Institute of Human Genetics, Saarland University, 66421 Homburg, Germany.
Chair for Clinical Bioinformatics, Saarland University, 66123 Saarbrücken, Germany.
Nucleic Acids Res. 2019 Apr 23;47(7):3353-3364. doi: 10.1093/nar/gkz097.
While the number of human miRNA candidates continuously increases, only a few of them are completely characterized and experimentally validated. Toward determining the total number of true miRNAs, we employed a combined in silico high- and experimental low-throughput validation strategy. We collected 28 866 human small RNA sequencing data sets containing 363.7 billion sequencing reads and excluded falsely annotated and low quality data. Our high-throughput analysis identified 65% of 24 127 mature miRNA candidates as likely false-positives. Using northern blotting, we experimentally validated miRBase entries and novel miRNA candidates. By exogenous overexpression of 108 precursors that encode 205 mature miRNAs, we confirmed 68.5% of the miRBase entries with the confirmation rate going up to 94.4% for the high-confidence entries and 18.3% of the novel miRNA candidates. Analyzing endogenous miRNAs, we verified the expression of 8 miRNAs in 12 different human cell lines. In total, we extrapolated 2300 true human mature miRNAs, 1115 of which are currently annotated in miRBase V22. The experimentally validated miRNAs will contribute to revising targetomes hypothesized by utilizing falsely annotated miRNAs.
尽管人类 miRNA 候选物的数量不断增加，但只有少数得到了完全的描述和实验验证。为了确定真正的 miRNAs 的总数，我们采用了结合计算高通量和实验低通量的验证策略。我们收集了 28,866 个人类小 RNA 测序数据集，包含 3637 亿个测序读段，并排除了错误注释和低质量的数据。我们的高通量分析确定了 24127 个成熟 miRNA 候选物中的 65%可能是假阳性。我们使用 northern blot 实验验证了 miRBase 条目和新的 miRNA 候选物。通过过表达编码 205 个成熟 miRNA 的 108 个前体，我们验证了 miRBase 条目中的 68.5%，其中高可信度条目的验证率上升到 94.4%，而新的 miRNA 候选物的验证率为 18.3%。分析内源性 miRNAs 时，我们验证了 12 种不同人类细胞系中 8 个 miRNA 的表达。总共推断出 2300 个真正的人类成熟 miRNA，其中 1115 个目前在 miRBase V22 中注释。这些经过实验验证的 miRNAs 将有助于修正利用错误注释的 miRNAs 假设的靶基因。
