MiR&moRe2: A Bioinformatics Tool to Characterize microRNAs and microRNA-Offset RNAs from Small RNA-Seq Data.

MicroRNA-offset RNAs (moRNAs) are microRNA-like small RNAs generated by microRNA precursors. To date, little is known about moRNAs and bioinformatics tools to inspect their expression are still missing. We developed miR&moRe2, the first bioinformatics method to consistently characterize microRNAs, moRNAs, and their isoforms from small RNA sequencing data. To illustrate miR&moRe2 discovery power, we applied it to several published datasets. MoRNAs identified by miR&moRe2 were in agreement with previous research findings. Moreover, we observed that moRNAs and new microRNAs predicted by miR&moRe2 were downregulated upon the silencing of the microRNA-biogenesis pathway. Further, in a sizeable dataset of human blood cell populations, tens of novel miRNAs and moRNAs were discovered, some of them with significantly varied expression levels among the cell types. Results demonstrate that miR&moRe2 is a valid tool for a comprehensive study of small RNAs generated from microRNA precursors and could help to investigate their biogenesis and function.

A missense mutation in the rabbit melanocortin 4 receptor (MC4R) gene is associated with finishing weight in a meat rabbit line.

In this study we resequenced 1729 bp of the rabbit melanocortin 4 receptor (MC4 R) gene in 31 rabbits from different breeds/lines and identified ten polymorphisms: one was an indel and 9 were single nucleotide polymorphisms (SNPs). The indel and 5 SNPs were in the 5'-flanking region, 3 were synonymous SNPs and one was a missense mutation (c.101G>A; p.G34D), located in a conserved position of the extracellular tail of the MC4 R protein. The missense mutation was analyzed in a panel of 74 rabbits of different breeds and in 516 performance tested rabbits of a commercial paternal line under selection for growth efficiency. Association analysis indicated that rabbits with the less frequent genotype in this population (DD) had a lighter weight at 70 postnatal days than animals with genotype GD (P < 0.10) and animals with genotype GG (P < 0.05). This is the third study on candidate genes, after those on GH1 and IGF2 that reported a marker associated with finishing weight. Therefore, it seems that a candidate gene approach in rabbit based on previous information accumulated in other livestock species could be useful to identify genes explaining a fraction of variability of performance traits with potential application on rabbit breeding and selection.

Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients.

Background: SARS-CoV-2 induces a spectrum of clinical conditions ranging from asymptomatic infection to life threatening severe disease. Host microRNAs have been involved in the cytokine storm driven by SARS-CoV-2 infection and proposed as candidate biomarkers for COVID-19. Methods: To discover signatures of circulating miRNAs associated with COVID-19, disease severity and mortality, small RNA-sequencing was performed on serum samples collected from 89 COVID-19 patients (34 severe, 29 moderate, 26 mild) at hospital admission and from 45 healthy controls (HC). To search for possible sources of miRNAs, investigation of differentially expressed (DE) miRNAs in relevant human cell types in vitro. Results: COVID-19 patients showed upregulation of miRNAs associated with lung disease, vascular damage and inflammation and downregulation of miRNAs that inhibit pro-inflammatory cytokines and chemokines, angiogenesis, and stress response. Compared with mild/moderate disease, patients with severe COVID-19 had a miRNA signature indicating a profound impairment of innate and adaptive immune responses, inflammation, lung fibrosis and heart failure. A subset of the DE miRNAs predicted mortality. In particular, a combination of high serum miR-22-3p and miR-21-5p, which target antiviral response genes, and low miR-224-5p and miR-155-5p, targeting pro-inflammatory factors, discriminated severe from mild/moderate COVID-19 (AUROC 0.88, 95% CI 0.80-0.95, p<0.0001), while high leukocyte count and low levels of miR-1-3p, miR-23b-3p, miR-141-3p, miR-155-5p and miR-4433b-5p predicted mortality with high sensitivity and specificity (AUROC 0.95, 95% CI 0.89-1.00, p<0.0001). In vitro experiments showed that some of the DE miRNAs were modulated directly by SARS-CoV-2 infection in permissive lung epithelial cells. Conclusions: We discovered circulating miRNAs associated with COVID-19 severity and mortality. The identified DE miRNAs provided clues on COVID-19 pathogenesis, highlighting signatures of impaired interferon and antiviral responses, inflammation, organ damage and cardiovascular failure as associated with severe disease and death.

MiR-26a-5p as a Reference to Normalize MicroRNA qRT-PCR Levels in Plasma Exosomes of Pediatric Hematological Malignancies.

