Machine Learning Identifies Cellular and Exosomal MicroRNA Signatures of Lyssavirus Infection in Human Stem Cell-Derived Neurons

Despite being vaccine preventable, rabies (lyssavirus) still has a significant impact on global mortality, disproportionally affecting children under 15 years of age. This neurotropic virus is deft at avoiding the immune system while travelling through neurons to the brain. Until recently, research efforts into the role of non-coding RNAs in rabies pathogenicity and detection have been hampered by a lack of human in vitro neuronal models. Here, we utilized our previously described human stem cell-derived neural model to investigate the effect of lyssavirus infection on microRNA (miRNA) expression in human neural cells and their secreted exosomes. Conventional differential expression analysis identified 25 cellular and 16 exosomal miRNAs that were significantly altered (FDR adjusted P-value <0.05) in response to different lyssavirus strains. Supervised machine learning algorithms determined 6 cellular miRNAs (miR-99b-5p, miR-346, miR-5701, miR-138-2-3p, miR-651-5p, and miR-7977) were indicative of lyssavirus infection (100% accuracy), with the first four miRNAs having previously established roles in neuronal function, or panic and impulsivity-related behaviors. Another 4-miRNA signatures in exosomes (miR-25-3p, miR-26b-5p, miR-218-5p, miR-598-3p) can independently predict lyssavirus infected cells with >99% accuracy. Identification of these robust lyssavirus miRNA signatures offers further insight into neural lineage responses to infection and provides a foundation for utilizing exosome miRNAs in the development of next-generation molecular diagnostics for rabies.

Herpesviridae and microRNAs

MicroRNAs (miRNAs), first discovered in the year 1993 in the nematode C. elegans, are small, approximately 22-nucleotide-long, non-coding RNAs that regulate gene expression. Cellular miRNAs have been implicated in the control of many biological processes, and their dysregulation is associated with different diseases. They can be significantly up/downregulated upon infection or disease, serving as excellent biomarkers and therapeutic targets. Several human DNA viruses, including many herpesviruses, have now been reported to encode viral miRNAs. There are a variety of possible interactions and mechanisms of viral microRNAs (vmiRNAs) which are yet to be remains obscure. Viral miRNAs can function as orthologs of cellular miRNAs and regulate their expression. Additionally, viruses have also developed vmiRNA mechanisms to avoid being targeted by the host miRNAs. Herpes Simplex Viruses (HSV-1 & HSV-2) cause genital and oral herpes, establishing lifelong latent infections in their hosts, and it is one of the most prevalent sexually transmitted infections (STIs) worldwide. vmiRNAs play essential roles in Herpesvirus biology. In this chapter, we will discuss the current knowledge about miRNAs and their role in different stages of Herpesvirus infection. It will also elaborate the biomarkers, therapeutic potential of these molecules, and the prospective areas of future research.

Roles of Macrophage Polarization and Macrophage-Derived miRNAs in Pulmonary Fibrosis

This mini-review summarizes the current evidence for the role of macrophage activation and polarization in inflammation and immune response pertinent to interstitial lung disease, specifically pulmonary fibrosis. In the fibrosing lung, the production and function of inflammatory and fibrogenic mediators involved in the disease development have been reported to be regulated by the effects of polarized M1/M2 macrophage populations. The M1 and M2 macrophage phenotypes were suggested to correspond with the pro-inflammatory and pro-fibrogenic signatures, respectively. These responses towards tissue injury followed by the development and progression of lung fibrosis are further regulated by macrophage-derived microRNAs (miRNAs). Besides cellular miRNAs, extracellular exosomal-miRNAs derived from M2 macrophages have also been proposed to promote the progression of pulmonary fibrosis. In a future perspective, harnessing the noncoding miRNAs with a key role in the macrophage polarization is, therefore, suggested as a promising therapeutic strategy for this debilitating disease.

