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Methods in Molecular Biology (Clifton,... 2022Tiny single-stranded noncoding RNAs with size 19-27 nucleotides serve as microRNAs (miRNAs), which have emerged as key gene regulators in the last two decades. miRNAs...
Tiny single-stranded noncoding RNAs with size 19-27 nucleotides serve as microRNAs (miRNAs), which have emerged as key gene regulators in the last two decades. miRNAs serve as one of the hallmarks in regulatory pathways with critical roles in human diseases. Ever since the discovery of miRNAs, researchers have focused on how mature miRNAs are produced from precursor mRNAs. Experimental methods are faced with notorious challenges in terms of experimental design, since it is time consuming and not cost-effective. Hence, different computational methods have been employed for the identification of miRNA sequences where most of them labeled as miRNA predictors are in fact pre-miRNA predictors and provide no information about the putative miRNA location within the pre-miRNA. This chapter provides an update and the current state of the art in this area covering various methods and 15 software suites used for prediction of mature miRNA.
Topics: Computational Biology; Humans; MicroRNAs; RNA Precursors; Software
PubMed: 34432279
DOI: 10.1007/978-1-0716-1170-8_9 -
Trends in Genetics : TIG Apr 2022Alterations in microRNAs (miRNAs) expression are causative in the initiation and progression of human cancers. The molecular events responsible for the widespread... (Review)
Review
Alterations in microRNAs (miRNAs) expression are causative in the initiation and progression of human cancers. The molecular events responsible for the widespread differential expression of miRNAs in malignancy are exemplified by their location in cancer-associated genomic regions, epigenetic mechanisms, transcriptional dysregulation, chemical modifications and editing, and alterations in miRNA biogenesis proteins. The classical miRNA function is synonymous with post-transcriptional repression of target protein genes. However, several studies have reported miRNAs functioning outside this paradigm and some of these novel modes of regulation of gene expression have been implicated in cancers. Here, we summarize key aspects of miRNA involvement in cancer, with a special focus on these lesser-studied mechanisms of action.
Topics: Epigenesis, Genetic; Gene Expression; Humans; MicroRNAs; Neoplasms
PubMed: 34728089
DOI: 10.1016/j.tig.2021.10.002 -
Bioscience Reports Oct 2021MicroRNAs (miRNAs) play important roles in a variety of human diseases, including breast cancer. A number of miRNAs are up- and down-regulated in breast cancer. However,...
MicroRNAs (miRNAs) play important roles in a variety of human diseases, including breast cancer. A number of miRNAs are up- and down-regulated in breast cancer. However, little is known about miRNA similarity and similarity network in breast cancer. Here, a collection of 272 breast cancer-associated miRNA precursors (pre-miRNAs) were utilized to calculate similarities of sequences, target genes, pathways and functions and construct a combined similarity network. Well-characterized miRNAs and their similarity network were highlighted. Interestingly, miRNA sequence-dependent similarity networks were not identified in spite of sequence-target gene association. Similarity networks with minimum and maximum number of miRNAs originate from pathway and mature sequence, respectively. The breast cancer-associated miRNAs were divided into seven functional classes (classes I-VII) followed by disease enrichment analysis and novel miRNA-based disease similarities were found. The finding would provide insight into miRNA similarity, similarity network and disease heterogeneity in breast cancer.
Topics: Biomarkers, Tumor; Breast Neoplasms; Databases, Genetic; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; MicroRNAs
PubMed: 34612484
DOI: 10.1042/BSR20211123 -
International Journal of Molecular... Oct 2023The main complications causing practically 75% of all maternal deaths are severe bleeding, infections, and high blood pressure during pregnancy (preeclampsia (PE) and... (Review)
Review
The main complications causing practically 75% of all maternal deaths are severe bleeding, infections, and high blood pressure during pregnancy (preeclampsia (PE) and eclampsia). The usefulness of ncRNAs as clinical biomarkers has been explored in an extensive range of human diseases including pregnancy-related diseases such as PE. Immunological dysregulation show that the Th1/17:Th2/Treg ratio is "central and causal" to PE. However, there is evidence of the involvement of placenta-expressed miRNAs and lncRNAs in the immunological regulation of crucial processes of placenta development and function during pregnancy. Abnormal expression of these molecules is related to immune physiopathological processes that occur in PE. Therefore, this work aims to describe the importance of miRNAs and lncRNAs in immune dysregulation in PE. Interestingly, multiple ncRNAS are involved in the immune dysregulation of PE participating in type 1 immune response regulation, immune microenvironment regulation in placenta promoting inflammatory factors, trophoblast cell invasion in women with Early-Onset PE (EOPE), placental development, and angiogenesis, promotion of population of M1 and M2, proliferation, invasion, and migration of placental trophoblast cells, and promotion of invasion and autophagy through vias such as PI3K/AKT/mTOR, VEGF/VEGFR1, and TLR9/STAT3.
