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Human MicroRNAs Interacting With SARS-CoV-2 RNA Sequences: Computational Analysis and Experimental Target Validation.

ABSTRACT: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel RNA virus affecting humans, causing a form of acute pulmonary respiratory disorder named COVID-19, declared a pandemic by the World Health Organization. MicroRNAs (miRNA) play an emerging and important role in the interplay between viruses and host cells. Although the impact of host miRNAs on SARS-CoV-2 infection has been predicted, experimental data are still missing. This study started by a bioinformatics prediction of cellular miRNAs potentially targeting viral RNAs; then, a number of criteria also based on experimental evidence and virus biology were applied, giving rise to eight promising binding miRNAs. Their interaction with viral sequences was experimentally validated by transfecting luciferase-based reporter plasmids carrying viral target sequences or their inverted sequences into the lung A549 cell line. Transfection of the reporter plasmids resulted in a reduction of luciferase activity for five out of the eight potential binding sites, suggesting responsiveness to endogenously expressed miRNAs. Co-transfection of the reporter plasmids along with miRNA mimics led to a further and strong reduction of luciferase activity, validating the interaction between miR-219a-2-3p, miR-30c-5p, miR-378d, miR-29a-3p, miR-15b-5p, and viral sequences. miR-15b was also able to repress plasmid-driven Spike expression. Intriguingly, the viral target sequences are fully conserved in more recent variants such as United Kingdom variant B.1.1.7 and South Africa 501Y.V2. Overall, this study provides a first experimental evidence of the interaction between specific cellular miRNAs and SARS-CoV-2 sequences, thus contributing to understanding the molecular mechanisms underlying virus infection and pathogenesis to envisage innovative therapeutic interventions and diagnostic tools.

SUBMITTER: Siniscalchi C

PROVIDER: S-EPMC8215607 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:MicroRNAs (miRNAs) are post-transcriptional regulators involved in numerous biological processes including the pathogenesis of Alzheimer's disease (AD). A key gene of AD, ADAM10, controls the proteolytic processing of APP and the formation of the amyloid plaques and is known to be regulated by miRNA in hepatic cancer cell lines. To predict miRNAs regulating ADAM10 expression concerning AD, we developed a computational approach.MiRNA binding sites in the human ADAM10 3' untranslated region were predicted using the RNA22, RNAhybrid and miRanda programs and ranked by specific selection criteria with respect to AD such as differential regulation in AD patients and tissue-specific expression. Furthermore, target genes of miR-103, miR-107 and miR-1306 were derived from six publicly available miRNA target site prediction databases. Only target genes predicted in at least four out of six databases in the case of miR-103 and miR-107 were compared to genes listed in the AlzGene database including genes possibly involved in AD. In addition, the target genes were used for Gene Ontology analysis and literature mining. Finally, we used a luciferase assay to verify the potential effect of these three miRNAs on ADAM10 3'UTR in SH-SY5Y cells.Eleven miRNAs were selected, which have evolutionary conserved binding sites. Three of them (miR-103, miR-107, miR-1306) were further analysed as they are linked to AD and most strictly conserved between different species. Predicted target genes of miR-103 (p-value = 0.0065) and miR-107 (p-value = 0.0009) showed significant overlap with the AlzGene database except for miR-1306. Interactions between miR-103 and miR-107 to genes were revealed playing a role in processes leading to AD. ADAM10 expression in the reporter assay was reduced by miR-1306 (28%), miR-103 (45%) and miR-107 (52%).Our approach shows the requirement of incorporating specific, disease-associated selection criteria into the prediction process to reduce the amount of false positive predictions. In summary, our method identified three miRNAs strongly suggested to be involved in AD, which possibly regulate ADAM10 expression and hence offer possibilities for the development of therapeutic treatments of AD.

Project description:BACKGROUND: In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution. RESULTS: Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells. CONCLUSIONS: The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.

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Human MicroRNAs Interacting With SARS-CoV-2 RNA Sequences: Computational Analysis and Experimental Target Validation.

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