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BMC Immunology Feb 2023A comprehensive dissection of the role of microRNAs (miRNAs) in gene regulation and subsequent cell functions requires a specific and efficient knockdown or...
BACKGROUND
A comprehensive dissection of the role of microRNAs (miRNAs) in gene regulation and subsequent cell functions requires a specific and efficient knockdown or overexpression of the miRNA of interest; these are achieved by transfecting the cell of interest with a miRNA inhibitor or a miRNA mimic, respectively. Inhibitors and mimics of miRNAs with a unique chemistry and/or structural modifications are available commercially and require different transfection conditions. Here, we aimed to investigate how various conditions affect the transfection efficacy of two miRNAs with high and low endogenous expression, miR-15a-5p and miR-20b-5p respectively, in human primary cells.
RESULTS
MiRNA inhibitors and mimics from two commonly used commercial vendors were employed, i.e., mirVana (Thermo Fisher Scientific) and locked nucleic acid (LNA) miRNA (Qiagen). We systematically examined and optimized the transfection conditions of such miRNA inhibitors and mimics to primary endothelial cells and monocytes using either a lipid-based carrier (lipofectamine) for delivery or an unassisted uptake. Transfection of LNA inhibitors with either phosphodiester (PE)- or phosphorothioate (PS)-modified nucleotide bonds, delivered using a lipid-based carrier, efficiently downregulated the expression levels of miR-15a-5p already 24 h following transfection. MirVana miR-15a-5p inhibitor displayed a less efficient inhibitory effect, which was not improved 48 h following a single transfection or two consecutive transfections. Interestingly, LNA-PS miR-15a-5p inhibitor efficiently reduced the levels of miR-15a-5p when delivered without a lipid-based carrier in both ECs and monocytes. When using a carrier, mirVana and LNA miR-15a-5p and miR-20b-5p mimics showed similar efficiency 48 h following transfection to ECs and monocytes. None of the miRNA mimics effectively induced overexpression of the respective miRNA when given to primary cells without a carrier.
CONCLUSION
LNA miRNA inhibitors efficiently downregulated the cellular expression of miRNA, such as miR-15a-5p. Furthermore, our findings suggest that LNA-PS miRNA inhibitors can be delivered in the absence of a lipid-based carrier, whereas miRNA mimics need the aid of a lipid-based carrier to achieve sufficient cellular uptake.
Topics: Humans; Endothelial Cells; Workflow; MicroRNAs; Gene Expression Regulation
PubMed: 36792999
DOI: 10.1186/s12865-023-00540-9 -
Methods in Molecular Biology (Clifton,... 2021MicroRNAs control plant development and are key regulators of plant responses to biotic and abiotic stresses. Thus, their expression must be carefully controlled since...
MicroRNAs control plant development and are key regulators of plant responses to biotic and abiotic stresses. Thus, their expression must be carefully controlled since both excess and deficiency of a given microRNA may be deleterious to plant cell. MicroRNA expression regulation can occur at several stages of their biogenesis pathway. One of the most important of these regulatory checkpoints is transcription efficiency. mirEX database is a tool for exploration and visualization of plant pri-miRNA expression profiles. It includes results obtained using high-throughput RT-qPCR platform designed to monitor pri-miRNA expression in different miRNA biogenesis mutants and developmental stages of Arabidopsis, barley, and Pellia plants. A step-by-step instruction for browsing the database and detailed protocol for high-throughput RT-qPCR experiments, including list of primers designed for the amplification of pri-miRNAs, are presented.
Topics: Arabidopsis; Gene Expression Regulation, Plant; Hordeum; MicroRNAs
PubMed: 32797451
DOI: 10.1007/978-1-0716-0743-5_5 -
Biology Open Dec 2023MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate gene expression. An important step in miRNA biogenesis occurs when primary miRNAs are bound and cleaved by...
MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate gene expression. An important step in miRNA biogenesis occurs when primary miRNAs are bound and cleaved by the microprocessor to generate precursor miRNAs. Regulation at this step is essential and one such regulator includes m6A RNA methylation, an RNA modification found on primary miRNAs that is installed by METTL3 and bound by hnRNPA2B1. Our lab has recently discovered that the Cajal body marker protein coilin also participates in miRNA biogenesis and hypothesized that coilin may be influencing miRNA biogenesis through m6A RNA methylation. Here we report that coilin suppression reduces m6A on primary Let7a and miR-21. We also found that coilin suppression reduced the protein expression of hnRNPA2B1 and METTL3. We observed an interaction between coilin and ectopically expressed METTL3 and found that coilin suppression reduced the nucleoplasmic portion of METTL3 and blunted ectopic METTL3 phosphorylation. Finally, coilin suppression disrupted the greater METTL3 complex with cofactors METTL14 and WTAP. Collectively, our work has uncovered a role for coilin in mediating m6A RNA methylation and provides an avenue by which coilin participates in miRNA biogenesis.
Topics: Methylation; Phosphorylation; MicroRNAs; Cell Nucleus
PubMed: 38050869
DOI: 10.1242/bio.060116 -
Genome Biology Apr 2021Recent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding...
BACKGROUND
Recent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, our team recently discovered that plant primary transcripts of microRNAs (pri-miRs) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. Here we investigate whether this regulatory feedback loop is present in Drosophila melanogaster.
RESULTS
We perform a survey of ribosome profiling data and reveal that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, we show that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, we show that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes.
CONCLUSION
Taken together, these results reveal that several Drosophila pri-miRs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet undescribed primary-microRNA-encoded peptides in Drosophila and their regulatory potential on genome expression.
Topics: Animals; Drosophila; Female; Gene Expression Profiling; Gene Expression Regulation; Male; MicroRNAs; Mutation; Nucleic Acid Conformation; Open Reading Frames; Peptides; Phenotype; Protein Biosynthesis; RNA Interference; RNA, Long Noncoding
PubMed: 33892772
DOI: 10.1186/s13059-021-02345-8 -
Cellular and Molecular Neurobiology Oct 2023Migraine is a common primary headache disorder, affecting about 14% of the population. Importantly, it was indicated as the second cause of disability globally and the... (Review)
Review
Migraine is a common primary headache disorder, affecting about 14% of the population. Importantly, it was indicated as the second cause of disability globally and the leading cause among young women. Despite the widespread prevalence, migraine remains underdiagnosed and undertreated. The possible solution may be microRNAs-small, non-coding molecules. Until now, multiple studies have shown the great value of microRNA in both the diagnosis and treatment of different human diseases. Furthermore, a significant role in neurological disorders has been suggested. Little research regarding the utility of microRNA in migraine has been conducted, however, the results so far appear to be promising. We performed an electronic article search through PubMed and Embase Database to further explore the topic. After the analysis, according to PRISMA 2020 guidelines, we included 21 studies. The dysregulation was observed in migraine in general, as well as in different types and phases; thus, miRNAs emerge as promising diagnostic biomarkers. Additionally, some studies showed the influence of the intervention with miRNA levels on neuroinflammation and the expression of peptides, which are crucial in migraine pathogenesis. This review aims to summarize the current knowledge about the role of miRNAs in migraine and encourage to further research in this field.Kindly check and confirm the edit made in the title.I checked and confirm.
