Discovery of Novel MicroRNAs in Female Reproductive Tract Using Next Generation Sequencing

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.

微小RNA（microRNAs, miRNAs）是一类介导转录后基因沉默的小型非编码RNA。目前已鉴定出超过700种人类miRNA，其中许多在疾病中发生突变或表达失调。本研究通过对100余份源自人类女性生殖器官（涵盖正常与疾病状态）的组织或细胞系的小RNA组（<30 nt）进行深度测序，开展新型miRNA的鉴定工作。所分析的标本包括卵巢上皮与卵巢癌组织、子宫内膜与子宫内膜异位瘤、子宫肌层与子宫平滑肌肿瘤。将未比对至已知miRNA的测序读段定位至人类基因组，提取其侧翼序列。对这些延伸的序列区域进行计算机模拟折叠，以识别RNA发夹结构。若某序列可形成最低自由能低于-25 kcal的茎环结构，且其预测的Drosha与Dicer剪切位点可产生与实际序列匹配的成熟miRNA，则该序列被认定为推定新型miRNA。当推定的新型发夹结构对应一组与推定成熟miRNA高度相似但3'端存在异质性的序列时，可进一步提升该候选物的置信度。若某推定新型miRNA满足上述全部标准，且在本研究数据集内存在其"star"序列，则可确认为新型miRNA。本研究共鉴定出7种经确证的新型miRNA，以及51种置信度较高但未检测到星序列的候选新型miRNA。这些新型miRNA可在多个样本中被检测到，但表达水平较低，且未表现出组织或细胞类型特异性。截至目前，本研究是同类研究中采用最大规模联合分析样本集以鉴定新型miRNA的工作。