Targeted comparative RNA interference analysis reveals differential requirement of genes essential for cell proliferation
Targeted comparative RNA interference analysis reveals differential requirement of genes essential for cell proliferation
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DOI：
10.1091/mbc.e06-04-0340
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发表时间：
2006-11-01
影响因子：
3.3
通讯作者：
Dutta, Anindya
中科院分区：
文献类型：
--
作者：
Machida, Yuichi J.;Chen, Yuefeng;Dutta, Anindya
细胞系的遗传和表观遗传组成的差异对于剖析特定基因在细胞生物学中的作用非常有用。靶向比较RNAi（TARCOR）分析使用针对靶向基因集的高通量RNA干扰（RNAi）和表型的严格定量来鉴定具有不同遗传背景的细胞系之间的增殖的差异要求的基因。为了证明这种分析的实用性，我们在乳腺上皮细胞系MCF 10A和前列腺癌细胞系PC 3中平行检测了257个生长调节基因。出乎意料的高数量的基因（25%）的消耗差异影响两种细胞系的增殖。敲除许多基因，使PC 3（p53-）不受影响，但抑制MCF 10A（p53+）增殖，诱导MCF 10A细胞中的p53。参与核糖体生物发生的EBNA 1BP 2是这种基因的一个例子，其缺失以p53依赖性方式将MCF 10A阻滞在G1/S。因此，TARCOR可用于鉴定细胞类型特异性基因和参与增殖的途径，也可用于探索细胞系的异质性。特别是，我们的数据强调了在哺乳动物细胞中进行siRNA筛选时考虑遗传状态的重要性。
Differences in the genetic and epigenetic make up of cell lines have been very useful for dissecting the roles of specific genes in the biology of a cell. Targeted comparative RNAi (TARCOR) analysis uses high throughput RNA interference (RNAi) against a targeted gene set and rigorous quantitation of the phenotype to identify genes with a differential requirement for proliferation between cell lines of different genetic backgrounds. To demonstrate the utility of such an analysis, we examined 257 growth-regulated genes in parallel in a breast epithelial cell line, MCF10A, and a prostate cancer cell line, PC3. Depletion of an unexpectedly high number of genes (25%) differentially affected proliferation of the two cell lines. Knockdown of many genes that spare PC3 (p53-) but inhibit MCF10A (p53+) proliferation induces p53 in MCF10A cells. EBNA1BP2, involved in ribosome biogenesis, is an example of such a gene, with its depletion arresting MCF10A at G1/S in a p53-dependent manner. TARCOR is thus useful for identifying cell type-specific genes and pathways involved in proliferation and also for exploring the heterogeneity of cell lines. In particular, our data emphasize the importance of considering the genetic status, when performing siRNA screens in mammalian cells.