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European Heart Journal May 2023Epicardium and epicardium-derived cells are critical players in myocardial fibrosis. Mesenchymal stem cell-derived extracellular vesicles (EVs) have been studied for...
AIMS
Epicardium and epicardium-derived cells are critical players in myocardial fibrosis. Mesenchymal stem cell-derived extracellular vesicles (EVs) have been studied for cardiac repair to improve cardiac remodelling, but the actual mechanisms remain elusive. The aim of this study is to investigate the mechanisms of EV therapy for improving cardiac remodelling and develop a promising treatment addressing myocardial fibrosis.
METHODS AND RESULTS
Extracellular vesicles were intrapericardially injected for mice myocardial infarction treatment. RNA-seq, in vitro gain- and loss-of-function experiments, and in vivo studies were performed to identify targets that can be used for myocardial fibrosis treatment. Afterward, a lipid nanoparticle-based long non-coding RNA (lncRNA) therapy was prepared for mouse and porcine models of myocardial infarction treatment. Intrapericardial injection of EVs improved adverse myocardial remodelling in mouse models of myocardial infarction. Mechanistically, Tcf21 was identified as a potential target to improve cardiac remodelling. Loss of Tcf21 function in epicardium-derived cells caused increased myofibroblast differentiation, whereas forced Tcf21 overexpression suppressed transforming growth factor-β signalling and myofibroblast differentiation. LncRNA-Tcf21 antisense RNA inducing demethylation (TARID) that enriched in EVs was identified to up-regulate Tcf21 expression. Formulated lncRNA-TARID-laden lipid nanoparticles up-regulated Tcf21 expression in epicardium-derived cells and improved cardiac function and histology in mouse and porcine models of myocardial infarction.
CONCLUSION
This study identified Tcf21 as a critical target for improving cardiac fibrosis. Up-regulating Tcf21 by using lncRNA-TARID-laden lipid nanoparticles could be a promising way to treat myocardial fibrosis. This study established novel mechanisms underlying EV therapy for improving adverse remodelling and proposed a lncRNA therapy for cardiac fibrosis.
Topics: Mice; Animals; Swine; RNA, Long Noncoding; RNA, Antisense; Ventricular Remodeling; Myocardial Infarction; Fibrosis; Demethylation
PubMed: 36916305
DOI: 10.1093/eurheartj/ehad114 -
RNA (New York, N.Y.) Mar 2020The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in...
The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in vitrotranscribed RNAs, predominantly using phage RNA polymerases (RNAPs), is well established. However, transcripts synthesized with RNAPs are known to display an immune-stimulatory activity in vivo that is often undesirable. Previous studies have identified double-stranded RNA (dsRNA), a major by-product of the in vitro transcription (IVT) process, as a trigger of cellular immune responses. Here we describe the characterization of a high-temperature IVT process using thermostable T7 RNAPs to synthesize functional mRNAs that demonstrate reduced immunogenicity without the need for a post-synthesis purification step. We identify features that drive the production of two kinds of dsRNA by-products-one arising from 3' extension of the run-off product and one formed by the production of antisense RNAs-and demonstrate that at a high temperature, T7 RNAP has reduced 3'-extension of the run-off product. We show that template-encoded poly(A) tailing does not affect 3'-extension but reduces the formation of the antisense RNA by-products. Combining high-temperature IVT with template-encoded poly(A) tailing prevents the formation of both kinds of dsRNA by-products generating functional mRNAs with reduced immunogenicity.
Topics: Bacteriophage T7; DNA-Directed RNA Polymerases; Immunity, Cellular; RNA; RNA, Antisense; RNA, Double-Stranded; RNA, Messenger; Transcription, Genetic
PubMed: 31900329
DOI: 10.1261/rna.073858.119 -
Science (New York, N.Y.) Dec 2008RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of...
RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. We show that peaks of promoter-proximal polymerase reside on approximately 30% of human genes, transcription extends beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.
