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1.
PLoS One ; 18(4): e0283589, 2023.
Article in English | MEDLINE | ID: covidwho-2291680

ABSTRACT

Non-coding RNAs (ncRNAs) can control the flux of genetic information; affect RNA stability and play crucial roles in mediating epigenetic modifications. A number of studies have highlighted the potential roles of both virus-encoded and host-encoded ncRNAs in viral infections, transmission and therapeutics. However, the role of an emerging type of non-coding transcript, circular RNA (circRNA) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has not been fully elucidated so far. Moreover, the potential pathogenic role of circRNA-miRNA-mRNA regulatory axis has not been fully explored as yet. The current study aimed to holistically map the regulatory networks driven by SARS-CoV-2 related circRNAs, miRNAs and mRNAs to uncover plausible interactions and interplay amongst them in order to explore possible therapeutic options in SARS-CoV-2 infection. Patient datasets were analyzed systematically in a unified approach to explore circRNA, miRNA, and mRNA expression profiles. CircRNA-miRNA-mRNA network was constructed based on cytokine storm related circRNAs forming a total of 165 circRNA-miRNA-mRNA pairs. This study implies the potential regulatory role of the obtained circRNA-miRNA-mRNA network and proposes that two differentially expressed circRNAs hsa_circ_0080942 and hsa_circ_0080135 might serve as a potential theranostic agents for SARS-CoV-2 infection. Collectively, the results shed light on the functional role of circRNAs as ceRNAs to sponge miRNA and regulate mRNA expression during SARS-CoV-2 infection.


Subject(s)
COVID-19 , MicroRNAs , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Precision Medicine , COVID-19/genetics , SARS-CoV-2/genetics
2.
Biomed Pharmacother ; 156: 113845, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-2242820

ABSTRACT

Ischemic stroke is a prevalent disease that seriously threatens human health. It is characterized by high morbidity, mortality, disability, and recurrence rates, causing a significant economic burden on individuals and society. Circular RNA, a novel non-coding RNA, not only serves as the sponge for microRNAs and proteins but also promotes transcription of their parental genes and translates into peptides. In recent years, circRNAs have emerged as key regulators in ischemic stroke. This article aims to provide new ideas about the pathogenesis and progression of ischemic stroke by reviewing the roles of circRNAs in cerebral ischemic injury and summarizing the association between circRNAs and risk factors for ischemic stroke.


Subject(s)
Ischemic Stroke , MicroRNAs , Humans , RNA, Circular/genetics , Ischemic Stroke/genetics , Ischemic Stroke/prevention & control , MicroRNAs/genetics , MicroRNAs/metabolism
3.
Cells ; 12(2)2023 01 14.
Article in English | MEDLINE | ID: covidwho-2245472

ABSTRACT

The existence of circular RNA (circRNA) research in mainstream science can be attributed to the contemporary synergism of big data and keen attention to detail by several research groups worldwide. Since the re-emergence of these non-canonical RNA transcripts, seminal advances have been made in understanding their biogenesis, interactome, and functions in diverse fields and a myriad of human diseases. However, most research outputs to date have focused on the ability of highly stable circRNAs to interact with, and impact signalling through, microRNAs. This is likely to be the result of seminal papers in the field ascribing a few remarkable circRNAs as "miRNA sponges". However, the stoichiometric ratio between the (often-lowly-expressed) circRNA and their (commonly-more-abundant) target is rarely in favour of a biologically relevant and functional consequence of these interactions. It is time for yet another revolution in circRNA research to uncover functions beyond their documented ability to bind miRNAs. This Special Issue aims to highlight non-canonical functions for this non-canonical family of RNA molecules.


Subject(s)
MicroRNAs , RNA, Circular , Humans , RNA, Circular/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Signal Transduction
4.
Funct Integr Genomics ; 23(1): 33, 2023 Jan 10.
Article in English | MEDLINE | ID: covidwho-2242834

ABSTRACT

Human diseases have been a critical threat from the beginning of human history. Knowing the origin, course of action and treatment of any disease state is essential. A microscopic approach to the molecular field is a more coherent and accurate way to explore the mechanism, progression, and therapy with the introduction and evolution of technology than a macroscopic approach. Non-coding RNAs (ncRNAs) play increasingly important roles in detecting, developing, and treating all abnormalities related to physiology, pathology, genetics, epigenetics, cancer, and developmental diseases. Noncoding RNAs are becoming increasingly crucial as powerful, multipurpose regulators of all biological processes. Parallel to this, a rising amount of scientific information has revealed links between abnormal noncoding RNA expression and human disorders. Numerous non-coding transcripts with unknown functions have been found in addition to advancements in RNA-sequencing methods. Non-coding linear RNAs come in a variety of forms, including circular RNAs with a continuous closed loop (circRNA), long non-coding RNAs (lncRNA), and microRNAs (miRNA). This comprises specific information on their biogenesis, mode of action, physiological function, and significance concerning disease (such as cancer or cardiovascular diseases and others). This study review focuses on non-coding RNA as specific biomarkers and novel therapeutic targets.