Topics: Humans; Pregnancy; Female; Placenta; Phosphatidylinositol 3-Kinases; Pre-Eclampsia; RNA, Long Noncoding; MicroRNAs; Trophoblasts
PubMed: 37894897
DOI: 10.3390/ijms242015215 -
Nature Mar 2023Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs). Human DICER (hDICER, also known as DICER1) is specialized for cleaving small...
Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs). Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.
Topics: Humans; Cryoelectron Microscopy; DEAD-box RNA Helicases; MicroRNAs; Mutation; Ribonuclease III; RNA Precursors; RNA, Double-Stranded; Substrate Specificity
PubMed: 36813958
DOI: 10.1038/s41586-023-05723-3 -
Intervirology 2023Herpes simplex virus 1 (HSV-1), an important human pathogen, is capable of latent infection in neurons and productive (lytic) infection in other tissue cells. Once... (Review)
Review
BACKGROUND
Herpes simplex virus 1 (HSV-1), an important human pathogen, is capable of latent infection in neurons and productive (lytic) infection in other tissue cells. Once infected with HSV-1, the immune system of the organism cannot eliminate the virus and carries it lifelong. HSV-1 possesses approximately 150 kb of double-stranded linear genomic DNA and can encode at least 70 proteins and 37 mature microRNAs (miRNAs) derived from 18 precursor miRNAs (pre-miRNAs).
SUMMARY
These HSV-1-encoded miRNAs are widely involved in multiple processes in the life cycle of the virus and the host cell, including viral latent and lytic infection, as well as host cell immune signaling, proliferation, and apoptosis.
KEY MESSAGE
In this review, we focused primarily on recent advances in HSV-1-encoded miRNA expression, function, and mechanism, which may provide new research ideas and feasible research methods systemically and comprehensively.
Topics: Humans; MicroRNAs; Herpesvirus 1, Human; Virus Latency; Signal Transduction
PubMed: 37285807
DOI: 10.1159/000531348 -
Scientific Reports Apr 2021The aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule...
The aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Förster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA-target binding studies.
Topics: Aminoglycosides; Fluorescence Resonance Energy Transfer; Humans; Kinetics; MicroRNAs; Protein Binding; Surface Plasmon Resonance
PubMed: 33931703
DOI: 10.1038/s41598-021-88922-0 -
Cell Reports Dec 2023Argonaute (AGO) proteins execute microRNA (miRNA)-mediated gene silencing. However, it is unclear whether all 4 mammalian AGO proteins (AGO1, AGO2, AGO3, and AGO4) are...
Argonaute (AGO) proteins execute microRNA (miRNA)-mediated gene silencing. However, it is unclear whether all 4 mammalian AGO proteins (AGO1, AGO2, AGO3, and AGO4) are required for miRNA activity. We generate Ago1, Ago3, and Ago4-deficient mice (Ago134) and find AGO1/3/4 to be redundant for miRNA biogenesis, homeostasis, or function, a role that is carried out by AGO2. Instead, AGO1/3/4 regulate the expansion of type 2 immunity via precursor mRNA splicing in CD4 T helper (Th) lymphocytes. Gain- and loss-of-function experiments demonstrate that nuclear AGO3 interacts directly with SF3B3, a component of the U2 spliceosome complex, to aid global mRNA splicing, and in particular the isoforms of the gene Nisch, resulting in a dysregulated Nisch isoform ratio. This work uncouples AGO1, AGO3, and AGO4 from miRNA-mediated RNA interference, identifies an AGO3:SF3B3 complex in the nucleus, and reveals a mechanism by which AGO proteins regulate inflammatory diseases.
Topics: Animals; Mice; Argonaute Proteins; Imidazoline Receptors; Mammals; MicroRNAs; RNA Interference; RNA Precursors; RNA Splicing; RNA, Messenger
PubMed: 38096048
DOI: 10.1016/j.celrep.2023.113515 -
Experimental Eye Research Nov 2023Keratoconus (KC) is a corneal thinning disorder and a leading cause of corneal transplantation worldwide. Exosomes are small, secreted extracellular vesicles...
