Search Results (93)

The selective regulation of gene expression at the RNA level represents a rapidly evolving field offering substantial clinical potential. This review examines the molecular mechanisms of intracellular enzymatic systems that utilize single-stranded nucleic acids to downregulate specific RNA targets. The analysis encompasses antisense oligonucleotides and synthetic mimics of small interfering RNA (siRNA), microRNA (miRNA), transfer RNA-derived small RNA (tsRNA), and PIWI-interacting RNA (piRNA), elucidating their intricate interactions with crucial cellular machinery, specifically RNase H1, RNase P, AGO, and PIWI proteins, mediating their biological effects. The functional and structural characteristics of these endonucleases are examined in relation to their mechanisms of action and resultant therapeutic outcomes. This comprehensive analysis illuminates the interactions between single-stranded nucleic acids and their endonuclease partners, covering antisense inhibition pathways as well as RNA interference processes. This field of research has important implications for advancing targeted RNA modulation strategies across various disease contexts. Full article

Long non-coding RNAs (lncRNAs) are a class of non-coding RNA molecules with transcripts longer than 200 bp, which were initially thought to be noise from genomic transcription without biological function. However, since the discovery of H19 in 1980 and Xist in 1990, increasing evidence has shown that lncRNAs regulate gene expression at epigenetic, transcriptional, and post-transcriptional levels through specific regulatory actions and are involved in the development of cancer and other diseases. Despite many lncRNAs being expressed at lower levels than those of protein-coding genes with less sequence conservation across species, lncRNAs have become an intense area of RNA research. They exert diverse biological functions such as inducing chromatin remodeling, recruiting transcriptional machinery, acting as competitive endogenous RNAs for microRNAs, and modulating protein–protein interactions. Epithelial–mesenchymal transition (EMT) is a developmental process, associated with embryonic development, wound healing, and cancer progression. In the context of oncogenesis, the EMT program is transiently activated and confers migratory/invasive and cancer stem cell (CSC) properties to tumor cells, which are crucial for malignant progression, metastasis, and therapeutic resistance. Accumulating evidence has revealed that lncRNAs play crucial roles in the regulation of tumor epithelial/mesenchymal plasticity (EMP) and cancer stemness. Here, we summarize the emerging roles and molecular mechanisms of lncRNAs in regulating tumor cell EMP and their effects on tumor initiation and progression through regulation of CSCs. We also discuss the potential of lncRNAs as diagnostic and prognostic biomarkers and therapeutic targets. Full article

MicroRNAs (miRNAs) are a class of small regulatory RNA that are generated via core protein machinery. The miRNAs direct gene-silencing mechanisms to mediate an essential role in gene expression regulation. In mollusks, miRNAs have been demonstrated to be required to regulate gene expression in various biological processes, including normal development, immune responses, reproduction, and stress adaptation. In this study, we aimed to establishment the requirement of the miRNA pathway as part of the molecular response of exposure of Biomphalaria glabrata (snail host) to Schistosoma mansoni (trematode parasite). Initially, the core pieces of miRNA pathway protein machinery, i.e., Drosha, DGCR8, Exportin-5, Ran, and Dicer, together with the central RNA-induced silencing complex (RISC) effector protein Argonaute2 (Ago2) were elucidated from the B. glabrata genome. Following exposure of B. glabrata to S. mansoni miracidia, we identified significant expression up-regulation of all identified pieces of miRNA pathway protein machinery, except for Exportin-5, at 16 h post exposure. For Ago2, we went on to show that the Bgl-Ago2 protein was localized to regions surrounding the sporocysts in the digestive gland of infected snails 20 days post parasite exposure. In addition to documenting elevated miRNA pathway protein machinery expression at the early post-exposure time point, a total of 13 known B. glabrata miRNAs were significantly differentially expressed. Of these thirteen B. glabrata miRNAs responsive to S. mansoni miracidia exposure, five were significantly reduced in their abundance, and correspondingly, these five miRNAs were determined to putatively target six genes with significantly elevated expression and that have been previously associated with immune responses in other animal species, including humans. In conclusion, this study demonstrates the central importance of a functional miRNA pathway in snails, which potentially forms a critical component of the immune response of snails to parasite exposure. Further, the data reported in this study provide additional evidence of the complexity of the molecular response of B. glabrata to S. mansoni infection: a molecular response that could be targeted in the future to overcome parasite infection and, in turn, human schistosomiasis. Full article

