based knockout of MRCKalpha (CDC42BPA), MRCKbeta (CDC42BPB), either alone or in combination, in the invasive breast cancer cell line MDA-MB-231. The combinatory double knockout was performed twice as two biological replicates to increase confidence in our data. The knockout cells were then prepared and sent for RNA-seq using by BGISEQ-500, and differential gene expression profile was obtained. Overall design: mRNA from vector control, MRCKalpha (CDC42BPA) knockout, MRCKbeta (CDC42BPB) knockout and MRCKalpha/MRCKbeta (CDC42BPA/CDC42BPB) double knockout MDA-MB-231 cells

研究目的：MRCK激酶家族（MRCK family of kinases）即MRCKα与MRCKβ，是Rho GTP酶（Rho GTPase）信号通路的下游效应因子，可通过调控肌动蛋白细胞骨架（actin cytoskeleton）介导细胞迁移。据此推测，MRCK家族或许也参与癌细胞侵袭与转移过程。但目前仍缺乏关键的体内实验数据，且对MRCK所调控的信号通路缺乏系统性认知，无法为该假说提供实证支撑。因此，本研究拟对分别敲除CDC42BPA、CDC42BPB，或同时敲除两者的MDA-MB-231细胞开展转录组测序（RNA-seq），以解析差异表达基因，并探究CDC42BPA与CDC42BPB是否为转移性乳腺癌的致病相关因子。实验方法：我们在侵袭性乳腺癌细胞系MDA-MB-231中，采用CRISPR技术分别或联合敲除MRCKα（CDC42BPA）、MRCKβ（CDC42BPB）。为提升实验数据的可信度，双基因敲除组设置了两次生物学重复。随后制备敲除细胞样本，使用BGISEQ-500平台进行RNA-seq测序，最终获得差异基因表达谱。整体实验设计：本实验设置四组样本，分别为空载载体对照组、MRCKα（CDC42BPA）敲除组、MRCKβ（CDC42BPB）敲除组，以及MRCKα/MRCKβ（CDC42BPA/CDC42BPB）双敲除组的MDA-MB-231细胞。