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.

The Epstein-Barr virus (EBV) is an oncogenic human Herpes virus involved in the pathogenesis of nasal NK/T-cell lymphoma. EBV encodes microRNAs (miRNAs) and induces changes in the host cellular miRNA profile. MiRNAs are short non-coding RNAs of about 19–25 nt length that regulate gene expression by post-transcriptional mechanisms and are frequently deregulated in human malignancies including cancer. The microRNA profiles of EBV-positive NK/T-cell lymphoma, non-infected T-cell lymphoma and normal thymus were established by deep sequencing of small RNA libraries. The comparison of the EBV-positive NK/T-cell vs. EBV-negative T-cell lymphoma revealed 15 up- und 16 down-regulated miRNAs. In contrast, the majority of miRNAs was repressed in the lymphomas compared to normal tissue. We also identified 10 novel miRNAs from known precursors and two so far unknown miRNAs. The sequencing results were confirmed for selected miRNAs by quantitative Real-Time PCR (qRT-PCR). We show that the proinflammatory cytokine interleukin 1 alpha (IL1A) is a target for miR-142-3p and the oncogenic BCL6 for miR-205. MiR-142-3p is down-regulated in the EBV-positive vs. EBV-negative lymphomas. MiR-205 was undetectable in EBV-negative lymphoma and strongly down-regulated in EBV-positive NK/T-cell lymphoma as compared to thymus. The targets were confirmed by reporter assays and by down-regulation of the proteins by ectopic expression of the cognate miRNAs. Taken together, our findings demonstrate the relevance of deregulated miRNAs for the post-transcriptional gene regulation in nasal NK/T-cell lymphomas.

MicroRNAs (miRNAs) are short 18–23 nucleotide long noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA. Our previous miRNA profiling of diffuse large B-cell lymphoma (DLBCL) revealed a mutation in the seed sequence of miR-142-3p. Further analysis now showed that miR-142 was mutated in 11 (19.64%) of the 56 DLBCL cases. Of these, one case had a mutation in both alleles, with the remainder being heterozygous. Four mutations were found in the mature miR-142-5p, four in the mature miR-142-3p, and three mutations affected the miR-142 precursor. Two mutations in the seed sequence redirected miR-142-3p to the mRNA of the transcriptional repressor ZEB2 and one of them also targeted the ZEB1 mRNA. However, the other mutations in the mature miR-142-3p did not influence either the ZEB1 or ZEB2 3′ untranslated region (3′ UTR). On the other hand, the mutations affecting the seed sequence of miR-142-3p resulted in a loss of responsiveness in the 3′ UTR of the known miR-142-3p targets RAC1 and ADCY9. In contrast to the mouse p300 gene, the human p300 gene was not found to be a target for miR-142-5p. In one case with a mutation of the precursor, we observed aberrant processing of the miR-142-5p. Our data suggest that the mutations in miR-142 probably lead to a loss rather than a gain of function. This is the first report describing mutations of a miRNA gene in a large percentage of a distinct lymphoma subtype.

Objective: To elucidate the potential regulatory function of miR-23a/b-3p on spermatogenesis-specific genes. Design: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) validation, Northern blot, dual luciferase assay, and Western blot confirmation. Setting: University research and clinical institutes. Patient(s): A total of 115 men presenting at an infertility clinic. Intervention(s): None. Main Outcome Measure(s): Significant higher abundance levels of miR-23a/b-3p and lower abundance levels of PFKFB4, HMMR, SPATA6, and TEX15 in oligoasthenozoospermic men compared with those in normozoospermic men. Result(s): In oligoasthenozoospermic men, the abundance levels of miR-23a/b-3p were significantly higher when compared with controls as determined by RT-qPCR. After in silico prediction of potential targets of miR-23a/b-3p, PFKFB4, HMMR, SPATA6, and TEX15 have been identified as direct targets by dual luciferase assays. Mutations in the miR-23a/b-3p binding site within the 3 0 UTRs resulted in abrogated responsiveness to miR-23a/b-3p. PFKFB4, HMMR, SPATA6, and TEX15 mRNA and HMMR and SPATA6 protein levels were significantly lower in oligoasthenozoospermic men compared with in normozoospermic men. Correlation analysis showed that the sperm count, motility, and morphology were negatively correlated with miR-23a/b-3p and positively correlated with PFKFB4, HMMR, SPATA6, and TEX15 abundance levels (lower DCt, the higher abundance levels). Conclusion(s): This study establishes a link between up-regulation of miR-23a/b-3p and the coincident down-regulation of four expressed genes in the sperm of men with oligoasthenozoospermia, compared with men with normozoospermia. This study provides a novel insight into some of the mechanisms leading to male subfertility, offering a possible therapeutic target for treatment, or even for male contraception. (Fertil Steril Ò 2019;112:323-35. Ó2019 by American Society for Reproductive Medicine.) El resumen está disponible en Español al final del artículo.

The aim of this work was to establish the micro RNA profile of SNK 6 and SNT 16, two Epstein–Barr virus ( EBV )‐infected cell lines derived from nasal NK /T‐cell lymphoma ( NKTL ). The oncogenic EBV is strongly associated with the pathogenesis of nasal and extranodal NK /T‐cell lymphoma and expresses 44 mature micro RNA s and two noncoding EBV ‐encoded RNA s ( EBER s). mi RNA s are 19‐25nt noncoding RNA s that affect host and viral gene expression post‐transcriptionally. Deregulated mi RNA patterns are frequently linked to a variety of human cancers including lymphomas. mi RNA profiling of the two NK /T cell lines vs. primary cells revealed 10 and 4 up‐regulated and 10 and 12 down‐regulated mi RNA s in SNK 6 and SNT 16 cells respectively. The results were validated by qRT ‐ PCR for selected mi RNA s. Target gene analyses confirmed cullin 5 ( CUL 5) and sphingosin‐1‐phosphate receptor 1 (S1 PR 1) as targets for the down‐regulated hsa‐miR‐148a and viral ebv‐miR‐ BART 16 respectively. As recently demonstrated for the regulation of IL 1‐alpha by miR‐142‐3p, coexpression of the EBER s selectively exerted corepression of S1 PR 1 by BART 16 but not of CUL 5 by miR‐148a, indicating selective corepression by the EBER s.