Genetic features of alcohol dependence have been extensively investigated in recent years. A large body of studies has underlined the important role of genetic variants not only in metabolic pathways but also in the neurobiology of alcohol dependence, mediated by the neuronal circuits regulating reward and craving. Serotonin transporter (5-HTT), encoded by the SLC6A4 gene (Solute carrier family 6-neurotransmitter transporter-member 4), is targeted by antidepressant drugs such as selective serotonin reuptake inhibitors (SSRIs) and plays a pivotal role in serotoninergic transmission; it has been associated with psychiatric diseases and alcohol dependence. Transcriptional regulation and expression of 5-HTT depend not only on epigenetic modifications, among which DNA methylation (CpG and non-CpG) is primarily involved, but also on sequence variations occurring in intron/exon regions and in untranslated regions in 5′ and 3′, being the first sequences important for the splicing machinery and the last for the binding of transcription factors and micro RNAs. This work intends to shed light on the role of sequence variations known to affect the expression or function of 5-HTT in alcohol-dependent individuals. We found a statistically significant difference in the allelic (p = 0.0083) and genotypic (p = 0.0151) frequencies of the tri-allelic polymorphism, with higher function alleles and genotypes more represented in the control population. Furthermore, we identified three haplotypes more frequent in subjects with AUD (p < 0.0001) and one more frequent in the control population (p < 0.0001). The results obtained for the tri-allelic polymorphism in alcohol dependence confirm what is already present in part of the literature. The role of haplotypes requires further studies to be clarified. Full article

Mirtrons represent a subclass of microRNAs (miRNAs) that rely on the splicing machinery for their maturation. However, the molecular details of this Drosha-independent processing are still not fully understood; as an example, the Microprocessor complex cannot process the mirtronic pre-miRNA from the transcript even if splice site mutations are present. To investigate the influence of alternative splicing sites on mirtron formation, we generated Enhanced Green Fluorescent Protein (EGFP) reporters containing artificial introns to compare the processing of canonical miRNAs and mirtrons. Although mutations of both splice sites generated a complex pattern of alternative transcripts, mirtron formation was always severely affected as opposed to the normal processing of the canonical hsa-mir-33b miRNA. However, we also detected that while its formation was also hindered, the mirtron-derived hsa-mir-877-3p miRNA was less affected by certain mutations than the hsa-mir-877-5p species. By knocking down Drosha, we showed that this phenomenon is not dependent on Microprocessor activity but rather points toward the potential stability difference between the miRNAs from the different arms. Our results indicate that when the major splice sites are mutated, mirtron formation cannot be rescued by nearby alternative splice sites, and stability differences between 5p and 3p species should also be considered for functional studies of mirtrons. Full article

During infection, adenoviruses inhibit the cellular RNA interference (RNAi) machinery by saturating the RNA-induced silencing complex (RISC) of the host cells with large amounts of virus-derived microRNAs (mivaRNAs) that bind to the key component of the complex, Argonaute 2 (AGO2). In the present study, we investigated AGO2 as a prominent player at the intersection between human adenovirus 5 (HAdV-5) and host cells because of its ability to interfere with the HAdV-5 life cycle. First, the ectopic expression of AGO2 had a detrimental effect on the ability of the virus to replicate. In addition, in silico and in vitro analyses suggested that endogenous microRNAs (miRNAs), particularly hsa-miR-7-5p, have similar effects. This miRNA was found to be able to target the HAdV-5 DNA polymerase mRNA. The inhibitory effect became more pronounced upon overexpression of AGO2, likely due to elevated AGO2 levels, which abolished the competition between cellular miRNAs and mivaRNAs for RISC incorporation. Collectively, our data suggest that endogenous miRNAs would be capable of significantly inhibiting viral replication if adenoviruses had not developed a mechanism to counteract this function. Eventually, AGO2 overexpression-mediated relief of the RISC-saturating action of mivaRNAs strongly enhanced the effectiveness of artificial miRNAs (amiRNAs) directed against the HAdV-5 preterminal protein (pTP) mRNA, suggesting a substantial benefit of co-expressing amiRNAs and AGO2 in RNAi-based strategies for the therapeutic inhibition of adenoviruses. Full article

