The Role of MicroRNAs in the Regulation of Gene Expression

在哺乳动物细胞中，RNA干扰(RNAi)可以由合成的双链RNAs介导，称为小干扰RNAs(SiRNAs)，通过介导完全互补的mRNA转录本的切割。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个非翻译区(UTRs)相互作用，但几乎没有实验数据支持这一点，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个UTRs的错配siRNA比针对编码区的siRNA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。为了解决miRNA表达改变是否会导致癌症过程，Caplen博士(遗传学分部基因沉默科(GSS)科长)参与了CCR(实验室)Curt Harriss博士小组进行的一项研究。(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。在这项合作研究中(Yanaihara等人，2006年癌细胞9：189-198)，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microRNAs的去调控，并发现了一个与不良预后相关的新的microRNA标记物hsa-miR-155。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阵列平台(Ambion/ABI)在GSS中建立了自己的基于miRNA微阵列的分析，并开始对乳腺癌细胞系进行广泛的miRNA表达分析。本工作鉴定了一些在不同乳腺癌细胞系中差异表达的miRNAs。后续研究的重点是确定这些差异表达的miRNAs的假定靶标。最后，我们检查了一个基因组区域，人类染色体8q24，通常与Burkitts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，以寻找以前未确定的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。对这些新型miRNAs的进一步分子和遗传学研究正在人类和小鼠系统中进行。

In mammalian cells, RNA interference (RNAi) can be mediated by synthetic duplex RNAs, termed small interfering RNAs (siRNAs, by mediating the cleavage of 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 untranslated regions (UTRs) of transcripts but little experimental 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, (Section head, Gene Silencing Section (GSS), Genetics Branch), became involved in a study conducted by Dr. Curt Harriss 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 this collaborative study (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. To further study the role of miRNA expression in cancer we have now established our own miRNA microarray based analysis within GSS using a commercial array platform (Ambion/ABI) and have begun extensive analysis of the miRNA expression breast cancer cell lines. This work has identified a number of miRNAs differential expressed in different breast cancer cell line. Follow up studies are focused on characterizing the putative targets of these differentially expressed miRNAs. Finally, we have examined a genomic region, human chromosome 8q24, commonly associated with Burkitts 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 in both human and mouse systems.