HCG18, LEF1AS1 and lncCEACAM21 as biomarkers of disease severity in the peripheral blood mononuclear cells of COVID-19 patients.

BACKGROUND: Even after 3 years from SARS-CoV-2 identification, COVID-19 is still a persistent and dangerous global infectious disease. Significant improvements in our understanding of the disease pathophysiology have now been achieved. Nonetheless, reliable and accurate biomarkers for the early stratification of COVID-19 severity are still lacking. Long noncoding RNAs (LncRNAs) are ncRNAs longer than 200 nucleotides, regulating the transcription and translation of protein-coding genes and they can be found in the peripheral blood, thus holding a promising biomarker potential. Specifically, peripheral blood mononuclear cells (PBMCs) have emerged as a source of indirect biomarkers mirroring the conditions of tissues: they include monocytes, B and T lymphocytes, and natural killer T cells (NKT), being highly informative for immune-related events. METHODS: We profiled by RNA-Sequencing a panel of 2906 lncRNAs to investigate their modulation in PBMCs of a pilot group of COVID-19 patients, followed by qPCR validation in 111 hospitalized COVID-19 patients. RESULTS: The levels of four lncRNAs were found to be decreased in association with COVID-19 mortality and disease severity: HLA Complex Group 18-242 and -244 (HCG18-242 and HCG18-244), Lymphoid Enhancer Binding Factor 1-antisense 1 (LEF1-AS1) and lncCEACAM21 (i.e. ENST00000601116.5, a lncRNA in the CEACAM21 locus). Interestingly, these deregulations were confirmed in an independent patient group of hospitalized patients and by the re-analysis of publicly available single-cell transcriptome datasets. The identified lncRNAs were expressed in all of the PBMC cell types and inversely correlated with the neutrophil/lymphocyte ratio (NLR), an inflammatory marker. In vitro, the expression of LEF1-AS1 and lncCEACAM21 was decreased upon THP-1 monocytes exposure to a relevant stimulus, hypoxia. CONCLUSION: The identified COVID-19-lncRNAs are proposed as potential innovative biomarkers of COVID-19 severity and mortality.

Circular RNA regulatory role in pathological cardiac remodelling.

Cardiac remodelling involves structural, cellular and molecular alterations in the heart after injury, resulting in progressive loss of heart function and ultimately leading to heart failure. Circular RNAs (circRNAs) are a recently rediscovered class of non-coding RNAs that play regulatory roles in the pathogenesis of cardiovascular diseases, including heart failure. Thus, a more comprehensive understanding of the role of circRNAs in the processes governing cardiac remodelling may set the ground for the development of circRNA-based diagnostic and therapeutic strategies. In this review, the current knowledge about circRNA origin, conservation, characteristics and function is summarized. Bioinformatics and wet-lab methods used in circRNA research are discussed. The regulatory function of circRNAs in cardiac remodelling mechanisms such as cell death, cardiomyocyte hypertrophy, inflammation, fibrosis and metabolism is highlighted. Finally, key challenges and opportunities in circRNA research are discussed, and orientations for future work to address the pharmacological potential of circRNAs in heart failure are proposed.

Recommendations for detection, validation, and evaluation of RNA editing events in cardiovascular and neurological/neurodegenerative diseases.

RNA editing, a common and potentially highly functional form of RNA modification, encompasses two different RNA modifications, namely adenosine to inosine (A-to-I) and cytidine to uridine (C-to-U) editing. As inosines are interpreted as guanosines by the cellular machinery, both A-to-I and C-to-U editing change the nucleotide sequence of the RNA. Editing events in coding sequences have the potential to change the amino acid sequence of proteins, whereas editing events in noncoding RNAs can, for example, affect microRNA target binding. With advancing RNA sequencing technology, more RNA editing events are being discovered, studied, and reported. However, RNA editing events are still often overlooked or discarded as sequence read quality defects. With this position paper, we aim to provide guidelines and recommendations for the detection, validation, and follow-up experiments to study RNA editing, taking examples from the fields of cardiovascular and brain disease. We discuss all steps, from sample collection, storage, and preparation, to different strategies for RNA sequencing and editing-sensitive data analysis strategies, to validation and follow-up experiments, as well as potential pitfalls and gaps in the available technologies. This paper may be used as an experimental guideline for RNA editing studies in any disease context.

Addressing the unsolved challenges in microRNA-based biomarker development: Suitable endogenous reference microRNAs for SARS-CoV-2 infection severity.