Recently, myriad studies have defined the versatile abilities of gasotransmitters and their synthesizing enzymes to play a “Maestro” role in orchestrating several oncological and non-oncological circuits and, thus, nominated them as possible therapeutic targets. Although a significant amount of work has been conducted on the role of nitric oxide (NO) and carbon monoxide (CO) and their inter-relationship in the field of oncology, research about hydrogen sulfide (H₂S) remains in its infancy. Recently, non-coding RNAs (ncRNAs) have been reported to play a dominating role in the regulation of the endogenous machinery system of H₂S in several pathological contexts. A growing list of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are leading the way as upstream regulators for H₂S biosynthesis in different mammalian cells during the development and progression of human diseases; therefore, their targeting can be of great therapeutic benefit. In the current review, the authors shed the light onto the biosynthetic pathways of H₂S and their regulation by miRNAs and lncRNAs in various oncological and non-oncological disorders. Full article

Novel components of the mitochondrial fission machinery, mitochondrial dynamics proteins of 49 kDa (MiD49) and 51 kDa (MiD51), have been recently described, and their potential therapeutic targets for treating cardiovascular disease have been shown, including acute myocardial infarction (AMI), anthracycline cardiomyopathy and pulmonary arterial hypertension (PAH). Here, we examined the role of MiD49 and MiD51 in atherosclerosis. MiD49/51 expression was increased in the aortic valve endothelial cells (ECs) of high-fat diet-induced atherosclerosis in ApoE^-/-mice and IL-8-induced human umbilical vein endothelial cells (HUVECs), which accelerated dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. Silencing MiD49/51 reduced atherosclerotic plaque size, increased collagen content, and decreased the IL-8-induced adhesion and proliferation of HUVECs. MiD51 upregulation resulted from decreased microRNA (miR)-107 expression and increased hypoxia-inducible factor-1a (HIF-1a) expression. Treatment with miR-107 mimics decreased atherosclerotic plaque size by reducing HIF-1α and MiD51 production. Both MiD49 and MiD51 were involved in atherosclerotic plaque formation through Drp1-mediated mitochondrial fission, and the involvement of MiD51 in this process was the result of decreased miR-107 expression and increased HIF-1α expression. The miR-107–HIF-1α–MiD51 pathway might provide new therapeutic targets for atherosclerosis. Full article

Extracellular vesicles (EVs)/exosomes are nanosized membrane-bound structures that are released by virtually all cells. EVs have attracted great attention in the scientific community since the discovery of their roles in cell-to-cell communication. EVs’ enclosed structure protects bioactive molecules from degradation in the extracellular space and targets specific tissues according to the topography of membrane proteins. Upon absorption by recipient cells, EV cargo can modify the transcription machinery and alter the cellular functions of these cells, playing a role in disease pathogenesis. EVs have been tested as the delivery system for the mRNA COVID-19 vaccine. Recently, different therapeutic strategies have been designed to use EVs as a delivery system for microRNAs and mRNA. In this review, we will focus on the exciting and various platforms related to using EVs as delivery vehicles, mainly in gene editing using CRISPR/Cas9, cancer therapy, drug delivery, and vaccines. We will also touch upon their roles in disease pathogenesis. Full article

