Discovery of novel microRNAs in female reproductive tract using next generation sequencing.
Discovery of novel microRNAs in female reproductive tract using next generation sequencing.
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DOI：
10.1371/journal.pone.0009637
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发表时间：
2010-03-10
期刊：
影响因子：
3.7
通讯作者：
Gunaratne PH
中科院分区：
文献类型：
--
作者：
Creighton CJ;Benham AL;Zhu H;Khan MF;Reid JG;Nagaraja AK;Fountain MD;Dziadek O;Han D;Ma L;Kim J;Hawkins SM;Anderson ML;Matzuk MM;Gunaratne PH
microRNA（miRNAs）是一类介导转录后基因沉默的非编码小RNA。目前已经鉴定了超过700种人类miRNA，其中许多在疾病中发生突变或失调。在这里，我们报告了通过对来自正常和疾病状态下的人类女性生殖器官的100多个组织或细胞系的小RNAome（<30 nt）进行深度测序来鉴定新的miRNA。这些标本包括卵巢上皮和卵巢癌，子宫内膜和子宫肌瘤，子宫肌层和子宫平滑肌肿瘤。将不与已知miRNA比对的序列读数各自映射到基因组以提取侧翼序列。这些延伸的序列区域在计算机中折叠以鉴定RNA发夹。证明具有形成具有低最小自由能（<-25 kcal）的茎环结构的能力并且预测的Drosha和Dicer切割位点产生与实际序列匹配的成熟miRNA序列的序列被认为是推定的新型miRNA。当推定的新发夹组装了一系列与推定的成熟miRNA高度相似但具有异质性3′端的序列时，获得了额外的置信度。一种新的miRNA符合这些标准，并在我们的收藏中有它的“星星”序列。我们发现了7种不同的已证实的新的miRNAs，以及51种额外的新的miRNAs，它们代表了高度可信的预测，但没有可检测到的星星序列。我们的新型miRNAs在多个样本中均可检测到，但表达水平较低，并且对任何一种组织或细胞类型都没有特异性。到目前为止，这项研究代表了一起分析以鉴定新型miRNA的最大样本集。
MicroRNAs (miRNAs) are small non-coding RNAs that mediate post-transcriptional gene silencing. Over 700 human miRNAs have currently been identified, many of which are mutated or de-regulated in diseases. Here we report the identification of novel miRNAs through deep sequencing the small RNAome (<30 nt) of over 100 tissues or cell lines derived from human female reproductive organs in both normal and disease states. These specimens include ovarian epithelium and ovarian cancer, endometrium and endometriomas, and uterine myometrium and uterine smooth muscle tumors. Sequence reads not aligning with known miRNAs were each mapped to the genome to extract flanking sequences. These extended sequence regions were folded in silico to identify RNA hairpins. Sequences demonstrating the ability to form a stem loop structure with low minimum free energy (<−25 kcal) and predicted Drosha and Dicer cut sites yielding a mature miRNA sequence matching the actual sequence were considered putative novel miRNAs. Additional confidence was achieved when putative novel hairpins assembled a collection of sequences highly similar to the putative mature miRNA but with heterogeneous 3′-ends. A confirmed novel miRNA fulfilled these criteria and had its “star” sequence in our collection. We found 7 distinct confirmed novel miRNAs, and 51 additional novel miRNAs that represented highly confident predictions but without detectable star sequences. Our novel miRNAs were detectable in multiple samples, but expressed at low levels and not specific to any one tissue or cell type. To date, this study represents the largest set of samples analyzed together to identify novel miRNAs.
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