Single cell gene expression profiling of nasal ciliated cells reveals distinctive biological processes related to epigenetic mechanisms in patients with severe COVID-19

Objective: To explore the molecular processes associated with cellular regulatory programs in patients with COVID-19, including gene activation or repression mediated by epigenetic mechanisms. We hypothesized that a comprehensive gene expression profiling of nasopharyngeal epithelial cells might expand our understanding of the pathogenic mechanisms of severe COVID-19. Methods: We used single-cell RNA sequencing (scRNAseq) profiling of ciliated cells (n = 12,725) from healthy controls (SARS-CoV-2 negative n = 13) and patients with mild/moderate (n = 13) and severe (n = 14) COVID-19. ScRNAseq data at the patient level were used to perform gene set and pathway enrichment analyses. We prioritized candidate miRNA-target interactions and epigenetic mechanisms. Results: We found that mild/moderate COVID-19 compared to healthy controls had upregulation of gene expression signatures associated with mitochondrial function, misfolded proteins, and membrane permeability. In addition, we found that compared to mild/moderate disease, severe COVID-19 had downregulation of epigenetic mechanisms, including DNA and histone H3K4 methylation and chromatin remodelling regulation. Furthermore, we found 11-ranked miRNAs that may explain miRNA-dependent regulation of histone methylation, some of which share seed sequences with SARS-CoV-2 miRNAs. Conclusion: Our results may provide novel insights into the epigenetic mechanisms mediating the clinical course of SARS-CoV-2 infection.Fil: Diambra, Luis Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; ArgentinaFil: Alonso, Andrés Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Sookoian, Silvia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Pirola, Carlos Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Interaction and cross-talk between non-coding RNAs.

Non-coding RNA (ncRNA) has been shown to regulate diverse cellular processes and functions through controlling gene expression. Long non-coding RNAs (lncRNAs) act as a competing endogenous RNAs (ceRNAs) where microRNAs (miRNAs) and lncRNAs regulate each other through their biding sites. Interactions of miRNAs and lncRNAs have been reported to trigger decay of the targeted lncRNAs and have important roles in target gene regulation. These interactions form complicated and intertwined networks. Certain lncRNAs encode miRNAs and small nucleolar RNAs (snoRNAs), and may regulate expression of these small RNAs as precursors. SnoRNAs have also been reported to be precursors for PIWI-interacting RNAs (piRNAs) and thus may regulate the piRNAs as a precursor. These miRNAs and piRNAs target messenger RNAs (mRNAs) and regulate gene expression. In this review, we will present and discuss these interactions, cross-talk, and co-regulation of ncRNAs and gene regulation due to these interactions

Expression analysis of miRNA hsa-let7b-5p in naso-oropharyngeal swabs of COVID-19 patients supports its role in regulating ACE2 and DPP4 receptors

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the novel coronavirus responsible for worldwide coronavirus disease (COVID-19). We previously observed that Angiotensin-converting enzyme 2 (ACE2) and Dipeptidyl peptidase-4 (DPP4) are significantly overexpressed in naso-oropharyngeal swabs (NPS) of COVID-19 patients, suggesting their putative functional role in the disease progression. ACE2 and DPP4 overexpression in COVID-19 patients may be associated to epigenetic mechanism, such as miRNA differential expression. We investigated if hsa-let7b-5p, reported to target both ACE2 and DPP4 transcripts, could be involved in the regulation of these genes. We verified that the inhibition and overexpression of hsa-let7b-5p matched to a modulation of both ACE2 and DPP4 levels. Then, we observed a statistically significant downregulation (FC = -1.5; p < 0.05) of hsa-let7b-5p in the same COVID-19 and control samples of our previous study. This is the first study that shows hsa-let7b-5p low expression in naso-oropharyngeal swabs of COVID-19 patients and demonstrates a functional role of this miR in regulating ACE2 and DPP4 levels. These data suggest the involvement of hsa-let7b-5p in the regulation of genes necessary for SARS-CoV-2 infections and its putative role as a therapeutic target for COVID-19