Aging is a risk factor for many non-communicable diseases such as cardiovascular and neurodegenerative diseases. Extracellular vesicles and particles (EVP) carry microRNAs that may play a role in age-related diseases and may induce oxidative stress. We hypothesized that aging could impact EVP miRNA and impair redox homeostasis, contributing to chronic age-related diseases. Our aims were to investigate the microRNA profiles of circulating total EVPs from aged and young adult animals and to evaluate the pro- and antioxidant machinery in circulating total EVPs. Plasma from 3- and 21-month-old male Wistar rats were collected, and total EVPs were isolated. MicroRNA isolation and microarray expression analysis were performed on EVPs to determine the predicted regulation of targeted mRNAs. Thirty-one mature microRNAs in circulating EVPs were impacted by age and were predicted to target molecules in canonical pathways directly related to cardiovascular diseases and oxidative status. Circulating total EVPs from aged rats had significantly higher NADPH oxidase levels and myeloperoxidase activity, whereas catalase activity was significantly reduced in EVPs from aged animals. Our data shows that circulating total EVP cargo—specifically microRNAs and oxidative enzymes—are involved in redox imbalance in the aging process and can potentially drive cardiovascular aging and, consequently, cardiac disease. Full article

Penicillium expansum is the most popular post-harvest pathogen and causes blue mold disease in pome fruit and leads to significant economic losses worldwide every year. However, the fundamental regulation mechanisms of growth in P. expansum are unclear. Recently, non-coding RNAs (ncRNAs) have attracted more attention due to critical roles in normalizing gene expression and maintaining cellular genotypes in organisms. However, the research related to ncRNAs in P. expansum have not been reported. Therefore, to provide an overview of ncRNAs on composition, distribution, expression changes, and potential targets in the growth process, a comparative transcriptomic analysis was performed on spores and mycelia of P. expansum in the present study. A total of 2595 novel mRNAs, 3362 long non-coding RNAs (lncRNAs), 10 novel microRNAs (miRNAs), 86 novel small interfering RNAs (siRNAs), and 11,238 circular RNAs (circRNAs) were predicted and quantified. Of these, 1482 novel mRNAs, 5987 known mRNAs, 2047 lncRNAs, 40 miRNAs, 38 novel siRNAs, and 9235 circRNAs were differentially expressed (DE) in response to the different development stages. Afterward, the involved functions and pathways of DE RNAs were revealed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database enrichment analysis. The interaction networks between mRNAs, lncRNAs, and miRNAs were also predicted based on their correlation coefficient of expression profiles. Among them, it was found that miR168 family members may play important roles in fungal growth due to their central location in the network. These findings will contribute to a better understanding on regulation machinery at the RNA level on fungal growth and provide a theoretical basis to develop novel control strategies against P. expansum. Full article

With the emergence of the novel betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there has been an urgent need for the development of fast-acting antivirals, particularly in dealing with different variants of concern (VOC). SARS-CoV-2, like other RNA viruses, depends on host cell machinery to propagate and misregulate metabolic pathways to its advantage. Herein, we discovered that the immunometabolic microRNA-185 (miR-185) restricts SARS-CoV-2 propagation by affecting its entry and infectivity. The antiviral effects of miR-185 were studied in SARS-CoV-2 Spike protein pseudotyped virus, surrogate virus (HCoV-229E), as well as live SARS-CoV-2 virus in Huh7, A549, and Calu-3 cells. In each model, we consistently observed microRNA-induced reduction in lipid metabolism pathways-associated genes including SREBP2, SQLE, PPARG, AGPAT3, and SCARB1. Interestingly, we also observed changes in angiotensin-converting enzyme 2 (ACE2) levels, the entry receptor for SARS-CoV-2. Taken together, these data show that miR-185 significantly restricts host metabolic and other pathways that appear to be essential to SAR-CoV-2 replication and propagation. Overall, this study highlights an important link between non-coding RNAs, immunometabolic pathways, and viral infection. miR-185 mimics alone or in combination with other antiviral therapeutics represent possible future fast-acting antiviral strategies that are likely to be broadly antiviral against multiple variants as well as different virus types of potential pandemics. Full article