Keratoconus (KC) is a corneal thinning disorder and a leading cause of corneal transplantation worldwide. Exosomes are small, secreted extracellular vesicles (30-150 nm) that mediate cellular communication via their protein, lipid, and nucleic acid content. We aimed to characterize the exosomes secreted by primary corneal fibroblasts from subjects with or without KC. Using human keratoconus stromal fibroblast cells (HKC, n = 4) and healthy stromal fibroblasts (HCF, n = 4), we collected and isolated exosomes using serial ultracentrifugation. Using nanoparticle tracking analysis (NTA) with ZetaView®, we compared the size and concentration of isolated exosomes. Different exosomal markers were identified and quantified using a transmission electron microscope (TEM) (CD81) and Western blot (CD9 and CD63). Exosomal miRNA profiles were determined by qRT-PCR using Exiqon Human panel I miRNA assays of 368 pre-selected miRNAs. Proteomic profiles were determined using a label-free spectral counting method with mass spectrometry. Differential expression analysis for miRNAs and proteins was done using student's t-test with a significance cutoff of p-value ≤0.05. We successfully characterized exosomes isolated from HCFs using several complementary techniques. We found no significant differences in the size, quantity, or morphology between exosomes secreted by HCFs with or without KC. Expression of CD81 was confirmed by immuno-EM, and expression of CD63 and CD9 with western blots in all exosome samples. We detected the expression of 72-144 miRNAs (threshold cycle Ct < 36) in all exosome samples. In HKC-derived exosome samples, miR-328-3p, miR-532-5p, miR-345-5p, and miR-424-5p showed unique expression, while let-7c-5p and miR-665 have increased expression. Protein profiling identified 157 proteins in at least half of the exosome samples, with 38 known exosomal proteins. We identified 12 up- and 2 down-regulated proteins in HKC-derived exosomes. The proteins are involved in membrane-bounded vesicles, cytoskeletal, calcium binding, and nucleotide binding. These proteins are predicted to be regulated by NRF2, miR-205, and TGF-β1, which are involved in KC pathogenesis. We successfully characterized the HKC-derived exosomes and profiled their miRNA and protein contents, suggesting their potential role in KC development. Further studies are necessary to determine if and how these exosomes with differential protein/miRNA profiles contribute to the pathogenesis of KC.
Topics: Humans; Keratoconus; Exosomes; Proteomics; MicroRNAs; Stromal Cells
PubMed: 37714423
DOI: 10.1016/j.exer.2023.109642 -
Clinical and Translational Medicine Nov 2023MicroRNAs (miRNAs) have been implicated in the pathobiology of preeclampsia, a common hypertensive disorder of pregnancy. In a nested matched case-control cohort within...
BACKGROUND
MicroRNAs (miRNAs) have been implicated in the pathobiology of preeclampsia, a common hypertensive disorder of pregnancy. In a nested matched case-control cohort within the Vitamin D Antenatal Asthma Reduction Trial (VDAART), we previously identified peripheral blood mRNA signatures related to preeclampsia and vitamin D status (≤30 ng/mL) during gestation from 10 to 18 weeks, using differential expression analysis.
METHODS
Using quantitative PCR arrays, we conducted profiling of circulating miRNAs at 10-18 weeks of gestation in the same VDAART cohort to identify differentially expressed (DE) miRNAs associated with preeclampsia and vitamin D status. For the validation of the expression of circulating miRNA signatures in the placenta, the HTR-8/SVneo trophoblast cell line was used. Targets of circulating miRNA signatures in the preeclampsia mRNA signatures were identified by consensus ranking of miRNA-target prediction scores from four sources. The connected component of target signatures was identified by mapping to the protein-protein interaction (PPI) network and hub targets were determined. As experimental validation, we examined the gene and protein expression of IGF1R, one of the key hub genes, as a target of the DE miRNA, miR-182-5p, in response to a miR-182-5p mimic in HTR-8/SVneo cells.
RESULTS
Pregnant women with preeclampsia had 16 circulating DE miRNAs relative to normal pregnancy controls that were also DE under vitamin D insufficiency (9/16 = 56% upregulated, FDR < .05). Thirteen miRNAs (13/16 = 81.3%) were detected in HTR-8/SVneo cells. Overall, 16 DE miRNAs had 122 targets, of which 87 were unique. Network analysis demonstrated that the 32 targets of DE miRNA signatures created a connected subnetwork in the preeclampsia module with CXCL8, CXCL10, CD274, MMP9 and IGF1R having the highest connectivity and centrality degree. In an in vitro validation experiment, the introduction of an hsa-miR-182-5p mimic resulted in significant reduction of its target IGF1R gene and protein expression within HTR-8/SVneo cells.
CONCLUSIONS
The integration of the circulating DE miRNA and mRNA signatures associated preeclampsia added additional insights into the subclinical molecular signature of preeclampsia. Our systems and network biology approach revealed several biological pathways, including IGF-1, that may play a role in the early pathophysiology of preeclampsia. These pathways and signatures also denote potential biomarkers for the early stages of preeclampsia and suggest possible preventive measures.
Topics: Humans; Female; Pregnancy; Transcriptome; Pre-Eclampsia; Circulating MicroRNA; MicroRNAs; Vitamin D; Biomarkers; RNA, Messenger
PubMed: 37905457
DOI: 10.1002/ctm2.1446