Topics: Humans; Female; MicroRNAs; Migraine Disorders
PubMed: 37432603
DOI: 10.1007/s10571-023-01387-9 -
Neurochemistry International Nov 2014Chronic pain is a debilitating syndrome caused by a variety of disorders, and represents a major clinical problem because of the lack of adequate medication. In chronic... (Review)
Review
Chronic pain is a debilitating syndrome caused by a variety of disorders, and represents a major clinical problem because of the lack of adequate medication. In chronic pain, massive changes in gene expression are observed in a variety of cells, including neurons and glia, in the overall somatosensory system from the sensory ganglia to the higher central nervous system. The protein expressions of hundreds of genes are thought to be post-transcriptionally regulated by a single type of microRNA in a sequence-specific manner. Recently, critical roles of microRNAs in the pathophysiology of chronic pain have been emerging. Genome-wide screenings of microRNA expression changes have been reported in a variety of painful conditions, including peripheral nerve injury, inflammatory diseases, cancer and spinal cord injury. The data obtained suggest that a wide range of microRNAs change their expressions in individual pain conditions, although the pathological significance of individual microRNAs as causal mediators in distinct pain conditions remains to be revealed for a limited number of microRNAs. Insights into the roles of microRNAs in chronic pain will enhance our understanding of the pathophysiology of chronic pain and allow prompt therapeutic application of microRNA-related drugs against intractable persistent pain.
Topics: Animals; Brain Chemistry; Chronic Pain; Humans; MicroRNAs; Spinal Cord
PubMed: 24905749
DOI: 10.1016/j.neuint.2014.05.010 -
RNA (New York, N.Y.) Nov 2020MicroRNA expression is important for gene regulation and deregulated microRNA expression is often observed in diseases such as cancer. The processing of primary microRNA...
MicroRNA expression is important for gene regulation and deregulated microRNA expression is often observed in diseases such as cancer. The processing of primary microRNA transcripts is an important regulatory step in microRNA biogenesis. Due to low expression level and association with chromatin, primary microRNAs are challenging to study in clinical samples where input material is limited. Here, we present a high-sensitivity targeted method to determine processing efficiency of several hundred primary microRNAs from total RNA that requires relatively few RNA sequencing reads. We validate the method using RNA from HeLa cells and show the applicability to clinical samples by analyzing RNA from normal liver and hepatocellular carcinoma. We identify 24 primary microRNAs with significant changes in processing efficiency from normal liver to hepatocellular carcinoma, among those the highly expressed miRNA-122 and miRNA-21, demonstrating that differential processing of primary microRNAs is occurring and could be involved in disease. With our method presented here we provide means to study pri-miRNA processing in disease from clinical samples.
Topics: Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; HeLa Cells; High-Throughput Nucleotide Sequencing; Humans; Liver Neoplasms; MicroRNAs; Sequence Analysis, RNA
PubMed: 32669295
DOI: 10.1261/rna.076240.120 -
International Journal of Molecular... Jan 2023This review aims to consider retrospectively the available data on the coding properties of pri-microRNAs and the regulatory functions of their open reading frames... (Review)
Review
This review aims to consider retrospectively the available data on the coding properties of pri-microRNAs and the regulatory functions of their open reading frames (ORFs) and the encoded peptides (miPEPs). Studies identifying miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed together with a brief description of the methods to identify pri-miRNA ORFs and the encoded protein products. Generally, miPEPs have been identified in many plant species of several families and in a few animal species. Importantly, molecular mechanisms of the miPEP action are often quite different between flowering plants and metazoan species. Requirement for the additional studies in these directions is highlighted by alternative findings concerning negative or positive regulation of pri-miRNA/miRNA expression by miPEPs in plants and animals. Additionally, the question of how miPEPs are distributed in non-flowering plant taxa is very important for understanding the evolutionary origin of such micropeptides. Evidently, further extensive studies are needed to explore the functions of miPEPs and the corresponding ORFs and to understand the full set of their roles in eukaryotic organisms. Thus, we address the most recent integrative views of different genomic, physiological, and molecular aspects concerning the expression of miPEPs and their possible fine functions.
Topics: Animals; MicroRNAs; Open Reading Frames; Retrospective Studies; Plants; Micropeptides
PubMed: 36768436
DOI: 10.3390/ijms24032114 -
Cells Sep 2022PPARα is a ligand-activated transcription factor that shows protective effects against metabolic disorders, inflammation and apoptosis. Primary biliary cholangitis and...