Since its discovery in 1989, RNA interference (RNAi) has become a widely used tool for the in vitro downregulation of specific gene expression in molecular biological research. This basically involves a complementary RNA that binds a target sequence to affect its transcription or translation process. Currently, various small RNAs, such as small interfering RNA (siRNA), micro RNA (miRNA), small hairpin RNA (shRNA), and PIWI interacting RNA (piRNA), are available for application on in vitro cell culture, to regulate the cells’ gene expression by mimicking the endogenous RNAi-machinery. In addition, several biochemical, physical, and viral methods have been established to deliver these RNAs into the cell or nucleus. Since each RNA and each delivery method entail different off-target effects, limitations, and compatibilities, it is crucial to understand their basic mode of action. This review is intended to provide an overview of different nucleic acids and delivery methods for planning, interpreting, and troubleshooting of RNAi experiments. Full article

Within regenerative endodontics, exciting opportunities exist for the development of next-generation targeted biomaterials that harness epigenetic machinery, including microRNAs (miRNAs), histone acetylation, and DNA methylation, which are used to control pulpitis and to stimulate repair. Although histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) induce mineralisation in dental pulp cell (DPC) populations, their interaction with miRNAs during DPC mineralisation is not known. Here, small RNA sequencing and bioinformatic analysis were used to establish a miRNA expression profile for mineralising DPCs in culture. Additionally, the effects of a HDACi, suberoylanilide hydroxamic acid (SAHA), and a DNMTi, 5-aza-2′-deoxycytidine (5-AZA-CdR), on miRNA expression, as well as DPC mineralisation and proliferation, were analysed. Both inhibitors increased mineralisation. However, they reduced cell growth. Epigenetically-enhanced mineralisation was accompanied by widespread changes in miRNA expression. Bioinformatic analysis identified many differentially expressed mature miRNAs that were suggested to have roles in mineralisation and stem cell differentiation, including regulation of the Wnt and MAPK pathways. Selected candidate miRNAs were demonstrated by qRT-PCR to be differentially regulated at various time points in mineralising DPC cultures treated with SAHA or 5-AZA-CdR. These data validated the RNA sequencing analysis and highlighted an increased and dynamic interaction between miRNA and epigenetic modifiers during the DPC reparative processes. Full article

Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis and high rates of recurrence and metastasis. The origin and molecular mechanism underlying and driving its clinical and biological behavior remain largely unknown. Recently, we and others have revealed the role of microRNAs, DNA methylation, and histone modifications in contributing to the pathogenesis of uLMS. However, the connection between reversible m⁶A RNA methylation and uLMS pathogenesis remains unclear. In this study, we assessed the role and mechanism of FTO m⁶A RNA demethylase in the pathogenesis of uLMS. Immunohistochemistry analysis revealed that the levels of RNA demethylases FTO and ALKBH5 were aberrantly upregulated in uLMS tissues compared to adjacent myometrium with a significant change by histochemical scoring assessment (p < 0.01). Furthermore, the inhibition of FTO demethylase with its small, potent inhibitor (Dac51) significantly decreased the uLMS proliferation dose-dependently via cell cycle arrest. Notably, RNA-seq analysis revealed that the inhibition of FTO with Dac51 exhibited a significant decrease in cell-cycle-related genes, including several CDK members, and a significant increase in the expression of CDKN1A, which correlated with a Dac51-exerted inhibitory effect on cell proliferation. Moreover, Dac51 treatment allowed the rewiring of several critical pathways, including TNFα signaling, KRAS signaling, inflammation response, G₂M checkpoint, and C-Myc signaling, among others, leading to the suppression of the uLMS phenotype. Moreover, transcription factor (TF) analyses suggested that epitranscriptional alterations by Dac51 may alter the cell cycle-related gene expression via TF-driven pathways and epigenetic networks in uLMS cells. This intersection of RNA methylation and other epigenetic controls and pathways provides a framework to better understand uterine diseases, particularly uLMS pathogenesis with a dysregulation of RNA methylation machinery. Therefore, targeting the vulnerable epitranscriptome may provide an additional regulatory layer for a promising and novel strategy for treating patients with this aggressive uterine cancer. Full article