Topics: Cell Line; CpG Islands; DNA-Directed RNA Polymerases; Genome, Human; Humans; Nucleosomes; Promoter Regions, Genetic; RNA Polymerase II; RNA, Antisense; RNA, Messenger; Sequence Analysis, RNA; Transcription Initiation Site; Transcription, Genetic
PubMed: 19056941
DOI: 10.1126/science.1162228 -
Biochimie Nov 2011Although control of cellular function has classically been considered the responsibility of proteins, research over the last decade has elucidated many roles for RNA in... (Review)
Review
Although control of cellular function has classically been considered the responsibility of proteins, research over the last decade has elucidated many roles for RNA in regulation of not only the proteins that control cellular functions but also for the cellular functions themselves. In parallel to this advancement in knowledge about the regulatory roles of RNA there has been an explosion of knowledge about the role that epigenetics plays in controlling not only long-term cellular fate but also the short-term regulatory control of genes. Of particular interest is the crossover between these two worlds, a world where RNA can act out its part and subsequently elicit chromatin modifications that alter cellular function. Two main categories of RNA are examined here, non-coding RNA and antisense RNA both of which perform vital functions in controlling numerous genes, proteins and RNA itself. As the activities of non-coding and antisense RNA in both normal and aberrant cellular function are elucidated, so does the number of possible targets for pharmacopeic intervention.
Topics: Animals; Chromatin Assembly and Disassembly; Epigenesis, Genetic; Gene Expression Regulation; Genomic Imprinting; Humans; Pluripotent Stem Cells; RNA, Antisense; RNA, Untranslated; Transcription, Genetic; X Chromosome Inactivation
PubMed: 21843589
DOI: 10.1016/j.biochi.2011.07.031 -
RNA Biology 2014The symbiotic α-rhizobia Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium etli and the related plant pathogen Agrobacterium tumefaciens are important model... (Review)
Review
The symbiotic α-rhizobia Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium etli and the related plant pathogen Agrobacterium tumefaciens are important model organisms for studying plant-microbe interactions. These metabolically versatile soil bacteria are characterized by complex lifestyles and large genomes. Here we summarize the recent knowledge on their small non-coding RNAs (sRNAs) including conservation, function, and interaction of the sRNAs with the RNA chaperone Hfq. In each of these organisms, an inventory of hundreds of cis- and trans-encoded sRNAs with regulatory potential was uncovered by high-throughput approaches and used for the construction of 39 sRNA family models. Genome-wide analyses of hfq mutants and co-immunoprecipitation with tagged Hfq revealed a major impact of the RNA chaperone on the physiology of plant-associated α-proteobacteria including symbiosis and virulence. Highly conserved members of the SmelC411 family are the AbcR sRNAs, which predominantly regulate ABC transport systems. AbcR1 of A. tumefaciens controls the uptake of the plant-generated signaling molecule GABA and is a central regulator of nutrient uptake systems. It has similar functions in S. meliloti and the human pathogen Brucella abortus. As RNA degradation is an important process in RNA-based gene regulation, a short overview on ribonucleases in plant-associated α-proteobacteria concludes this review.
Topics: Alphaproteobacteria; Base Pairing; Gene Expression Regulation, Bacterial; Multigene Family; Plants; RNA Stability; RNA, Antisense; RNA, Bacterial; RNA, Messenger; RNA, Small Untranslated; RNA-Binding Proteins; Transcriptome
PubMed: 25003187
DOI: 10.4161/rna.29625 -
Human Molecular Genetics Sep 2014Recent years have seen the increasing understanding of the crucial role of RNA in the functioning of the eukaryotic genome. These discoveries, fueled by the achievements... (Review)
Review
Recent years have seen the increasing understanding of the crucial role of RNA in the functioning of the eukaryotic genome. These discoveries, fueled by the achievements of the FANTOM, and later GENCODE and ENCODE consortia, led to the recognition of the important regulatory roles of natural antisense transcripts (NATs) arising from what was previously thought to be 'junk DNA'. Roughly defined as non-coding regulatory RNA transcribed from the opposite strand of a coding gene locus, NATs are proving to be a heterogeneous group with high potential for therapeutic application. Here, we attempt to summarize the rapidly growing knowledge about this important non-coding RNA subclass.