Plasma exosomal microRNAs (miRNAs) are considered as valid circulating biomarkers for cancer diagnosis and prognosis. Quantitative real-time polymerase chain reaction (qRT-PCR), the most commonly used technique to assess circulating miRNA levels, requires a normalization step involving uniformly expressed endogenous miRNAs. However, there is still no consensus on reference miRNAs for plasma exosomal miRNA abundance normalization. In this study, we identified a panel of miRNAs with stable abundance by analyzing public plasma exosome RNA-seq data and selected miR-486-5p, miR-26a-5p, miR-423-5p and miR191-5p as candidate normalizers. Next, we tested the abundance variation of these miRNAs by qRT-PCR in plasma exosomes of healthy donors and pediatric patients with anaplastic large cell lymphoma, Burkitt lymphoma, Hodgkin lymphoma and mature B-cell acute lymphoblastic leukemia. MiR-486-5p and miR-26a-5p showed the most stable levels, both between healthy controls and patients and among the malignancies analyzed. In light of previous reports on miRNA stability in different exosome isolation methods, our data indicated that miR-26a-5p is a bona fide reference miRNA for qRT-PCR normalization to evaluate miRNA abundance from circulating plasma exosomes in studies of hematological malignancies.

Increased Tenascin C, Osteopontin and HSP90 Levels in Plasmatic Small Extracellular Vesicles of Pediatric ALK-Positive Anaplastic Large Cell Lymphoma: New Prognostic Biomarkers?

Over the past 15 years, several biological and pathological characteristics proved their significance in pediatric anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL) prognostic stratification. However, the identification of new non-invasive disease biomarkers, relying on the most important disease mechanisms, is still necessary. In recent years, plasmatic circulating small extracellular vesicles (S-EVs) gathered great importance both as stable biomarker carriers and active players in tumorigenesis. In the present work, we performed a comprehensive study on the proteomic composition of plasmatic S-EVs of pediatric ALCL patients compared to healthy donors (HDs). By using a mass spectrometry-based proteomics approach, we identified 50 proteins significantly overrepresented in S-EVs of ALCL patients. Gene Ontology enrichment analysis disclosed cellular components and molecular functions connected with S-EV origin and vesicular trafficking, whereas cell adhesion, glycosaminoglycan metabolic process, extracellular matrix organization, collagen fibril organization and acute phase response were the most enriched biological processes. Of importance, consistently with the presence of nucleophosmin (NPM)-ALK fusion protein in ALCL cells, a topological enrichment analysis based on Reactome- and Kyoto Encyclopedia of Genes and Genomes (KEGG)-derived networks highlighted a dramatic increase in proteins of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in ALCL S-EVs, which included heat shock protein 90-kDa isoform alpha 1 (HSP90AA1), osteopontin (SPP1/OPN) and tenascin C (TNC). These results were validated by Western blotting analysis on a panel of ALCL and HD cases. Further research is warranted to better define the role of these S-EV proteins as diagnostic and, possibly, prognostic parameters at diagnosis and for ALCL disease monitoring.

Low miR-214-5p Expression Correlates With Aggressive Subtypes of Pediatric ALCL With Non-Common Histology.

The unsatisfactory cure rate of relapsing ALK-positive Anaplastic Large-Cell Lymphoma (ALCL) of childhood calls for the identification of new prognostic markers. Here, the small RNA landscape of pediatric ALK-positive ALCL was defined by RNA sequencing. Overall, 121 miRNAs were significantly dysregulated in ALCL compared to non-neoplastic lymph nodes. The most up-regulated miRNA was miR-21-5p, whereas miR-19a-3p and miR-214-5p were reduced in ALCL. Characterization of miRNA expression in cases that relapsed after first line therapy disclosed a significant association between miR-214-5p down-regulation and aggressive non-common histology. Our results suggest that miR-214-5p level may help to refine the prognostic stratification of pediatric ALK-positive ALCL.

RNY4 in Circulating Exosomes of Patients With Pediatric Anaplastic Large Cell Lymphoma: An Active Player?

Emerging evidence indicates that extracellular vesicles, particularly exosomes, play a role in several biological processes and actively contribute to cancer development and progression, by carrying and delivering proteins, transcripts and small RNAs (sRNAs). There is high interest in studying exosomes of cancer patients both to develop non-invasive liquid biopsy tests for risk stratification and to elucidate their possible involvement in disease mechanisms. We profiled by RNA-seq the sRNA content of circulating exosomes of 20 pediatric patients with Anaplastic Large Cell Lymphoma (ALCL) and five healthy controls. Our analysis disclosed that non-miRNA derived sRNAs constitute the prominent fraction of sRNA loaded in exosomes and identified 180 sRNAs significantly more abundant in exosomes of ALCL patients compared to controls. YRNA fragments, accounting for most of exosomal content and being significantly increased in ALCL patients, were prioritized for further investigation by qRT-PCR. Quantification of RNY4 fragments and full-length sequences disclosed that the latter are massively loaded into exosomes of ALCL patients with more advanced and aggressive disease. These results are discussed in light of recent findings on the role of RNY4 in the modulation of tumor microenvironment.