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RNA介导，称为小干扰RNA（siRNA），其有助于切割完全互补的mRNA转录物。微小RNA（miRNAs）是内源性的小RNA，其有助于抑制具有部分互补区域的mRNA，但也可以降低转录水平。由于miRNA被认为主要与转录物的3'UTR相互作用，但几乎没有实验数据支持这一点，我们试图询问模拟miRNA的错配siRNA是否影响作为靶位点位置的函数的同源mRNA水平。我们的研究发现，靶向两种内源性转录物的3'UTR的错配siRNA比靶向编码区的siRNA产生更大的mRNA水平降低。我们的研究结果证明了靶位点定位在内源性mRNA中对于与RNAi相关的小RNA的重要性，这些研究发表在2006年的FEBS Letters上。为了解决改变的miRNA表达是否有助于癌症过程，GSS部门负责人Caplen博士参与了由CCR（实验室）Curt Harris博士小组进行的研究。人类致癌作用（LHC），CCR，NCI）与爱荷华州大学的Carlo Croce博士合作。Carlo Croce博士和其他人先前已经表明，不同类型的癌症与独特的microRNA模式相关，这些模式可能在诊断和确定预后方面有价值。在LHC的Yanaihara博士进行的合作研究中，CCR（Yanaihara等人，2006 Cancer Cell 9：189-198），来自与邻近正常组织匹配的104对原发性肺癌的差异microRNA表达数据证实了早期论文中报道的肺癌中microRNA的失调，并发现了与不良预后相关的新microRNA标记物hsa-miR-155。作为GSS与娜塔莉亚博士和鲍勃斯蒂芬斯（ABCC，NCI-Fredrick/SAIC Frederick Inc.）之间广泛合作的一部分，NCI，NIH）已经预测了hsa-miR-155的可能的mRNA靶标。hsa-miR-155与肺癌之间的关系需要更密切地检查，使用更大的患者群体，但验证这些结果可能会导致批准肺癌的诊断或预后测试。此外，识别miR-155的分子靶点可能会导致基于中断该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阵列平台（Ambion/ABI）在GSS中建立了基于miRNA微阵列的分析，并且已经开始在基线条件下以及在遗传和药物操作之后对miRNA表达乳腺癌细胞系进行广泛分析。最后，GGS已经检查了一个基因组区域，人类染色体8 q24，通常与伯基特淋巴瘤和一些实体瘤相关，这是由于基因重排和/或缺失和扩增，以证明以前未鉴定的miRNA的存在。许多研究表明，miRNA可能聚集在已知的基因组不稳定区域内，我们最近回顾了这一特定的主题领域，特别强调小鼠基因组（Huppi et al.，癌症生物学研讨会）。在与娜塔莉亚Volfovsky博士和鲍勃斯蒂芬斯（ABCC，NCI-Fredrick/SAIC Frederick Inc. NCI，NIH）12个可能的miRNA前体序列被鉴定为定位在人类chr. 8 q24区域内，其中7个被实验验证。进一步的分子和遗传学研究正在调查这些新的miRNAs，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博士在2006年AACR会议上展示了描述这些miRNAs鉴定的初步数据，说明了这项工作的重要性。

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.