A functional screen identifies miR-34a as a candidate neuroblastoma tumor suppressor gene
A functional screen identifies miR-34a as a candidate neuroblastoma tumor suppressor gene
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DOI：
10.1158/1541-7786.mcr-07-2102
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发表时间：
2008-05-01
影响因子：
5.2
通讯作者：
Maris, John M.
中科院分区：
文献类型：
--
作者：
Cole, Kristina A.;Attiyeh, Edward F.;Maris, John M.
MicroRNA是一种小的非编码RNA，通过转录沉默在调节许多细胞功能中起关键作用。它们被认为是几种人类肿瘤中的癌基因和肿瘤抑制基因（oncomirs）。我们使用了一个综合的基因组学和功能筛选策略，以确定潜在的oncomirs在儿科肿瘤神经母细胞瘤。我们首先确定了在我们和其他人定义为在高危神经母细胞瘤中经常缺失（1 p36，3 p22和11 q23 -24）或获得（17 q23）的染色体区域内的microRNA。然后，我们将microRNA前体模拟物或抑制剂瞬时转染到一组六个神经母细胞瘤细胞系中，我们对这些基因组畸变进行了表征。大多数转染没有表现出表型效应，但是miR-34 a（1 p36）和miR-34 c（11 q23）模拟物在具有1 p36半合子缺失的细胞系中表现出显著的生长抑制。相反，在没有1 p36缺失的细胞系中，这些模拟物没有生长抑制作用。定量逆转录-PCR显示，在模拟加回后，细胞系中miR-34 a表达缺失与1 p36畸变和表型效应完全相关。在1 p36缺失的原发性肿瘤（n = 54）中，miR-34 a的表达也降低（P = 0.009），但在30个成神经细胞瘤细胞系中，miR-34 a位点的重测序未发现突变。流式细胞仪时间序列分析显示，miR-34 a生长抑制的可能机制是通过细胞周期停滞，随后是细胞凋亡。BCL 2和MYCN被鉴定为miR-34 a靶标和肿瘤抑制表型效应的可能介质。这些数据支持miR-34 a作为人成神经细胞中的肿瘤抑制基因。
MicroRNAs are small noncoding RNAs that have critical roles in regulating a number of cellular functions through transcriptional silencing. They have been implicated as oncogenes and tumor suppressor genes (oncomirs) in several human neoplasms. We used an integrated genomics and functional screening strategy to identify potential oncomirs in the pediatric neoplasm neuroblastoma. We first identified microRNAs that map within chromosomal regions that we and others have defined as frequently deleted (1p36, 3p22, and 11q23-24) or gained (17q23) in high-risk neuroblastoma. We then transiently transfected microRNA precursor mimics or inhibitors into a panel of six neuroblastoma cell lines that we characterized for these genomic aberrations. The majority of transfections showed no phenotypic effect, but the miR-34a (1p36) and miR-34c (11q23) mimics showed dramatic growth inhibition in cell lines with 1p36 hemizygous deletion. In contrast, there was no growth inhibition by these mimics in cell lines without 1p36 deletions. Quantitative reverse transcription-PCR showed a perfect correlation of absent miR-34a expression in cell lines with a 1p36 aberration and phenotypic effect after mimetic add-back. Expression of miR-34a was also decreased in primary tumors (n = 54) with 1p36 deletion (P = 0.009), but no mutations were discovered in resequencing of the miR-34a locus in 30 neuroblastorna cell lines. Flow cytometric time series analyses showed that the likely mechanism of miR-34a growth inhibition is through cell cycle arrest followed by apoplosis. BCL2 and MYCN were identified as miR-34a targets and likely mediators of the tumor suppressor phenotypic effect. These data support miR-34a as a tumor suppressor gene in human neuroblastorna.