A Genomic Portrait of Hepatitis C Virus and MicroRNA-122

Hepatitis C virus (HCV) uniquely requires the liver specific microRNA-122 (miR- 122) for replication, yet global effects on endogenous microRNA (miRNA) targets during infection are unexplored. In this body of work, we employed highthroughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during HCV infection. We demonstrate robust AGO binding on the 5\u27 untranslated region of HCV RNA at known and predicted miR-122 sites, thereby establishing conclusive biochemical evidence of endogenous miR-122 action on HCV RNA that firmly agrees with previous genetic evidence. We further characterize novel AGO binding on HCV RNA to determine its dependence on miR-122, miRNAs generally, replication competence and time. These results establish an unbiased interaction landscape between HCV RNA and cellular miRNAs, mostly miR-122. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 sponge effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. Based on these results, we describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets. This in turn may provide an environment fertile for the long-term oncogenic potential of HCV. This last point presented a fitting entree into miR-122 biology, given its known tumor suppressive activity in the liver. To conclude this work, we performed AGO-CLIP in miR-122 knockout mouse livers as well as in human liver samples, to determine the in vivo targetome for this miRNA across two species. Surprisingly, we discovered widespread and non-canonical miR-122 binding throughout the transcriptome. Furthermore, a substantial fraction of this binding was not conserved between mouse and human transcriptomes, despite the fact that miR-122 is highly conserved. These results, in concert with AGOCLIP in HCV infected cells, point to a model where HCV may have evolved the use of miR-122 for its high abundance and its well buffered capacity to be inhibited with minimal detrimental effects to the host, and perhaps benefits for the virus. In sum, this thesis reveals how miR-122 is redistributed in the cell following HCV infection. As a molecular mechanism, chronic inhibition of miR-122 by HCV RNA is proposed to impact, and may very well help induce, the complex constellation of liver diseases that characterize this infection in humans

Identification of Prognostic Cancer Biomarkers through the Application of RNA-Seq Technologies and Bioinformatics

MicroRNAs (miRNAs) are short single-stranded RNAs that function as the guide sequence of the post-transcriptional regulatory process known as the RNA-induced silencing complex (RISC), which targets mRNA sequences for degradation through complementary binding to the guide miRNA. Changes in miRNA expression have been reported as correlated with numerous biological processes, including embryonic development, cellular differentiation, and disease manifestation. In the latter case, dysregulation has been observed in response to infection by human papillomavirus (HPV), which has also been established as both oncogenic in cervical cancers and oropharyngeal cancers and favorable for overall patient survival after tumor formation. The identification of dysregulated miRNAs associated with both HPV infection and cancer survival requires large datasets of high-throughput sequencing data, which were obtained through The Cancer Genome Atlas. By analyzing this public data, we have identified a series of proposed mechanisms for cancer formation and survival that is mediated through the miRNA-RISC regulatory mechanism in response to HPV infection. We have also identified a diverse set of miRNA biomarkers that have been incorporated into linear expression-based risk signatures that are prognostic for overall patient survival after tumor diagnosis in HPV-related cancers. The tools that were used to identify both miRNA biomarkers and proposed targets in public datasets, such as The Cancer Genome Atlas, have since been incorporated into an web-accessible resource, OncomiR.org, to streamline the process of biomarker identification for the cancer research community

Computational identification of hepatitis C virus associated microRNA-mRNA regulatory modules in human livers

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) is a major cause of chronic liver disease by infecting over 170 million people worldwide. Recent studies have shown that microRNAs (miRNAs), a class of small non-coding regulatory RNAs, are involved in the regulation of HCV infection, but their functions have not been systematically studied. We propose an integrative strategy for identifying the miRNA-mRNA regulatory modules that are associated with HCV infection. This strategy combines paired expression profiles of miRNAs and mRNAs and computational target predictions. A miRNA-mRNA regulatory module consists of a set of miRNAs and their targets, in which the miRNAs are predicted to coordinately regulate the level of the target mRNA.</p> <p>Results</p> <p>We simultaneously profiled the expression of cellular miRNAs and mRNAs across 30 HCV positive or negative human liver biopsy samples using microarray technology. We constructed a miRNA-mRNA regulatory network, and using a graph theoretical approach, identified 38 miRNA-mRNA regulatory modules in the network that were associated with HCV infection. We evaluated the direct miRNA regulation of the mRNA levels of targets in regulatory modules using previously published miRNA transfection data. We analyzed the functional roles of individual modules at the systems level by integrating a large-scale protein interaction network. We found that various biological processes, including some HCV infection related canonical pathways, were regulated at the miRNA level during HCV infection.</p> <p>Conclusion</p> <p>Our regulatory modules provide a framework for future experimental analyses. This report demonstrates the utility of our approach to obtain new insights into post-transcriptional gene regulation at the miRNA level in complex human diseases.</p

