Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling.
Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling.
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捕获microRNA结合的mRNA将肿瘤抑制器miR-34a识别为生长因子信号传导的调节剂。
DOI：
10.1371/journal.pgen.1002363
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发表时间：
2011-11
期刊：
影响因子：
4.5
通讯作者：
Lieberman J
中科院分区：
文献类型：
--
作者：
Lal A;Thomas MP;Altschuler G;Navarro F;O'Day E;Li XL;Concepcion C;Han YC;Thiery J;Rajani DK;Deutsch A;Hofmann O;Ventura A;Hide W;Lieberman J
使用一种简单的生物化学方法来分离用转染的生物素化microRNA拉下的mRNA，以鉴定肿瘤抑制基因miR-34 a的直接靶基因。该方法重新鉴定了K562和HCT 116癌细胞系中表达的大多数已知miR-34 a调控基因。在两种细胞系中，982个基因的转录物在具有miR-34 a的下拉中富集。尽管数量如此之大，验证实验表明，在两种细胞系中鉴定的基因中，约90%可以直接由miR-34 a调控。因此，miR-34 a能够调控数百个基因。用miR-34 a拉低的转录物高度富集它们在生长因子信号传导和细胞周期进程中的作用。这些基因形成相互作用的基因产物的密集网络，所述基因产物调节多个信号转导途径，所述多个信号转导途径协调对外部生长刺激的增殖反应。多个候选miR-34 a调节基因参与RAS-RAF-MAPK信号传导。异位miR-34 a表达降低了基础ERK和AKT磷酸化，增强了对血清生长因子撤除的敏感性，而miR-34 a基因缺陷的细胞则不太敏感。miR-34 a的14个新的直接靶点已经过实验验证，包括参与生长因子信号传导的基因（ARAF和PIK 3R 2）以及在细胞周期的各个阶段调节细胞周期进程的基因（细胞周期蛋白D3和G2、MCM 2和MCM 5、PLK 1和SMAD 4）。因此，miR-34 a通过干扰细胞分裂所需的生长因子信号转导和下游途径来缓和生长因子刺激的增殖和促存活作用。microRNA（miRNAs）是通过与具有部分互补序列的mRNA结合来调节基因表达的小RNA。miRNA降低mRNA靶标的稳定性或翻译，导致蛋白质表达减少。了解miRNA的生物学功能需要确定其靶点。在这里，我们开发了一种灵敏和特异的生化方法来识别候选microRNA靶点，这些靶点通过用标记的转染microRNA模拟物下拉而富集。该方法应用于miR-34 a，一种抑制细胞增殖的miRNA。我们发现miR-34 a可以潜在地调节数百个基因。对这些基因的计算分析表明，miR-34 a抑制对多种生长因子的促增殖反应具有新的功能。该功能补充了先前已知的miR-34 a在阻断细胞周期进展中的作用。因此，通过减少广泛的基因网络的表达，miR-34 a抑制生长因子信号传导及其下游后果，促进细胞存活和增殖。
A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ∼90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a–regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2) as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4). Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division. microRNAs (miRNAs) are small RNAs that regulate gene expression by binding to mRNAs bearing a partially complementary sequence. miRNAs decrease the stability or translation of mRNA targets, leading to reduced protein expression. Understanding the biological function of a miRNA requires identifying its targets. Here we developed a sensitive and specific biochemical method to identify candidate microRNA targets that are enriched by pull-down with a tagged, transfected microRNA mimic. The method was applied to miR-34a, a miRNA that inhibits cell proliferation. We found that miR-34a can potentially regulate hundreds of genes. Computational analysis of these genes suggested a novel function for miR-34a—suppression of the pro-proliferative response to diverse growth factors. This function complements the previously known role of miR-34a in blocking cell cycle progression. Thus, by reducing the expression of an extensive network of genes, miR-34a dampens growth factor signaling as well as its downstream consequences, promotion of cell survival and proliferation.
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影响因子：
64.5
作者：
Hafner M;Landthaler M;Burger L;Khorshid M;Hausser J;Berninger P;Rothballer A;Ascano M Jr;Jungkamp AC;Munschauer M;Ulrich A;Wardle GS;Dewell S;Zavolan M;Tuschl T
通讯作者：
Tuschl T
影响因子：
64.8
作者：
Baek, Daehyun;Villen, Judit;Shin, Chanseok;Camargo, Fernando D.;Gygi, Steven P.;Bartel, David P.
通讯作者：
Bartel, David P.
DOI：
10.1073/pnas.0909333107
发表时间：
2010-01-19
影响因子：
11.1
作者：
Gjerdrum, Christine;Tiron, Crina;Lorens, James B.
通讯作者：
Lorens, James B.
影响因子：
64.5
作者：
Lewis, BP;Shih, IH;Burge, CB
通讯作者：
Burge, CB
影响因子：
64.8
作者：
He, Lin;He, Xingyue;Hannon, Gregory J.
通讯作者：
Hannon, Gregory J.