Polysome-associated mRNA profiling of cancer cells in response to CXCL12 and IGF1

CXCL12 and IGF1 are key secreting molecules produced by cancer-associated fibroblasts in breast cancer. These factors promote the survival of disseminated cancer cells in the bone marrow. To assess the combined responses elicited by CXCL12 and IGF1, we examined the translating transcriptome of cancer cells in response to these two factors by Translating Ribosome Affinity Purification (TRAP)-RNAseq. MDA-MB-231 cells were engineered to express an EGFP-tagged version of ribosomal protein L10a. This allows the retrieval of polysome-associated mRNA by anti-GFP pull down (TRAP) and profiling the translating transcriptome by RNAseq. EGFP-L10a+ cancer cells were serum starved (0.2% serum) for 24 hours, and then treated with CXCL12 (30ng/mL) + IGF1 (10ng/mL) or CXCL12 (300ng/mL) + IGF1 (100ng/mL) for 6hrs. Two biological replicates were profiled for each condition.

CXCL12与IGF1是乳腺癌中癌症相关成纤维细胞（cancer-associated fibroblasts）分泌的关键效应分子。此类因子可促进播散性癌细胞在骨髓中的存活。为评估CXCL12与IGF1共同诱导的细胞应答，我们借助翻译核糖体亲和纯化（Translating Ribosome Affinity Purification，TRAP）-RNA测序技术，检测了癌细胞响应这两种因子的翻译转录组。我们对MDA-MB-231细胞进行工程改造，使其表达带有增强绿色荧光蛋白（EGFP）标签的核糖体蛋白L10a。借此可通过抗GFP下拉实验（即TRAP）富集与多聚核糖体结合的信使RNA（mRNA），并通过RNA测序分析翻译转录组。将表达EGFP-L10a的癌细胞置于0.2%血清浓度的培养基中饥饿培养24小时，随后分别采用30ng/mL CXCL12+10ng/mL IGF1，或300ng/mL CXCL12+100ng/mL IGF1处理6小时。每组实验设置两个生物学重复进行测序分析。