Deciphering molecular mechanisms of MEIS1-mediated prostate tumor suppression through RNAseq and ChIPseq - RNAseq data. Deciphering molecular mechanisms of MEIS1-mediated prostate tumor suppression through RNAseq and ChIPseq - RNAseq data

We conducted RNA-sequencing to determine MEIS1-mediated gene regulation in prostate cancer cells because phenotypic data has demonstrated that expression of MEIS1 is sufficient to slow tumor growth and metastatic colonization in vitro and in vivo. These analyses compared global gene expression of CWR22Rv1-Control and CWR22Rv1-LV-MEIS1, as well as the HOXB13ko and HOXB13ko-LV-MEIS1 cells to precisely delineate MEIS1 and HOXB13-regulated genes. Importantly, inclusion of HOXB13ko lines enabled determination of HOXB13-associated gene regulation, as well as identification of significant changes between LV-MEIS1 vs. Control that are HOXB13-independent and thus unrelated to tumor suppression. Are samples are from CWR-22Rv1 cells. Control denotes cells with constitutive expression of Cas9, but with no gRNA provided. Control cells have nearly undetectable levels of endogenous MEIS1 expression, but do express HOXB13. LV-MEIS1 denotes cells with exogenous lentiviral expression of MEIS1, and they still express endogenous HOXB13. HOXB13ko denotes cells where HOXB13 was knocked out using CRISPR, 6 cell clones were isolated with confirmed HOXB13 knockout by western blot and pooled together into one line, so these cells lack both MEIS1 and HOXB13. HOXB13ko-LV-MEIS1 denotes cells where the same lentiviral expression of MEIS1 was infected into the HOXB13ko cell pool, so these cells are positive for MEIS1 expression and negative for HOXB13. Overall design: mRNA from all four cell line derivatives (Control, LV-MEIS1, HOXB13ko, and HOXB13ko-LV-MEIS1) was sequenced in triplicate

本研究通过RNA测序（RNA-sequencing）解析前列腺癌细胞中MEIS1介导的基因调控机制。此前已有表型数据证实，MEIS1的表达足以在体外及体内环境中抑制肿瘤生长与转移定植。本次转录组分析比较了四组细胞的全局基因表达谱：CWR22Rv1-对照组（CWR22Rv1-Control）、CWR22Rv1过表达MEIS1组（CWR22Rv1-LV-MEIS1）、HOXB13敲除组（HOXB13ko）以及HOXB13敲除后过表达MEIS1组（HOXB13ko-LV-MEIS1），以精准界定受MEIS1与HOXB13调控的靶基因。值得注意的是，纳入HOXB13敲除细胞系不仅可解析HOXB13相关的基因调控网络，还能区分出不依赖HOXB13的LV-MEIS1与对照组间的显著表达差异，进而排除其与肿瘤抑制无关的调控通路。所有样本均来源于CWR22Rv1前列腺癌细胞。对照组（Control）为组成型表达Cas9但未导入向导RNA（guide RNA, gRNA）的细胞；该组细胞内源性MEIS1表达水平几乎无法检测，但仍可表达HOXB13。LV-MEIS1组细胞为通过慢病毒载体（lentiviral, LV）外源过表达MEIS1的细胞，该组仍保留内源性HOXB13的表达。HOXB13敲除组（HOXB13ko）为利用CRISPR技术敲除HOXB13的细胞：我们通过Western blot验证共分离得到6株HOXB13完全敲除的单克隆细胞，并将其混合为单一细胞系，因此该组细胞同时缺乏内源性MEIS1与HOXB13的表达。HOXB13ko-LV-MEIS1组细胞为在HOXB13敲除细胞池中导入上述慢病毒MEIS1表达载体构建得到的细胞，因此该组细胞仅表达MEIS1而不表达HOXB13。实验整体设计：对上述四组细胞系的mRNA进行三次生物学重复的RNA测序。