Topics: Gene Expression; Gene Targeting; Genome; Humans; RNA, Antisense; RNA, Untranslated; Transcription, Genetic
PubMed: 24838284
DOI: 10.1093/hmg/ddu207 -
Journal of Zhejiang University....Antisense RNA molecule represents a unique type of DNA transcript that comprises 19-23 nucleotides and is complementary to mRNA. Antisense RNAs play the crucial role in... (Review)
Review
Antisense RNA molecule represents a unique type of DNA transcript that comprises 19-23 nucleotides and is complementary to mRNA. Antisense RNAs play the crucial role in regulating gene expression at multiple levels, such as at replication, transcription, and translation. In addition, artificial antisense RNAs can effectively regulate the expression of related genes in host cells. With the development of antisense RNA, investigating the functions of antisense RNAs has emerged as a hot research field. This review summarizes our current understanding of antisense RNAs, particularly of the formation of antisense RNAs and their mechanism of regulating the expression of their target genes. In addition, we detail the effects and applications of antisense RNAs in antivirus and anticancer treatments and in regulating the expression of related genes in plants and microorganisms. This review is intended to highlight the key role of antisense RNA in genetic research and guide new investigators to the study of antisense RNAs.
Topics: Animals; Antineoplastic Agents; Antiviral Agents; Gene Expression Regulation; Genetic Research; Humans; MicroRNAs; RNA, Antisense; RNA, Long Noncoding; RNA, Small Interfering
PubMed: 30269442
DOI: 10.1631/jzus.B1700594 -
Biomedicine & Pharmacotherapy =... May 2020RNA therapy is a treatment that regulates cell proteins and cures diseases by affecting the metabolism of mRNAs in cells, which has cut a figure in the studies on... (Review)
Review
RNA therapy is a treatment that regulates cell proteins and cures diseases by affecting the metabolism of mRNAs in cells, which has cut a figure in the studies on various incurable illnesses like hereditary diseases, tumors, etc. In this review, we introduced the discovery and development of RNA therapy and discussed its classification, mechanisms, advantages, and challenges. Moreover, we highlighted how RNA therapy works in killing tumor cells as well as what progresses it has made in related researches. And the development of RNA anti-tumor drugs and the clinical trial process were also included.
Topics: Animals; Biomarkers, Tumor; Cellular Reprogramming; Genetic Therapy; Humans; Immunotherapy; Molecular Targeted Therapy; Neoplasm Grading; Neoplasm Staging; Neoplasms; RNA; RNA Interference; RNA, Antisense; RNA, Messenger; Translational Research, Biomedical
PubMed: 32062550
DOI: 10.1016/j.biopha.2020.109997 -
Hfq-Antisense RNA I Binding Regulates RNase E-Dependent RNA Stability and ColE1 Plasmid Copy Number.International Journal of Molecular... Apr 2024The mechanisms and consequences of gene regulation by Hfq on -encoded small RNAs (sRNAs) have been well studied and documented. Recent employment of Genomic SELEX to...
The mechanisms and consequences of gene regulation by Hfq on -encoded small RNAs (sRNAs) have been well studied and documented. Recent employment of Genomic SELEX to search for Hfq-binding motifs has indicated that Hfq might frequently regulate gene expression controlled by -antisense RNAs. Here, we use the classic ColE1 plasmid antisense RNA-based regulation model (i.e., RNA I) to study the role of Hfq in controlling antisense regulatory functions. We show that Hfq exhibits a high binding affinity for RNA I and that binding limits RNase E cleavage, thereby stabilizing RNA I and reducing the plasmid copy number. Full-length RNA I displays a binding affinity for Hfq in the sub-micromolar range. In vivo overexpression of Hfq prolongs RNA I stability and reduces the ColE1 plasmid copy number, whereas deletion of reduces RNA I stability and increases the plasmid copy number. RNA I predominantly binds to the proximal face of Hfq and exhibits competitive ability against a chromosome-borne proximal face-bound sRNA (DsrA) for Hfq binding. Through its strong promoter and high gene dosage features, plasmid-encoded antisense RNA I results in high RNA I expression, so it may antagonize the effects of -encoded RNAs in controlling target gene expression.
Topics: RNA, Antisense; DNA Copy Number Variations; Plasmids; RNA Stability; Endoribonucleases
PubMed: 38612765
DOI: 10.3390/ijms25073955 -
Essays in Biochemistry Oct 2021RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding... (Review)
Review
RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic.
Topics: Animals; Mice; Protein Biosynthesis; Proteins; RNA, Antisense; RNA, Long Noncoding; RNA, Messenger
PubMed: 34623427
DOI: 10.1042/EBC20200114