The Role of MicroRNAs in the Regulation of Gene Expressi

在哺乳动物细胞中，RNAi可以由合成的双链RNAs介导，称为小干扰RNAs(SiRNAs)，它帮助切割完全互补的mRNA转录产物。MicroRNAs(MiRNAs)是一种内源性小RNA，帮助翻译上抑制具有部分互补区的mRNAs，但也可能降低转录水平。由于miRNAs被认为主要与转录本的3‘UTRs相互作用，但几乎没有经验数据支持这一点，我们试图询问模仿miRNAs的不匹配siRNAs是否影响同源mRNA水平作为目标位置的函数。我们的研究发现，针对两个内源转录本的3‘UTRs的错配siRNAs比针对编码区的siRNAs产生的mRNA水平下降更大。我们的发现证明了内源性mRNAs中靶点位置对于与RNAi相关的小RNAs的重要性，这些研究发表在2006年的《FEBS快报》上。为了研究改变的miRNA表达是否有助于癌症过程，GSS科长卡普伦博士参与了Curt Harris博士在CCR(实验室)进行的研究。(LHC，CCR，NCI)与爱荷华州大学的卡洛·克罗奇博士合作。卡洛·克罗齐博士和其他人之前已经证明，不同类型的癌症与独特的microRNA模式有关，这些模式可能在诊断和确定预后方面有价值。在CCR LHC的Yanaihara博士(Yanaihara等人，2006年癌细胞9：189-198)进行的合作研究中，来自104对与邻近正常组织匹配的原发肺癌的差异microRNA表达数据证实了早期论文中报道的肺癌中microRNA的去调控，并发现了一种与不良预后相关的新的microRNA标记物hsa-miR-155。作为GSS与Natalia Volfovsky博士和Bob Stephens(ABCC，NCI-Fredrick/SAIC Frederick Inc.NCI，NIH)之间广泛合作的一部分，已经预测了hsa-miR-155的可能的信使核糖核酸靶标。Hsa-miR-155与肺癌之间的关系需要更密切地研究，使用更多的患者组，但验证这些结果可能会导致批准的肺癌诊断或预后测试。此外，识别miR-155的分子靶点可能会导致基于阻断miRNA的作用或抑制其下游靶点的靶向治疗。为了进一步研究miRNA表达在癌症中的作用，我们现在已经使用商业ARRY平台(Ambion/ABI)在GSS中建立了基于miRNA微阵列的分析，并开始广泛地分析在基线条件下以及在遗传和药物操作之后的乳腺癌细胞株的miRNA表达。最后，GGS检查了基因组区域，人类染色体8q24，通常与Burkitt淋巴瘤和一些实体肿瘤有关，这是基因重排和/或缺失和扩增的结果，以寻找以前未识别的miRNAs存在的证据。一些研究表明，miRNAs可能聚集在已知的基因组不稳定区域内，我们最近审查了这一特定主题领域，特别强调小鼠基因组(Huppi等人，癌症生物学研讨会)。在Natalia Volfovsky博士和Bob Stephens博士(ABCC，NCI-Fredrick/SAIC Frederick Inc.NCI，NIH)的合作下，12个可能的miRNA前体序列被鉴定为映射到人类ChR内。8q24区，其中7个经实验验证。对这些新的miRNAs的进一步分子和遗传学研究正在进行中，我们预计在2006年底之前提交一份描述这一发现的手稿。Konrad Huppi博士的选择说明了这项工作的重要性，他在2006年AACR会议上介绍了描述其中一些miRNAs识别的初步数据。

In mammalian cells RNAi can be mediated by synthetic duplex RNAs, termed small interfering RNAs (siRNAs), that assist in cleaving completely complementary mRNA transcripts. MicroRNAs (miRNAs) are endogenous small RNAs that assist in translationally repressing mRNAs with regions of partial complementarity, but may also reduce transcript levels. Since miRNAs are thought to predominantly interact with the 3' UTRs of transcripts but little experiemental data exists to support this, we sought to ask if mismatched siRNAs mimicking miRNAs affect cognate mRNA levels as a function of target site location. Our studies found that mismatched siRNAs targeting the 3' UTRs of two endogenous transcripts yield a greater reduction in mRNA levels than those targeting the coding region. Our findings demonstrated the importance of target site location within endogenous mRNAs for small RNAs associated with RNAi and these studies were published in FEBS Letters in 2006. To address if altered miRNA expression can contribute to the cancer process Dr. Caplen, the GSS section head, became involved in study conducted by Dr. Curt Harris's group at CCR (Lab. of Human Carcinogenesis, (LHC), CCR, NCI) in collaboration with Dr. Carlo Croce, University of Iowa. Dr. Carlo Croce and others have previously shown that different types of cancer are associated with unique microRNA patterns, and that these patterns may be valuable in diagnosing and ascertaining prognosis. In the collaborative study conducted by Dr. Yanaihara from LHC, CCR (Yanaihara et al., 2006 Cancer Cell 9: 189-198), differential microRNA expression data from 104 pairs of primary lung cancers matched against adjacent normal tissue confirmed the deregulation of microRNAs in lung cancer reported in earlier papers, and uncovered a new microRNA marker associated with poor prognosis, hsa-miR-155. As part of a broad ranging collaboration between GSS and Drs. Natalia Volfovsky and Bob Stephens (ABCC, NCI-Fredrick/SAIC Frederick Inc. NCI, NIH) possible mRNA targets for hsa-miR-155 have been predicted. The relationship between hsa-miR-155 and lung cancer, needs to be examined more closely, using larger groups of patients, but validating these results may lead to an approved diagnostic or prognostic test for lung cancer. Moreover, identification of the molecular target for miR-155 could lead to targeted therapies based either on interrupting that miRNA's effects or inhibiting its downstream targets. To further study the role of miRNA expression in cancer we have now established miRNA microarray based analysis in GSS using a commercial arry platform (Ambion/ABI) and have begun extensive analysis of the miRNA expression breast cancer cell lines under baseline conditions and following genetic and drug manipulation. Finally, GGS has examined a genomic region, human chromosome 8q24, commonly associated with Burkitt's lymphoma and some solid tumors as a result of genetic rearrangements and/or deletions and amplifications for evidence of the presence of previously unidentified miRNAs. A number of studies have suggested that miRNAs may be clustered within known areas of genomic instability and we have recently reviewed this particular subject area with a special emphasis on the mouse genome (Huppi et al., Seminars in Cancer Biology). In collaboration with Drs. Natalia Volfovsky and Bob Stephens (ABCC, NCI-Fredrick/SAIC Frederick Inc. NCI, NIH) 12 possible miRNA precursor sequences were identified as mapping to within the human chr. 8q24 region, of which, 7 were experimentally verified. Further molecular and genetic studies investigating these novel miRNAs are on going and we anticipate submission of a manuscript describing this finding before the end of 2006. The importance of this work is illustrated by Dr. Konrad Huppi selection to present preliminary data describing the identification of some of these miRNAs at the 2006 AACR meeting.