Genome-wide transcriptome profiling of SK-Mel-28, UACC-62 and HCT-116 cells stably expressing scrambled shRNA and RAB7 shRNA

Purpose: Asess the transcritpional changes induced upon RAB7 knock-down in melanoma (SK-Mel-28 and UACC-62) and in colon cancer (HCT-116) cell lines. Methods: mRNA profiles of tumor cell lines (SK-Mel-28, UACC-62, HCT-116) stably expressing scrambled shRNA or RAB7 shRNA (harvested at day 3 after lentiviral infection) were generated by deep sequencing, using three biological replicates per condition. The sequence reads that passed quality filters were analyzed with TopHat and Cufflinks. Validation of induced / silenced genes was performed by western blot. Results show a differential impact of RAB7 expression in the transcriptomic profile of melanoma vs non-melanoma cell lines, and support a lineage-specific role of this small GTPase in melanoma. Examination of the mRNA profiles RAB7-depleted vs wild type cells, performed in parallel in 3 different tumor cell lines (Melanomas: SK-Mel-28 and UACC-62, Non-melanoma: HCT-116) harvested at day 3 after lentiviral infection.

研究目的：评估在黑色素瘤（SK-Mel-28与UACC-62）及结肠癌（HCT-116）细胞系中敲低RAB7所诱导的转录组变化。
研究方法：针对稳定表达乱序短发夹RNA（short hairpin RNA, shRNA）或RAB7短发夹RNA（shRNA）的肿瘤细胞系（SK-Mel-28、UACC-62、HCT-116）开展深度测序以获取其mRNA表达谱；所有样本均于慢病毒感染后第3天收获，每组设置3次生物学重复。对通过质量过滤的测序reads，采用TopHat与Cufflinks进行数据分析。通过蛋白质印迹（western blot）对上调或沉默的基因进行验证。
研究结果显示，RAB7的表达对黑色素瘤与非黑色素瘤细胞系的转录组谱存在差异化影响，证实了该小GTP酶在黑色素瘤中发挥谱系特异性功能。本数据集同时对3种不同肿瘤细胞系（黑色素瘤：SK-Mel-28、UACC-62；非黑色素瘤：HCT-116）中RAB7敲低组与野生型组的mRNA表达谱进行分析，所有样本均采集于慢病毒感染后第3天。