Subject(s)
MicroRNAs , Neoplasms , RNA, Long Noncoding , Humans , RNA, Untranslated/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Biomarkers , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Circular/genetics , Neoplasms/genetics , Neoplasms/therapy
5.
Front Immunol ; 13: 1091797, 2022.
Article in English | MEDLINE | ID: covidwho-2227844

ABSTRACT

Owing to the success of linear mRNA coronavirus disease 2019 (COVID-19) vaccines, biopharmaceutical companies and research teams worldwide have attempted to develop more stable circular RNA (circRNA) vaccines and have achieved some preliminary results. This review aims to summarize key findings and important progress made in circRNA research, the in vivo metabolism and biological functions of circRNAs, and research progress and production process of circRNA vaccines. Further, considerations regarding the quality control of circRNA vaccines are highlighted herein, and the main challenges and problem-solving strategies in circRNA vaccine development and quality control are outlined to provide a reference for circRNA vaccine-related research.


Subject(s)
Biological Products , COVID-19 , Vaccines , Humans , RNA, Circular/genetics , COVID-19/prevention & control , RNA, Messenger , COVID-19 Vaccines
6.
Theranostics ; 12(14): 6422-6436, 2022.
Article in English | MEDLINE | ID: covidwho-2203053

ABSTRACT

Rationale: Messenger RNA (mRNA) vaccine outperforms other kinds of cancer immunotherapy due to its high response rates, easy preparation, and wide applicability, which is considered as one of the most promising forms of next-generation cancer therapies. However, the inherent instability and insufficient protein expression duration of mRNA limit the efficacy and widespread application of the vaccine. Methods: Here, we first tested the possibility of a novel circular RNA (circRNA) platform for protein expression and compare its duration with linear RNA. Then, we developed a lipid nanoparticle (LNP) system for circRNA delivery in vitro and in vivo. Next, the innate and adaptive immune response of circRNA-LNP complex was evaluated in vivo. The anti-tumor efficacy of circRNA-LNP was further confirmed in three tumor models. Finally, the possibility of combination therapy with circRNA-LNP and adoptive cell transfer therapy was further investigated in a late-stage tumor model. Results: We successfully increased the stability of the RNA vaccine by circularizing the linear RNA molecules to form highly stable circRNA molecules which exhibited durable protein expression ability. By encapsulating the antigen-coding circRNA in LNP enabling in vivo expression, we established a novel circRNA vaccine platform, which was capable of triggering robust innate and adaptive immune activation and showed superior anti-tumor efficacy in multiple mouse tumor models. Conclusions: Overall, our circRNA vaccine platform provides a novel prospect for the development of cancer RNA vaccines in a wide range of hard-to-treat malignancies.


Subject(s)
Cancer Vaccines , Nanoparticles , Neoplasms , Animals , Liposomes , Mice , Neoplasms/therapy , RNA/genetics , RNA, Circular/genetics , RNA, Messenger/genetics , Vaccines, Synthetic , mRNA Vaccines
7.
Front Immunol ; 13: 980231, 2022.
Article in English | MEDLINE | ID: covidwho-2142007

