Functional screening for miRNAs targeting Smad4 identified miR-199a as a negative regulator of TGF-β signalling pathway.
Functional screening for miRNAs targeting Smad4 identified miR-199a as a negative regulator of TGF-β signalling pathway.
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DOI：
10.1093/nar/gks667
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发表时间：
2012-10
影响因子：
14.9
通讯作者：
Yang X
中科院分区：
文献类型：
--
作者：
Zhang Y;Fan KJ;Sun Q;Chen AZ;Shen WL;Zhao ZH;Zheng XF;Yang X
转化生长因子-β（TGF-β）信号通路参与多种生物学过程。Smad 4是TGF-β信号传导的中心细胞转导子，其调节异常与多种人类疾病和发育障碍有关。然而，Smad 4失调的机制尚未完全了解。使用基于荧光素酶报告基因分析的功能筛选方法，我们从388个人类miRNAs的表达文库中鉴定出39个microRNAs（miRNAs）作为Smad 4的潜在调节因子。筛选得到生物信息学分析的支持，因为39种鉴定的miRNA中有24种也预测靶向Smad 4。MiR-199 a在多种人癌细胞系和胃癌组织中与Smad 4的表达呈负相关，并抑制Smad 4的表达，阻断TGF-β的典型转录反应。这些效应依赖于Smad 4 3′-非翻译区（UTR）中存在保守但不完美的种子配对的miR-199 a结合位点。miR-199 a的过表达显著抑制TGF-β诱导胃癌细胞生长停滞和凋亡的能力，并促进软琼脂中的锚定非依赖性生长，提示miR-199 a在人胃肿瘤发生中起致癌作用。总之，我们的功能筛选揭示了多种调节TGF-β信号传导的细胞反应性的miRNA，并揭示了miR-199 a通过直接靶向Smad 4在胃癌中的重要作用。
The transforming growth factor-β (TGF-β) signalling pathway participates in various biological processes. Dysregulation of Smad4, a central cellular transducer of TGF-β signalling, is implicated in a wide range of human diseases and developmental disorders. However, the mechanisms underlying Smad4 dysregulation are not fully understood. Using a functional screening approach based on luciferase reporter assays, we identified 39 microRNAs (miRNAs) as potential regulators of Smad4 from an expression library of 388 human miRNAs. The screening was supported by bioinformatic analysis, as 24 of 39 identified miRNAs were also predicted to target Smad4. MiR-199a, one of the identified miRNAs, was inversely correlated with Smad4 expression in various human cancer cell lines and gastric cancer tissues, and repressed Smad4 expression and blocked canonical TGF-β transcriptional responses in cell lines. These effects were dependent on the presence of a conserved, but not perfect seed paired, miR-199a-binding site in the Smad4 3′-untranslated region (UTR). Overexpression of miR-199a significantly inhibited the ability of TGF-β to induce gastric cancer cell growth arrest and apoptosis in vitro, and promoted anchorage-independent growth in soft agar, suggesting that miR-199a plays an oncogenic role in human gastric tumourigenesis. In conclusion, our functional screening uncovers multiple miRNAs that regulate the cellular responsiveness to TGF-β signalling and reveals important roles of miR-199a in gastric cancer by directly targeting Smad4.
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