Circulating cell-free microRNAs (miRNAs) are promising biomarkers for medical decision-making. Suitable endogenous controls are essential to ensure reproducibility. We aimed to identify and validate endogenous reference miRNAs for qPCR data normalization in samples from SARS-CoV-2-infected hospitalized patients. We used plasma samples (n = 170) from COVID-19 patients collected at hospital admission (COVID-Ponent project, www.clinicaltrials.gov/NCT04824677). First, 179 miRNAs were profiled using RT-qPCR. After stability assessment, candidates were validated using the same methodology. miRNA stability was analyzed using the geNorm, NormFinder and BestKeeper algorithms. Stability was further evaluated using an RNA-seq dataset derived from COVID-19 hospitalized patients, along with plasma samples from patients with critical COVID-19 profiled using RT-qPCR. In the screening phase, after strict control of expression levels, stability assessment selected eleven candidates (miR-17-5p, miR-20a-5p, miR-30e-5p, miR-106a-5p, miR-151a-5p, miR-185-5p, miR-191-5p, miR-423-3p, miR-425-5p, miR-484 and miR-625-5p). In the validation phase, all algorithms identified miR-106a-5p and miR-484 as top endogenous controls. No association was observed between these miRNAs and clinical or sociodemographic characteristics. Both miRNAs were stably detected and showed low variability in the additional analyses. In conclusion, a 2-miRNA panel composed of miR-106a-5p and miR-484 constitutes a first-line normalizer for miRNA-based biomarker development using qPCR in hospitalized patients infected with SARS-CoV-2.

circRNA-miRNA-mRNA Deregulated Network in Ischemic Heart Failure Patients.

Noncoding RNAs (ncRNAs), which include circular RNAs (circRNAs) and microRNAs (miRNAs), regulate the development of cardiovascular diseases (CVD). Notably, circRNAs can interact with miRNAs, influencing their specific mRNA targets' levels and shaping a competing endogenous RNAs (ceRNA) network. However, these interactions and their respective functions remain largely unexplored in ischemic heart failure (IHF). This study is aimed at identifying circRNA-centered ceRNA networks in non-end-stage IHF. Approximately 662 circRNA-miRNA-mRNA interactions were identified in the heart by combining state-of-the-art bioinformatics tools with experimental data. Importantly, KEGG terms of the enriched mRNA indicated CVD-related signaling pathways. A specific network centered on circBPTF was validated experimentally. The levels of let-7a-5p, miR-18a-3p, miR-146b-5p, and miR-196b-5p were enriched in circBPTF pull-down experiments, and circBPTF silencing inhibited the expression of HDAC9 and LRRC17, which are targets of miR-196b-5p. Furthermore, as suggested by the enriched pathway terms of the circBPTF ceRNA network, circBPTF inhibition elicited endothelial cell cycle arrest. circBPTF expression increased in endothelial cells exposed to hypoxia, and its upregulation was confirmed in cardiac samples of 36 end-stage IHF patients compared to healthy controls. In conclusion, circRNAs act as miRNA sponges, regulating the functions of multiple mRNA targets, thus providing a novel vision of HF pathogenesis and laying the theoretical foundation for further experimental studies.

Association of miR-144 levels in the peripheral blood with COVID-19 severity and mortality.

Coronavirus disease-2019 (COVID-19) can be asymptomatic or lead to a wide symptom spectrum, including multi-organ damage and death. Here, we explored the potential of microRNAs in delineating patient condition and predicting clinical outcome. Plasma microRNA profiling of hospitalized COVID-19 patients showed that miR-144-3p was dynamically regulated in response to COVID-19. Thus, we further investigated the biomarker potential of miR-144-3p measured at admission in 179 COVID-19 patients and 29 healthy controls recruited in three centers. In hospitalized patients, circulating miR-144-3p levels discriminated between non-critical and critical illness (AUCmiR-144-3p = 0.71; p = 0.0006), acting also as mortality predictor (AUCmiR-144-3p = 0.67; p = 0.004). In non-hospitalized patients, plasma miR-144-3p levels discriminated mild from moderate disease (AUCmiR-144-3p = 0.67; p = 0.03). Uncontrolled release of pro-inflammatory cytokines can lead to clinical deterioration. Thus, we explored the added value of a miR-144/cytokine combined analysis in the assessment of hospitalized COVID-19 patients. A miR-144-3p/Epidermal Growth Factor (EGF) combined score discriminated between non-critical and critical hospitalized patients (AUCmiR-144-3p/EGF = 0.81; p < 0.0001); moreover, a miR-144-3p/Interleukin-10 (IL-10) score discriminated survivors from nonsurvivors (AUCmiR-144-3p/IL-10 = 0.83; p < 0.0001). In conclusion, circulating miR-144-3p, possibly in combination with IL-10 or EGF, emerges as a noninvasive tool for early risk-based stratification and mortality prediction in COVID-19.