ABSTRACT

Coronavirus disease 2019 (COVID-19), an infectious acute respiratory disease caused by a newly emerging RNA virus, is a still-growing pandemic that has caused more than 6 million deaths globally and has seriously threatened the lives and health of people across the world. Currently, several drugs have been used in the clinical treatment of COVID-19, such as small molecules, neutralizing antibodies, and monoclonal antibodies. In addition, several vaccines have been used to prevent the spread of the pandemic, such as adenovirus vector vaccines, inactivated vaccines, recombinant subunit vaccines, and nucleic acid vaccines. However, the efficacy of vaccines and the onset of adverse reactions vary among individuals. Accumulating evidence has demonstrated that circular RNAs (circRNAs) are crucial regulators of viral infections and antiviral immune responses and are heavily involved in COVID-19 pathologies. During novel coronavirus infection, circRNAs not only directly affect the transcription process and interfere with viral replication but also indirectly regulate biological processes, including virus-host receptor binding and the immune response. Consequently, understanding the expression and function of circRNAs during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection will provide novel insights into the development of circRNA-based methods. In this review, we summarize recent progress on the roles and underlying mechanisms of circRNAs that regulate the inflammatory response, viral replication, immune evasion, and cytokines induced by SARS-CoV-2 infection, and thus highlighting the diagnostic and therapeutic challenges in the treatment of COVID-19 and future research directions.


Subject(s)
COVID-19 , Humans , RNA, Circular/genetics , SARS-CoV-2 , Vaccines, Inactivated , Virus Replication , Vaccines, Synthetic
8.
J Vis Exp ; (188)2022 10 21.
Article in English | MEDLINE | ID: covidwho-2110320

ABSTRACT

Circular RNAs (circRNAs) are a class of non-coding RNAs that are formed via back-splicing. These circRNAs are predominantly studied for their roles as regulators of various biological processes. Notably, emerging evidence demonstrates that host circRNAs can be differentially expressed (DE) upon infection with pathogens (e.g., influenza and coronaviruses), suggesting a role for circRNAs in regulating host innate immune responses. However, investigations on the role of circRNAs during pathogenic infections are limited by the knowledge and skills required to carry out the necessary bioinformatic analysis to identify DE circRNAs from RNA sequencing (RNA-seq) data. Bioinformatics prediction and identification of circRNAs is crucial before any verification, and functional studies using costly and time-consuming wet-lab techniques. To solve this issue, a step-by-step protocol of in silico prediction and characterization of circRNAs using RNA-seq data is provided in this manuscript. The protocol can be divided into four steps: 1) Prediction and quantification of DE circRNAs via the CIRIquant pipeline; 2) Annotation via circBase and characterization of DE circRNAs; 3) CircRNA-miRNA interaction prediction through Circr pipeline; 4) functional enrichment analysis of circRNA parental genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). This pipeline will be useful in driving future in vitro and in vivo research to further unravel the role of circRNAs in host-pathogen interactions.


Subject(s)
MicroRNAs , RNA, Circular , RNA, Circular/genetics , Sequence Analysis, RNA , MicroRNAs/genetics , Computational Biology/methods , Host-Pathogen Interactions/genetics , Gene Expression Profiling/methods
9.
Microb Genom ; 8(6)2022 06.
Article in English | MEDLINE | ID: covidwho-2042679

ABSTRACT

Emerging evidence has identified viral circular RNAs (circRNAs) in human cells infected by viruses, interfering with the immune system and inducing diseases including human cancer. However, the biogenesis and regulatory mechanisms of virus-encoded circRNAs in host cells remain unknown. In this study, we used the circRNA detection tool CIRI2 to systematically determine the virus-encoded circRNAs in virus-infected cancer cell lines and cancer patients, by analysing RNA-Seq datasets derived from RNase R-treated samples. Based on the thousands of viral circRNAs we identified, the biological characteristics and potential roles of viral circRNAs in regulating host cell function were determined. In addition, we developed a Viral-circRNA Database (http://www.hywanglab.cn/vcRNAdb/), which is open to all users to search, browse and download information on circRNAs encoded by viruses upon infection.


Subject(s)
RNA, Circular , Viruses , Cell Line , Humans , RNA/genetics , RNA/metabolism , RNA, Circular/genetics , Viruses/genetics
10.
J Clin Lab Anal ; 36(10): e24666, 2022 Oct.
Article in English | MEDLINE | ID: covidwho-1999875

