RNA-sequencing of isogenic primary, pre-malignant immortalized, and Ras-transformed human mammary epithelial cells. RNA-sequencing of isogenic primary, pre-malignant immortalized, and Ras-transformed human mammary epithelial cells

Understanding changes in gene expression during tumor initiation and progression is critical to understanding how genetic alterations drive malignancy. We used a genetically defined cell culture model to study the progression of normal human mammary epithelial cells (HMECs) to malignancy. Primary HMECs were immortalized through the expression of hTERT, p53DD, cyclin D1, CDK4R24C and c-MYCT58A. This immortalization conferred limitless replicative potential as well as migratory capacity. These pre-malignant cells were subsequently HRASG12V transformed, which converted the immortalized cells to a fully tumorigenic state with significantly increased invasive capacity. We analyzed the cells using RNA-sequencing, and we report dramatic mRNA expression changes during the pre-malignant immortalization of primary cells, and very few mRNA expression changes occurring during oncogenic Ras transformation. RNA signatures in pre-malignant immortalized and Ras-transformed cells are consistent with previously reported epithelial-to-mesenchymal transition (EMT) signatures. Overall design: RNA-sequencing in biological triplicate of three cell types (Primary HMECs, Immortalized HMECs, Transformed HMECs); Illumina HiSeq 2000 125bp PE (1 replicate) and Illumina HiSeq 4000 150bp PE (2 replicates)

解析肿瘤起始与进展过程中的基因表达变化，对于阐明遗传变异如何驱动恶性转化具有核心意义。本研究采用遗传特征明确的细胞培养模型，探究正常人类乳腺上皮细胞（human mammary epithelial cells, HMECs）向恶性表型转化的全过程。研究人员通过过表达hTERT、p53DD、细胞周期蛋白D1（cyclin D1）、CDK4R24C及c-MYCT58A，使原代HMECs实现永生化；该永生化过程赋予细胞无限增殖潜能与迁移能力。随后，通过HRASG12V转化上述永生化癌前细胞，将其转化为完全致瘤状态，且侵袭能力显著增强。本研究采用RNA测序（RNA-sequencing）对细胞样本进行分析，结果显示：原代细胞向永生化癌前状态的转化过程中，mRNA表达发生显著变化；而致癌性Ras转化阶段，mRNA表达仅出现极少量变化。癌前永生化细胞与Ras转化细胞的RNA表达特征，与此前报道的上皮间质转化（epithelial-to-mesenchymal transition, EMT）特征高度一致。实验整体设计：对三种细胞类型（原代HMECs、永生化HMECs、转化型HMECs）设置三次生物学重复，开展RNA测序；测序平台及参数为：Illumina HiSeq 2000（125bp双端测序，1个重复）、Illumina HiSeq 4000（150bp双端测序，2个重复）。