Detection and validation of miRNA targets in breast cancer

描述（由申请人提供）：乳腺癌是女性中最常见的癌症，也是美国女性癌症死亡的第二大原因，每年有30万例新诊断和4.1万例死亡。最近，一类新的基因表达调控因子，称为microRNAs（miRNAs）的功能障碍与乳腺癌的发生，发展和转移有关。miRNA是短的非编码RNA，其与细胞质中的3'非翻译区（3' UTR）中的互补序列结合并抑制基因表达。基于生物信息学分析，预测每个miRNA控制基因产物的网络，使得数百个转录物可能由单个miRNA调控。与靶3 'UTR的配对不需要序列之间的完美匹配。由于这些元件是简并的和小的，它们通常难以检测，因此绝大多数癌症相关的miRNA靶点仍然未知。 为了克服我们知识中的这些差距，我们开发了一种无偏倚的高通量筛选方法，称为3 'LIFE测定（基于发光的3' UTR中功能元件的鉴定）。3 'LIFE以前所未有的规模系统地绘制了3' UTR中的miRNA靶点，使我们能够研究乳腺癌发生和发展过程中由miRNA触发的遗传网络的动态。我们将使用3 'LIFE检测在由1，880个3' UTR组成的中试文库中检测miRNA靶标，并探测两种乳腺癌相关miRNA：let-7 c和miR-10 b。我们将绘制它们的相互作用网络，并将使用它们的结合要求，以便将靶标扩展到整个人类转录组。 然后，我们将在体内验证目标，并将我们的结果与使用人类乳腺条件性重编程细胞（HMCRC）系统的匹配正常和早期乳腺癌活检组织中的转录组和miRNA变化进行比较。我们的方法1）开创了一种检测miRNA：3 'UTR相互作用的创新方法，2）将检测由两种乳腺癌相关miRNA控制的遗传网络，3）将精确定位早期原发性乳腺癌的肿瘤特异性miRNA和转录组变化，以及4）将发现用于早期乳腺癌检测和筛查的生物标志物和潜在药物靶点。

DESCRIPTION (provided by applicant): Breast cancer is the most common cancer among women, and the second leading cause of cancer mortality in females in the U.S., with 300,000 new diagnoses and 41,000 fatalities annually. Recently, the dysfunction of a new class of gene expression regulators, named microRNAs (miRNAs), was linked to breast cancer initiation, progression and metastasis. miRNAs are short non-coding RNAs that bind to complementary sequences in the 3' UnTranslated Regions (3'UTRs) in the cytoplasm and repress gene expression. Based on bioinformatic analysis each miRNA is predicted to control a network of gene products, such that hundreds of transcripts are likely to be regulated by a single miRNA. The pairing with target 3'UTRs does not require a perfect match between the sequences. Since these elements are degenerate and small, they are generally difficult to detect, thus the vast majority of cancer relevant miRNA targets are still unknown. To overcome these gaps in our knowledge, we have developed an unbiased high-throughput screening method named 3'LIFE assay (Luminescence-based Identification of Functional Elements in 3'UTRs). 3'LIFE systematically maps miRNA targets in 3'UTRs with an unprecedented scale, allowing us to study the dynamics of genetic networks triggered by miRNAs during the initiation and the progression of breast cancer. We will use the 3'LIFE assay to detect miRNA targets in a pilot library composed of 1,880 3'UTRs, and probe two breast cancer relevant miRNAs: let-7c and miR-10b. We will map their network of interaction and will use their binding requirements in order to extend the targets to the entire human transcriptome. We will then validate the targets in vivo and compare our results to the transcriptome and miRNA changes in matched normal and early stage breast cancer biopsies using the human mammary conditionally reprogrammed cell (HMCRC) system. Our approach 1) pioneers an innovative method to detect miRNA:3'UTR interactions, 2) will detect the genetic network controlled by two breast cancer-relevant miRNAs, 3) will pinpoint tumor-specific miRNA and transcriptome changes of early stage primary breast cancers, and 4) will discover biomarkers and potential drug targets for early breast cancer detection and screening.