BACKGROUND
PPARα is a ligand-activated transcription factor that shows protective effects against metabolic disorders, inflammation and apoptosis. Primary biliary cholangitis and primary sclerosing cholangitis result in the intrahepatic accumulation of bile acids that leads to liver dysfunction and damage. Small, non-coding RNAs such as miR-155 and miR-21 are associated with silencing PPARα.
METHODS
The expression of miR-155, miR-21 and PPARα were evaluated using real-time PCR on liver tissue, as well as on human hepatocytes (HepG2) or cholangiocytes (NHCs) following exposure to lipopolysaccharide (LPS), glycodeoxycholic acid (GCDCA), lithocholic acid (LCA) and/or ursodeoxycholic acid (UDCA).
RESULTS
A reduction of PPARα in primary biliary cholangitis (PBC) livers was associated with miR-21 and miR-155 upregulation. Experimental overexpression of either miR-155 or miR-21 inhibited PPARα in hepatocytes, whereas, in cholangiocytes, only miR-21 suppressed PPARα. Both GCDCA and LCA induced the cell type-specific upregulation of miR-155 or miR-21. In HepG2, LPS-induced miR-155 expression was blocked by a cotreatment with UDCA and was associated with PPARα upregulation. In NHC cells, the expression of miR-21 was induced by LPS but did not affect PPARα expression.
CONCLUSIONS
Hepatic PPARα expression is reduced in PBC livers as a likely result of miR-155 overexpression. UDCA effectively reduced both baseline and LPS-induced miR-155 expression, thus preventing the suppression of PPARα.
Topics: Bile Acids and Salts; Glycodeoxycholic Acid; Humans; Ligands; Lipopolysaccharides; Lithocholic Acid; Liver Cirrhosis, Biliary; MicroRNAs; PPAR alpha; Transcription Factors; Ursodeoxycholic Acid
PubMed: 36139455
DOI: 10.3390/cells11182880 -
Cellular Signalling Feb 2024This study delves into the role of FBLN5 in pelvic organ prolapse (POP) and its molecular mechanisms, focusing on the FOSL1/miR-222/MEIS1/COL3A1 axis. Gene relationships...
This study delves into the role of FBLN5 in pelvic organ prolapse (POP) and its molecular mechanisms, focusing on the FOSL1/miR-222/MEIS1/COL3A1 axis. Gene relationships linked to POP were confirmed using bioinformatics databases like GEO and StarBase. Primary human uterosacral ligament fibroblasts (hUSLF) were extracted and subjected to mechanical stretching. Cellular cytoskeletal changes were examined via phalloidin staining, intracellular ROS levels with a ROS kit, cell apoptosis through flow cytometry, and cell senescence using β-galactosidase staining. FBLN5's downstream targets were identified, and the interaction between FOSL1 and miR-222 and miR-222 and MEIS1 were validated using assays. In rat models, the role of FBLN5 in POP was assessed using bladder pressure tests. Results indicated diminished FBLN5 expression in uterine prolapse. Enhanced FBLN5 countered mechanical damage in hUSLF cells by downregulating FOSL1. FOSL1 augmented miR-222, inhibiting MEIS1, which subsequently fostered COL3A1 transcription. In rat models, the absence of FBLN5 exacerbated POP by influencing the FOSL1/miR-222/MEIS1/COL3A1 pathway. FBLN5's protective role likely involves regulating the above axis and boosting COL3A1 expression. Further research is needed to validate the effectiveness and safety of this mechanism in human patients and to propose potential new treatment options.
Topics: Female; Humans; Rats; Animals; Reactive Oxygen Species; Pelvic Organ Prolapse; MicroRNAs; Collagen Type III; Extracellular Matrix Proteins
PubMed: 38056607
DOI: 10.1016/j.cellsig.2023.111000