Biological role of miRNA-146a at virus infections. Modern strategy of search of new safe pharmacological agents for treatment

Cellular microRNAs (miRNAs) were identified as a key player in the posttranscriptional regulation of cellular-genes regulatory pathways. Here, we review the current knowledge on the interaction between RNA viruses and cellular miRNAs. We also discuss how cell and tissue-specific expression of miRNAs can directly affect viral pathogenesis. They also emerged as a significant regulator of the immune response. In particular, miR-146a acts as an importance modulator of function and differentiation cells of the innate and adaptive immunity. It has been associated with disorder including cancer and viral infections. Given its significance in the regulation of key cellular processes, it is not surprising which virus infection have found ways to dysregulation of miRNAs. miR-146a has been identified in exosomes (exosomal miR-146a). After the exosomes release from donor cells, they are taken up by the recipient cell and probably the exosomal miR-146a is able to modulate the antiviral response in the recipient cell and result in making them more susceptible to virus infection. In this review, we discuss recent reports regarding miR-146a expression levels, target genes, function, and contributing role in the pathogenesis of the viral infection and provide a clue to develop the new preventive and therapeutic strategies for medical treatment viral disease, and СOVID-19.

Chandipura virus dysregulates the expression of hsa-miR-21-5p to activate NF-κB in human microglial cells

Background Chandipura virus (CHPV) is a negative single-stranded RNA virus of the Rhabdoviridae family. CHPV infection has been reported in Central and Western India. CHPV causes acute encephalitis with a case fatality rate of 70 % and mostly affects children below 15 years of age. CHPV infection in brain leads to neuronal apoptosis and activation of the microglial cells. The microRNAs (miRNAs) are small endogenous non-coding RNA that regulate the gene expression. Viral infections perturb the expression pattern of cellular miRNAs, which may in turn affect the expression pattern of downstream genes. This study aims to investigate hsa-miR-21-5p mediated regulation of PTEN, AKT, NF-ĸBp65, IL-6, TNF-α, and IL-1β, in human microglial cells during CHPV infection. Methods To understand the role of hsa-miR-21-5p in CHPV infection, the human microglial cells were infected with CHPV (MOI-0.1). Real-time PCR, western blotting, Luciferase assay, over-expression and knockdown techniques were used to understand the role of hsa-miR-21-5p in the regulation of PTEN, AKT and, NF-ĸBp65, IL-6, TNF-α, and IL-1β in this study. Results The hsa-miR-21-5p was found to be upregulated during CHPV infection in human microglial cells. This led to the downregulation of PTEN which promoted the phosphorylation of AKT and NF-ĸBp65. Over-expression of hsa-miR-21-5p led to the decreased expression of PTEN and promoted further phosphorylation of AKT and NF-ĸBp65 in human microglial cells. However, the inhibition of hsa-miR-21-5p using hsa-miR-21-5p inhibitor restored the expression. Conclusions This study supports the role of hsa-miR-21-5p in the regulation of pro-inflammatory genes in CHPV infected human microglial cells.

Mutations within the Open Reading Frame (ORF) including Ochre stop codon of the Surface Glycoprotein gene of SARS-CoV-2 virus erase potential seed location motifs of human non-coding microRNAs.

MicroRNA are short and non-coding RNA, 18-25 nucleotides in length. They are produced at the early stage of viral infection. The roles played by cellular miRNAs and miRNA-mediated gene-silencing in the COVID-19 epidemic period is critical in order to develop novel therapeutics. We analysed SARS-CoV-2 Surface Glycoprotein (S) nucleotide sequence originating from India as well as Iran, Australia, Germany, Italy, Russia, China, Japan and Turkey and identified mutation in potential seed location of several human miRNA. Seventy single nucleotide polymorphisms (SNP) were detected in the S gene out of which, 36, 32 and 2 were cases of transitions, transversions and deletions respectively. Eleven human miRNA targets were identified on the reference S gene sequence with a score >80 in the miRDB database. Mutation A845S erased a common binding site of 7 human miRNA (miR-195-5p, miR-16-5p, miR-15b-5p, miR-15a-5p, miR-497-5p, miR-424-5p and miR-6838-5p). A synonymous mutation altered the wild type Ochre stop codon within the S gene sequence (Italy) to Opal thereby changing the seed sequence of miR-511-3p. Similar (synonymous) mutations were detected at amino acid position 659 and 1116 of the S gene where amino acids serine and threonine were retained, abolishing potential seed location for miR-219a-1-3p and miR-20b-3p respectively. The significance of this finding in reference to the strategy to use synthetic miRNA combinations as a novel therapeutic tool is discussed.