MicroRNAs and HIV-1 Infection: Antiviral Activities and Beyond

AbstractCellular microRNAs (miRNAs) are an important class of small, non-coding RNAs that bind to host mRNAs based on sequence complementarity and regulate protein expression. They play important roles in controlling key cellular processes including cellular inception, differentiation and death. While several viruses have been shown to encode for viral miRNAs, controversy persists over the expression of a functional miRNA encoded in the human immunodeficiency virus type 1 (HIV-1) genome. However, it has been reported that HIV-1 infectivity is influenced by cellular miRNAs. Either through directly targeting the viral genome or by targeting host cellular proteins required for successful virus replication, multiple cellular miRNAs seem to modulate HIV-1 infection and replication. Perhaps as a survival strategy, HIV-1 may modulate proteins in the miRNA biogenesis pathway to subvert miRNA-induced antiviral effects. Global expression profiles of cellular miRNAs have also identified alterations of specific miRNAs post-HIV-1 infection both in vitro and in vivo (in various infected patient cohorts), suggesting potential roles for miRNAs in pathogenesis and disease progression. However, little attention has been devoted in understanding the roles played by these miRNAs at a cellular level. In this manuscript, we review past and current findings pertaining to the field of miRNA and HIV-1 interplay. In addition, we suggest strategies to exploit miRNAs therapeutically for curbing HIV-1 infectivity, replication and latency since they hold an untapped potential that deserves further investigation

MicroRNA Profile of HCV Spontaneous Clarified Individuals, Denotes Previous HCV Infection

Factors involved in the spontaneous cleareance of a hepatitis C (HCV) infection are related to both HCV and the interaction with the host immune system, but little is known about the consequences after a spontaneous resolution. The main HCV extrahepatic reservoir is the peripheral blood mononuclear cells (PBMCs), and their transcriptional profile provides us information of innate and adaptive immune responses against an HCV infection. MicroRNAs regulate the innate and adaptive immune responses, and they are actively involved in the HCV cycle. High Throughput sequencing was used to analyze the miRNA profiles from PBMCs of HCV chronic naïve patients (CHC), individuals that spontaneously clarified HCV (SC), and healthy controls (HC). We did not find any differentially expressed miRNAs between SC and CHC. However, both groups showed similar expression differences (21 miRNAs) with respect to HC. This miRNA signature correctly classifies HCV-exposed (CHC and SC) vs. HC, with the has-miR-21-3p showing the best performance. The potentially targeted molecular pathways by these 21 miRNAs mainly belong to fatty acids pathways, although hippo signaling, extracellular matrix (ECM) interaction, proteoglycans-related, and steroid biosynthesis pathways were also altered. These miRNAs target host genes involved in an HCV infection. Thus, an HCV infection promotes molecular alterations in PBMCs that can be detected after an HCV spontaneous resolution, and the 21-miRNA signature is able to identify HCV-exposed patients (either CHC or SC).Funding: This work has been funded by the Instituto de Salud Carlos III (Subdirección General de Evaluación) (grant number CP14/0010) Fondo de Investigación Sanitaria (FIS) (grant numbers MPY 1404/15, MPY 1144/16, and MPY 382/18), and Integrated Projects of Excellence (grant number PIE15/00079).S

Non-Coding RNAs Improve the Predictive Power of Network Medicine

Network Medicine has improved the mechanistic understanding of disease, offering quantitative insights into disease mechanisms, comorbidities, and novel diagnostic tools and therapeutic treatments. Yet, most network-based approaches rely on a comprehensive map of protein-protein interactions, ignoring interactions mediated by non-coding RNAs (ncRNAs). Here, we systematically combine experimentally confirmed binding interactions mediated by ncRNA with protein-protein interactions, constructing the first comprehensive network of all physical interactions in the human cell. We find that the inclusion of ncRNA, expands the number of genes in the interactome by 46% and the number of interactions by 107%, significantly enhancing our ability to identify disease modules. Indeed, we find that 132 diseases, lacked a statistically significant disease module in the protein-based interactome, but have a statistically significant disease module after inclusion of ncRNA-mediated interactions, making these diseases accessible to the tools of network medicine. We show that the inclusion of ncRNAs helps unveil disease-disease relationships that were not detectable before and expands our ability to predict comorbidity patterns between diseases. Taken together, we find that including non-coding interactions improves both the breath and the predictive accuracy of network medicine.Comment: Paper and S