Optimal endogenous controls enable reliable normalization of microRNA (miRNA) expression in reverse-transcription quantitative PCR (RT-qPCR). This is particularly important when miRNAs are considered as candidate diagnostic or prognostic biomarkers. Universal endogenous controls are lacking, thus candidate normalizers must be evaluated individually for each experiment. Here we present a strategy that we applied to the identification of optimal control miRNAs for RT-qPCR profiling of miRNA expression in T-cell acute lymphoblastic leukemia (T-ALL) and in normal cells of T-lineage. First, using NormFinder for an iterative analysis of miRNA stability in our miRNA-seq data, we established the number of control miRNAs to be used in RT-qPCR. Then, we identified optimal control miRNAs by a comprehensive analysis of miRNA stability in miRNA-seq data and in RT-qPCR by analysis of RT-qPCR amplification efficiency and expression across a variety of T-lineage samples and T-ALL cell line culture conditions. We then showed the utility of the combination of three miRNAs as endogenous normalizers (hsa-miR-16-5p, hsa-miR-25-3p, and hsa-let-7a-5p). These miRNAs might serve as first-line candidate endogenous controls for RT-qPCR analysis of miRNAs in different types of T-lineage samples: T-ALL patient samples, T-ALL cell lines, normal immature thymocytes, and mature T-lymphocytes. The strategy we present is universal and can be transferred to other RT-qPCR experiments.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy arising from T lymphocyte precursors. We have previously shown by miRNA-seq, that miRNAs from the mir-106a-363 cluster are overexpressed in pediatric T-ALL. In silico analysis indicated their potential involvement in the regulation of apoptosis. Here, we aimed to test the hypothesis on the pro-tumorigenic roles of these miRNAs in T-ALL cells in vitro. We demonstrate, for the first time, that hsa-miR-20b-5p and hsa-miR-363-3p from the mir-106a-363 cluster, when upregulated in T-ALL cells in vitro, protect leukemic cells from apoptosis, enhance proliferation, and contribute to growth advantage. We show, using dual luciferase reporter assays, Ago2-RNA immunoprecipitation, RT-qPCR, and Western blots, that the oncogenic effects of these upregulated miRNAs might, at least in part, be mediated by the downregulation of two important tumor suppressor genes, PTEN and BIM, targeted by both miRNAs. Additionally, we demonstrate the cooperative effects of these two miRNAs by simultaneous inhibition of both miRNAs as compared to the inhibition of single miRNAs. We postulate that hsa-miR-20b-5p and hsa-miR-363-3p from the mir-106a-363 cluster might serve as oncomiRs in T-ALL, by contributing to post-transcriptional repression of key tumor suppressors, PTEN and BIM.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy originating from T-cell precursors. The genetic landscape of T-ALL has been largely characterized by next-generation sequencing. Yet, the transcriptome of miRNAs (miRNome) of T-ALL has been less extensively studied. Using small RNA sequencing, we characterized the miRNome of 34 pediatric T-ALL samples, including the expression of isomiRs and the identification of candidate novel miRNAs (not previously annotated in miRBase). For the first time, we show that immunophenotypic subtypes of T-ALL present different miRNA expression profiles. To extend miRNome characteristics in T-ALL (to 82 T-ALL cases), we combined our small RNA-seq results with data available in Gene Expression Omnibus. We report on miRNAs most abundantly expressed in pediatric T-ALL and miRNAs differentially expressed in T-ALL versus normal mature T-lymphocytes and thymocytes, representing candidate oncogenic and tumor suppressor miRNAs. Using eight target prediction algorithms and pathway enrichment analysis, we identified differentially expressed miRNAs and their predicted targets implicated in processes (defined in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) of potential importance in pathogenesis of T-ALL, including interleukin-6-mediated signaling, mTOR signaling, and regulation of apoptosis. We finally focused on hsa-mir-106a-363 cluster and functionally validated direct interactions of hsa-miR-20b-5p and hsa-miR-363-3p with 3' untranslated regions of their predicted targets (PTEN, SOS1, LATS2), overrepresented in regulation of apoptosis. hsa-mir-106a-363 is a paralogue of prototypic oncogenic hsa-mir-17-92 cluster with yet unestablished role in the pathogenesis of T-ALL. Our study provides a firm basis and data resource for functional analyses on the role of miRNA-mRNA interactions in T-ALL.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous hereditary disease from a class of ciliopathies. In spite of the recent progress, the genetic basis of PCD in one-third of patients remains unknown. In search for new genes and/or mutations, whole-exome sequencing was performed in 120 unrelated Polish patients with PCD, in whom no genetic cause of PCD was earlier identified. Among a number of pathogenic variants in PCD genes, mutations in CFAP300 (alias C11orf70) were detected. Extended screening in the whole Polish PCD cohort revealed the relatively high frequency (3.6%) of otherwise rare c.[198_200 del_insCC] variant, indicating that it should be included in population-specific genetic tests for PCD in Slavic populations. Immunofluorescence analysis of the respiratory epithelial cells from patients with CFAP300 mutations revealed the absence or aberrant localization of outer and inner dynein arm markers, consistent with transmission electron microscope images indicating the lack of both dynein arms. Interestingly, the disparate localization of DNAH5 and DNALI1 proteins in patients with CFAP300 mutations suggested differential mechanisms for the trafficking of preassembled outer and inner dynein arms to the axoneme. The profile of CFAP300 expression during ciliogenesis in suspension culture was consistent with its role in cilia assembly. Gene silencing experiments, performed in a model organism, Schmidtea mediterranea (flatworm), pointed to the conserved role of CFAP300 in ciliary function.

MiRNAs form a class of noncoding RNAs, involved in post-transcriptional regulation of gene expression, broadly studied for their involvement in physiological and pathological context. Inhibition of mature miRNA transcripts, commonly used in miRNA loss-of-function experiments, may not be specific in case of miRNAs with high sequence homology, e.g. miRNAs from the same seed family. Phenotypic effects of miRNA repression might be biased by the repression of highly similar miRNAs. Another challenge is simultaneous inhibition of multiple miRNAs encoded within policistronic clusters, potentially co-regulating common biological processes. To elucidate roles of miRNA clusters and miRNAs with high sequence homology, it is of key importance to selectively repress only the miRNAs of interest. Targeting miRNAs on genomic level with CRISPR/dCas9-based methods is an attractive alternative to blocking mature miRNAs. Yet, so far no clear guidelines on the design of CRISPR inhibition (CRISPRi) experiments, specifically for miRNA repression, have been proposed. To address this need, here we propose a strategy for effective inhibition of miRNAs and miRNA clusters using CRISPRi. We provide clues on how to approach the challenges in using CRISPR/dCas in miRNA studies, which include prediction of miRNA transcription start sites (TSSs) and the design of single guide RNAs (sgRNAs). The strategy implements three TSS prediction online tools, dedicated specifically for miRNAs: miRStart, FANTOM 5 miRNA atlas, DIANA-miRGen, and CRISPOR tool for sgRNAs design; it includes testing and selection of optimal sgRNAs. We demonstrate that compared to siRNA/shRNA-based miRNA silencing, CRISPRi improves the repression specificity for miRNAs with highly similar sequence and contribute to higher uniformity of the effects of silencing the whole miRNA clusters. This strategy may be adapted for CRISPR-mediated activation (CRISPRa) of miRNA expression.