ABSTRACT

BACKGROUND: SARS-CoV-2 is one of the most contagious viruses in the Coronaviridae (CoV) family, which has become a pandemic. The aim of this study is to understand more about the role of hsa_circ_0004812 in the SARS-CoV-2 related cytokine storm and its associated molecular mechanisms. MATERIALS AND METHODS: cDNA synthesis was performed after total RNA was extracted from the peripheral blood mononuclear cells (PBMC) of 46 patients with symptomatic COVID-19, 46 patients with asymptomatic COVID-19, and 46 healthy controls. The expression levels of hsa_circ_0004812, hsa-miR-1287-5p, IL6R, and RIG-I were determined using qRT-PCR, and the potential interaction between these molecules was confirmed by bioinformatics tools and correlation analysis. RESULTS: hsa_circ_0004812, IL6R, and RIG-I are expressed higher in the severe symptom group compared with the negative control group. Also, the relative expression of these genes in the asymptomatic group is lower than in the severe symptom group. The expression level of hsa-miR-1287-5p was positively correlated with symptoms in patients. The results of the bioinformatics analysis predicted the sponging effect of hsa_circ_0004812 as a competing endogenous RNA on hsa-miR-1287-5p. Moreover, there was a significant positive correlation between hsa_circ_0004812, RIG-I, and IL-6R expressions, and also a negative expression correlation between hsa_circ_0004812 and hsa-miR-1287-5p and between hsa-miR-1287-5p, RIG-I, and IL-6R. CONCLUSION: The results of this in-vitro and in silico study show that hsa_circ_0004812/hsa-miR-1287-5p/IL6R, RIG-I can play an important role in the outcome of COVID-19.


Subject(s)
COVID-19 , MicroRNAs , Receptors, Cell Surface/metabolism , COVID-19/genetics , Cell Proliferation/physiology , Cytokine Release Syndrome , DNA, Complementary , Humans , Leukocytes, Mononuclear/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics , Receptors, Interleukin-6/genetics , Receptors, Interleukin-6/metabolism , SARS-CoV-2 , Up-Regulation/genetics
11.
Front Immunol ; 13: 939768, 2022.
Article in English | MEDLINE | ID: covidwho-1987496

ABSTRACT

Circular RNAs (circRNAs) as novel regulatory molecules have been recognized in diverse species, including viruses. The virus-derived circRNAs play various roles in the host biological process and the life cycle of the viruses. This review summarized the circRNAs from the DNA and RNA viruses and discussed the biogenesis of viral and host circRNAs, the potential roles of viral circRNAs, and their future perspective. This review will elaborate on new insights gained on viruses encoded circRNAs during virus infection.


Subject(s)
Host Microbial Interactions , RNA, Circular , Host Microbial Interactions/genetics , RNA, Circular/genetics
12.
Mol Biol Rep ; 49(7): 6971-6973, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1955990

ABSTRACT

Non-coding RNAs (ncRNAs) like miRNAs, siRNA, lncRNAs, circRNAs, piRNAs, snoRNAs, snRNAs etc. form a collective group of RNAs that is instrumental to the various functions of the genome. With the advent of cutting-edge molecular biology tools and techniques, scientists have unearthed several mechanisms through which these ncRNAs act. Although our understanding may still be limited, yet scientists have been able to establish ncRNAs as major regulators of genetic inter-plays that dictate various pathophysiological conditions. This special issue of Molecular Biology Reports features a collection of research and review articles on ncRNAs and their involvement in different pathophysiological conditions that include different types of cancers. It is expected that this special issue will motivate researchers in the field to delve deeper into the world of ncRNAs and attempt to develop new diagnostic and therapeutic interventions for challenging clinical conditions.


Subject(s)
MicroRNAs , RNA, Long Noncoding , MicroRNAs/genetics , RNA, Circular/genetics , RNA, Long Noncoding/genetics , RNA, Small Interfering , RNA, Small Nucleolar , RNA, Untranslated/genetics
13.
J Biol Chem ; 298(9): 102280, 2022 09.
Article in English | MEDLINE | ID: covidwho-1936718

ABSTRACT

Transmissible gastroenteritis virus (TGEV), a member of the coronavirus family, is the pathogen responsible for transmissible gastroenteritis, which results in mitochondrial dysfunction in host cells. Previously, we identified 123 differentially expressed circular RNAs (cRNA)from the TGEV-infected porcine intestinal epithelial cell line jejunum 2 (IPEC-J2). Previous bioinformatics analysis suggested that, of these, circBIRC6 had the potential to regulate mitochondrial function. Furthermore, mitochondrial permeability transition, a key step in the process of mitochondrial dysfunction, is known to be caused by abnormal opening of mitochondrial permeability transition pores (mPTPs) regulated by the voltage-dependent anion-selective channel protein 1 (VDAC)-Cyclophilin D (CypD) complex. Therefore, in the present study, we investigated the effects of circBIRC6-2 on mitochondrial dysfunction and opening of mPTPs. We found that TGEV infection reduced circBIRC6-2 levels, which in turn reduced mitochondrial calcium (Ca2+) levels, the decrease of mitochondrial membrane potential, and opening of mPTPs. In addition, we also identified ORFs and internal ribosomal entrance sites within the circBIRC6-2 RNA. We demonstrate circBIRC6-2 encodes a novel protein, BIRC6-236aa, which we show inhibits TGEV-induced opening of mPTPs during TGEV infection. Mechanistically, we identified an interaction between BIRC6-236aa and VDAC1, suggesting that BIRC6-236aa destabilizes the VDAC1-CypD complex. Taken together, the results suggest that the novel protein BIRC6-236aa encoded by cRNA circBIRC6-2 inhibits mPTP opening and subsequent mitochondrial dysfunction by interacting with VDAC1.


