Systematic validation of predicted microRNAs for cyclin D1.
Systematic validation of predicted microRNAs for cyclin D1.
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DOI：
10.1186/1471-2407-9-194
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发表时间：
2009-06-18
期刊：
影响因子：
3.8
通讯作者：
Mo YY
中科院分区：
文献类型：
--
作者：
Jiang Q;Feng MG;Mo YY
MicroRNAs是一种内源性的非编码小RNA分子，能够在转录后水平上沉默蛋白质编码基因。根据计算机辅助预测，单个microRNA可能有超过100个靶标。另一方面，一个单一的蛋白质编码基因可能成为许多潜在的microRNAs的靶标。然而，在这些预测的microRNA/mRNA相互作用中，只有相对较少的数量得到了实验验证，并且没有使用报告系统进行系统验证。在这项研究中，我们使用荧光素释放报告基因分析来验证能够沉默细胞周期蛋白D1(CCND1)的microRNAs，因为CCND1是一种众所周知的原癌基因，与多种类型的癌症有关。我们选择米兰达http://www.microRNA.org作为主要的预测方法。然后，我们将预测的58个microRNA前体中的51个克隆到pCDH-CMV-MCS-EF1-CopGFP中，并检测它们对携带CCND1基因3‘-非翻译区(UTR)的荧光素酶报告基因的影响。实时定量聚合酶链式反应显示，51个克隆的microRNA前体中有45个表达了较高水平的外源microRNA，这些外源microRNA用于我们的验证实验。通过任意截断35%的减少，我们确定了7个能够抑制Luc-CCND1-UTR活性的microRNAs。其中，4个是先前验证的靶标，其余3个在本研究中被验证为阳性。有趣的是，我们发现miR-503不仅抑制了荧光素酶的活性，而且在蛋白质和mRNA水平上都抑制了内源性的CCND1。此外，我们还发现miR-503能够减少S期细胞的数量，并引起细胞生长抑制，提示miR-503可能是一种潜在的肿瘤抑制因子。这项研究提供了更全面的microRNA/CCND1相互作用的图景，并进一步证明了实验靶标验证的重要性。
MicroRNAs are the endogenous small non-coding RNA molecules capable of silencing protein coding genes at the posttranscriptional level. Based on computer-aided predictions, a single microRNA could have over a hundred of targets. On the other hand, a single protein-coding gene could be targeted by many potential microRNAs. However, only a relatively small number of these predicted microRNA/mRNA interactions are experimentally validated, and no systematic validation has been carried out using a reporter system. In this study, we used luciferease reporter assays to validate microRNAs that can silence cyclin D1 (CCND1) because CCND1 is a well known proto-oncogene implicated in a variety of types of cancers. We chose miRanda http://www.microRNA.org as a primary prediction method. We then cloned 51 of 58 predicted microRNA precursors into pCDH-CMV-MCS-EF1-copGFP and tested for their effect on the luciferase reporter carrying the 3'-untranslated region (UTR) of CCND1 gene. Real-time PCR revealed the 45 of 51 cloned microRNA precursors expressed a relatively high level of the exogenous microRNAs which were used in our validation experiments. By an arbitrary cutoff of 35% reduction, we identified 7 microRNAs that were able to suppress Luc-CCND1-UTR activity. Among them, 4 of them were previously validated targets and the rest 3 microRNAs were validated to be positive in this study. Of interest, we found that miR-503 not only suppressed the luciferase activity, but also suppressed the endogenous CCND1 both at protein and mRNA levels. Furthermore, we showed that miR-503 was able to reduce S phase cell populations and caused cell growth inhibition, suggesting that miR-503 may be a putative tumor suppressor. This study provides a more comprehensive picture of microRNA/CCND1 interactions and it further demonstrates the importance of experimental target validation.
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