Advances in research on microRNAs related to the invasion and metastasis of nasopharyngeal carcinoma

: Nasopharyngeal carcinoma (NPC), which is associated with latent Epstein-Barr virus infection in most cases, is a unique epithelial malignancy arising from the nasopharyngeal mucosal lining. Accumulating evidence provides insights into the genetic and molecular aberrations that likely drive nasopharyngeal tumor development and progression. We review recent analyses of microRNAs (miRNAs), including Epstein-Barr virus-encoded miRNAs (EBV-encoded miRNAs) and dysregulated cellular miRNAs, that may be related to the metastasis of nasopharyngeal carcinoma. The studies summarized herein have greatly expanded our knowledge of the molecular biology of NPC involving miRNAs, and they may provide new biological targets for clinical diagnosis and reveal the potential of microRNA therapeutics. However, much information remains to be uncovered.

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

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.

Recovery of Depleted miR-146a in ALS Cortical Astrocytes Reverts Cell Aberrancies and Prevents Paracrine Pathogenicity on Microglia and Motor Neurons

Reactive astrocytes in Amyotrophic Lateral Sclerosis (ALS) change their molecular expression pattern and release toxic factors that contribute to neurodegeneration and microglial activation. We and others identified a dysregulated inflammatory miRNA profile in ALS patients and in mice models suggesting that they represent potential targets for therapeutic intervention. Such cellular miRNAs are known to be released into the secretome and to be carried by small extracellular vesicles (sEVs), which may be harmful to recipient cells. Thus, ALS astrocyte secretome may disrupt cell homeostasis and impact on ALS pathogenesis. Previously, we identified a specific aberrant signature in the cortical brain of symptomatic SOD1-G93A (mSOD1) mice, as well as in astrocytes isolated from the same region of 7-day-old mSOD1 mice, with upregulated S100B/HMGB1/Cx43/vimentin and downregulated GFAP. The presence of downregulated miR-146a on both cases suggests that it can be a promising target for modulation in ALS. Here, we upregulated miR-146a with pre-miR-146a, and tested glycoursodeoxycholic acid (GUDCA) and dipeptidyl vinyl sulfone (VS) for their immunoregulatory properties. VS was more effective in restoring astrocytic miR-146a, GFAP, S100B, HMGB1, Cx43, and vimentin levels than GUDCA, which only recovered Cx43 and vimentin mRNA. The miR-146a inhibitor generated typical ALS aberrancies in wild type astrocytes that were abolished by VS. Similarly, pre-miR-146a transfection into the mSOD1 astrocytes abrogated aberrant markers and intracellular Ca2+ overload. Such treatment counteracted miR-146a depletion in sEVs and led to secretome-mediated miR-146a enhancement in NSC-34-motor neurons (MNs) and N9-microglia. Secretome from mSOD1 astrocytes increased early/late apoptosis and FGFR3 mRNA in MNs and microglia, but not when derived from pre-miR-146a or VS-treated cells. These last strategies prevented the impairment of axonal transport and synaptic dynamics by the pathological secretome, while also averted microglia activation through either secretome, or their isolated sEVs. Proteomic analysis of the target cells indicated that pre-miR-146a regulates mitochondria and inflammation via paracrine signaling. We demonstrate that replenishment of miR-146a in mSOD1 cortical astrocytes with pre-miR-146a or by VS abrogates their phenotypic aberrancies and paracrine deleterious consequences to MNs and microglia. These results propose miR-146a as a new causal and emerging therapeutic target for astrocyte pathogenic processes in ALS.

The Impact of Dietary Compounds in Functional Foods on MicroRNAs Expression

MicroRNAs (miRNAs) are a class of non-coding endogenous RNA molecules that are involved in post-transcriptional gene silencing via binding to their target messenger RNA, leading to mRNA degradation or translational repression. MicroRNAs can be modulated by several factors including hormones, transcription factors, and dietary compounds. These biologically active compounds have positive impact on the progression of human pathology including non-communicable diseases, which indicating that administration of diet may have potential as therapeutic agents in modulating the risk of chronic diseases. Interestingly, evidence emerging in recent years suggests that dietary miRNAs can be absorbed in human circulation, modulated human gene expression and biological functions. The exploitation of the miRNA functioning within different origins, cellular miRNAs and dietary miRNAs will help us to understand the molecular machinery as well as the regulatory mechanisms involved in fundamentally important biological processes. Therefore, this knowledge may be applied of natural bioactive compounds in preventive or therapeutic approaches.