Subject(s)
Inhibitor of Apoptosis Proteins , Mitochondria , Mitochondrial Permeability Transition Pore , RNA, Circular , Transmissible gastroenteritis virus , Animals , Calcium/metabolism , Cell Line , Cyclophilin D/metabolism , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Mitochondria/virology , Mitochondrial Permeability Transition Pore/metabolism , RNA, Circular/genetics , RNA, Circular/metabolism , Swine , Transmissible gastroenteritis virus/genetics , Transmissible gastroenteritis virus/physiology , Voltage-Dependent Anion Channel 1/metabolism
14.
Antiviral Res ; 204: 105370, 2022 08.
Article in English | MEDLINE | ID: covidwho-1906743

ABSTRACT

Next-generation COVID-19 vaccines are critical due to the ongoing evolution of SARS-CoV-2 virus and rapid waning duration of the neutralizing antibody response against current vaccines. The mRNA vaccines mRNA-1273 and BNT162b2 were developed using linear transcripts encoding the prefusion-stabilized trimers (S-2P) of the wildtype spike, which have shown a reduced neutralizing activity against the variants of concern B.1.617.2 and B.1.1.529. Recently, a new version of spike trimer, termed VFLIP (five (V) prolines, Flexibly-Linked, Inter-Protomer disulfide) was developed. Based on the original amino acid sequence of the wildtype spike, VFLIP was genetically engineered by using five proline substitutions, a flexible cleavage site amino acid linker, and an inter-protomer disulfide bond. It has been suggested to possess native-like glycosylation, and greater pre-fusion trimeric stability as opposed to S-2P. Here, we report that the spike protein VFLIP-X, containing six rationally substituted amino acids to reflect emerging variants (K417N, L452R, T478K, E484K, N501Y and D614G), offers a promising candidate for a next-generation SARS-CoV-2 vaccine. Mice immunized by a circular mRNA (circRNA) vaccine prototype producing VFLIP-X had detectable neutralizing antibody titers for up to 7 weeks post-boost against SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs). In addition, a balance in TH1 and TH2 responses was achieved by immunization with VFLIP-X. Our results indicate that the VFLIP-X delivered by circRNA induces humoral and cellular immune responses, as well as broad neutralizing activity against SARS-CoV-2 variants.


Subject(s)
COVID-19 Vaccines , COVID-19 , RNA, Circular , SARS-CoV-2 , mRNA Vaccines , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines/genetics , Disulfides , Mice , Proline , Protein Subunits , RNA, Circular/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , mRNA Vaccines/genetics
15.
Cell ; 185(10): 1728-1744.e16, 2022 05 12.
Article in English | MEDLINE | ID: covidwho-1767964

ABSTRACT

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Macaca mulatta , Mice , RNA, Circular/genetics , SARS-CoV-2/genetics , Vaccines, Synthetic/genetics , mRNA Vaccines
16.
J Med Virol ; 94(7): 3203-3222, 2022 07.
Article in English | MEDLINE | ID: covidwho-1750409

ABSTRACT

Circular RNAs (circRNAs) are a newly recognized component of the transcriptome with critical roles in autoimmune diseases and viral pathogenesis. To address the importance of circRNA in RNA viral transcriptome, we systematically identified and characterized circRNAs encoded by the RNA genomes of betacoronaviruses using both bioinformatical and experimental approaches. We predicted 351, 224, and 2764 circRNAs derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus, respectively. We experimentally identified 75 potential SARS-CoV-2 circRNAs from RNA samples extracted from SARS-CoV-2-infected Vero E6 cells. A systematic comparison of viral and host circRNA features, including abundance, strand preference, length distribution, circular exon numbers, and breakpoint sequences, demonstrated that coronavirus-derived circRNAs had a spliceosome-independent origin. We further showed that back-splice junctions (BSJs) captured by inverse reverse-transcription polymerase chain reaction have different level of resistance to RNase R. Through northern blotting with a BSJ-spanning probe targeting N gene, we identified three RNase R-resistant bands that represent SARS-CoV-2 circRNAs that are detected cytoplasmic by single-molecule and amplified fluorescence in situ hybridization assays. Lastly, analyses of 169 sequenced BSJs showed that both back-splice and forward-splice junctions were flanked by homologous and reverse complementary sequences, including but not limited to the canonical transcriptional regulatory sequences. Our findings highlight circRNAs as an important component of the coronavirus transcriptome, offer important evaluation of bioinformatic tools in the analysis of circRNAs from an RNA genome, and shed light on the mechanism of discontinuous RNA synthesis.


Subject(s)
COVID-19 , Middle East Respiratory Syndrome Coronavirus , Humans , In Situ Hybridization, Fluorescence , Middle East Respiratory Syndrome Coronavirus/genetics , RNA, Circular/genetics , SARS-CoV-2/genetics , Spliceosomes/genetics
17.
Emerg Microbes Infect ; 11(1): 519-531, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1642257

ABSTRACT

ABSTRACTHost circular RNAs (circRNAs) play critical roles in the pathogenesis of viral infections. However, how viruses modulate the biogenesis of host proviral circRNAs to facilitate their replication remains unclear. We have recently shown that Middle East respiratory syndrome coronavirus (MERS-CoV) infection increases co-expression of circRNAs and their cognate messenger RNAs (mRNAs), possibly by hijacking specific host RNA binding proteins (RBPs). In this study, we systemically analysed the interactions between the representative circRNA-mRNA pairs upregulated upon MERS-CoV infection and host RBPs. Our analysis identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a key host factor that governed the expression of numerous MERS-CoV-perturbed circRNAs, including hsa_circ_0002846, hsa_circ_0002061, and hsa_circ_0004445. RNA immunoprecipitation assay showed that hnRNP C could bind physically to these circRNAs. Specific knockdown of hnRNP C by small interfering RNA significantly (P < 0.05 to P < 0.0001) suppressed MERS-CoV replication in human lung adenocarcinoma (Calu-3) and human small airway epithelial (HSAEC) cells. Both MERS-CoV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection increased the total and phosphorylated forms of hnRNP C to activate the downstream CRK-mTOR pathway. Treatment of MERS-CoV- (IC50: 0.618 µM) or SARS-CoV-2-infected (IC50: 1.233 µM) Calu-3 cells with the mTOR inhibitor OSI-027 resulted in significantly reduced viral loads. Collectively, our study identified hnRNP C as a key regulator of MERS-CoV-perturbed circRNAs and their cognate mRNAs, and the potential of targeting hnRNP C-related signalling pathways as an anticoronaviral strategy.


Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group C , Middle East Respiratory Syndrome Coronavirus , RNA, Circular/genetics , SARS-CoV-2 , Virus Replication , COVID-19 , Cognition , Heterogeneous-Nuclear Ribonucleoprotein Group C/genetics , Humans , Middle East Respiratory Syndrome Coronavirus/physiology , RNA, Messenger/genetics , SARS-CoV-2/physiology
18.
Cell Mol Life Sci ; 79(2): 75, 2022 Jan 17.
Article in English | MEDLINE | ID: covidwho-1630170

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new member of the Betacoronaviridae family, responsible for the recent pandemic outbreak of COVID-19. To start exploring the molecular events that follow host cell infection, we queried VirusCircBase and identified a circular RNA (circRNA) predicted to be synthesized by SARS-CoV-2, circ_3205, which we used to probe: (i) a training cohort comprised of two pools of cells from three nasopharyngeal swabs of SARS-CoV-2 infected (positive) or uninfected (negative, UCs) individuals; (ii) a validation cohort made up of 12 positive and 3 negative samples. The expression of circRNAs, miRNAs and miRNA targets was assayed through real-time PCR. CircRNA-miRNA interactions were predicted by TarpMiR, Analysis of Common Targets for circular RNAs (ACT), and STarMir tools. Enrichment of the biological processes and the list of predicted miRNA targets were retrieved from DIANA miRPath v3.0. Our results showed that the predicted SARS-CoV-2 circ_3205 was expressed only in positive samples and its amount positively correlated with that of SARS-CoV-2 Spike (S) mRNA and the viral load (r values = 0.80952 and 0.84867, Spearman's correlation test, respectively). Human (hsa) miR-298 was predicted to interact with circ_3205 by all three predictive tools. KCNMB4 and PRKCE were predicted as hsa-miR-298 targets. Interestingly, the function of both is correlated with blood coagulation and immune response. KCNMB4 and PRKCE mRNAs were upregulated in positive samples as compared to UCs (6 and 8.1-fold, p values = 0.049 and 0.02, Student's t test, respectively) and their expression positively correlated with that of circ_3205 (r values = 0.6 and 0.25, Spearman's correlation test, respectively). We propose that our results convincingly suggest that circ_3205 is a circRNA synthesized by SARS-CoV-2 upon host cell infection and that it may behave as a competitive endogenous RNA (ceRNA), sponging hsa-miR-298 and contributing to the upregulation of KCNMB4 and PRKCE mRNAs.


Subject(s)
COVID-19/genetics , COVID-19/metabolism , RNA, Circular/genetics , RNA, Viral , SARS-CoV-2/genetics , Computational Biology , Gene Expression Regulation, Viral , Gene Regulatory Networks , Humans , Large-Conductance Calcium-Activated Potassium Channel beta Subunits/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Nasopharynx/virology , Nerve Tissue Proteins/genetics , Protein Interaction Mapping , Protein Kinase C-epsilon/genetics , Reproducibility of Results
19.
Nucleic Acids Res ; 49(21): 12502-12516, 2021 12 02.
Article in English | MEDLINE | ID: covidwho-1546005

ABSTRACT

Circular RNAs (circRNAs) are noncoding RNAs that exist in all eukaryotes investigated and are derived from back-splicing of certain pre-mRNA exons. Here, we report the application of artificial circRNAs designed to act as antisense-RNAs. We systematically tested a series of antisense-circRNAs targeted to the SARS-CoV-2 genome RNA, in particular its structurally conserved 5'-untranslated region. Functional assays with both reporter transfections as well as with SARS-CoV-2 infections revealed that specific segments of the SARS-CoV-2 5'-untranslated region can be efficiently accessed by specific antisense-circRNAs, resulting in up to 90% reduction of virus proliferation in cell culture, and with a durability of at least 48 h. Presenting the antisense sequence within a circRNA clearly proved more efficient than in the corresponding linear configuration and is superior to modified antisense oligonucleotides. The activity of the antisense-circRNA is surprisingly robust towards point mutations in the target sequence. This strategy opens up novel applications for designer circRNAs and promising therapeutic strategies in molecular medicine.


Subject(s)
Genome, Viral/genetics , RNA, Antisense/genetics , RNA, Circular/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Replication/genetics , 5' Untranslated Regions/genetics , Animals , Antiviral Agents/metabolism , Base Sequence , COVID-19/prevention & control , COVID-19/virology , Cell Proliferation/genetics , Chlorocebus aethiops , Drug Design , HeLa Cells , Host-Pathogen Interactions/genetics , Humans , Nucleic Acid Conformation , RNA, Viral/chemistry , RNA-Seq/methods , SARS-CoV-2/physiology , Vero Cells
20.
Int J Mol Med ; 47(5)2021 05.
Article in English | MEDLINE | ID: covidwho-1448967

ABSTRACT

Circular RNAs (circRNAs) are a class of non­coding RNAs with a circular, covalent structure that lack both 5' ends and 3' poly(A) tails, which are stable and specific molecules that exist in eukaryotic cells and are highly conserved. The role of circRNAs in viral infections is being increasingly acknowledged, since circRNAs have been discovered to be involved in several viral infections (such as hepatitis B virus infection and human papilloma virus infection) through a range of circRNA/microRNA/mRNA regulatory axes. These findings have prompted investigations into the potential of circRNAs as targets for the diagnosis and treatment of viral infection­related diseases. The aim of the present review was to systematically examine and discuss the role of circRNAs in several common viral infections, as well as their potential as diagnostic markers and therapeutic targets.


Subject(s)
MicroRNAs/genetics , RNA, Circular/physiology , RNA, Messenger/genetics , Virus Diseases/genetics , Biomarkers/analysis , Humans , RNA, Circular/genetics , Virus Diseases/diagnosis , Virus Diseases/therapy , Virus Diseases/virology
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