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RNA (New York, N.Y.) Jan 2020Within the forensic science community, there is a continued push to develop novel tools to aid in criminal investigations. microRNA (miRNA) analysis has been the focus... (Review)
Review
Within the forensic science community, there is a continued push to develop novel tools to aid in criminal investigations. microRNA (miRNA) analysis has been the focus of many researcher's attention in the biomedical field since its discovery in 1993; however, the forensic application of miRNA analysis has only been suggested within the last 10 years and has been gaining considerable traction recently. The primary focus of the forensic application of miRNA analysis has been on body fluid identification to provide confirmatory universal analysis of unknown biological stains obtained from crime scenes or evidence items. There are, however, other forensic applications of miRNA profiling that have shown potential, yet are largely understudied, and warrant further investigation such as organ tissue identification, donor age estimation, and more. This review paper aims to evaluate the current literature and future potential of miRNA analysis within the forensic science field.
Topics: Forensic Anthropology; Forensic Medicine; Forensic Sciences; Humans; MicroRNAs
PubMed: 31658993
DOI: 10.1261/rna.072173.119 -
Proceedings of the National Academy of... Oct 2023The nuclear cleavage of a suboptimal primary miRNA hairpin by the Drosha/DGCR8 complex ("Microprocessor") can be enhanced by an optimal miRNA neighbor, a phenomenon...
The nuclear cleavage of a suboptimal primary miRNA hairpin by the Drosha/DGCR8 complex ("Microprocessor") can be enhanced by an optimal miRNA neighbor, a phenomenon termed cluster assistance. Several features and biological impacts of this new layer of miRNA regulation are not fully known. Here, we elucidate the parameters of cluster assistance of a suboptimal miRNA and also reveal competitive interactions amongst optimal miRNAs within a cluster. We exploit cluster assistance as a functional assay for suboptimal processing and use this to invalidate putative suboptimal substrates, as well as identify a "solo" suboptimal miRNA. Finally, we report complexity in how specific mutations might affect the biogenesis of clustered miRNAs in disease contexts. This includes how an operon context can buffer the effect of a deleterious processing variant, but reciprocally how a point mutation can have a nonautonomous effect to impair the biogenesis of a clustered, suboptimal, neighbor. These data expand our knowledge regarding regulated miRNA biogenesis in humans and represent a functional assay for empirical definition of suboptimal Microprocessor substrates.
Topics: Humans; MicroRNAs; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; Ribonuclease III
PubMed: 37788316
DOI: 10.1073/pnas.2306727120 -
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 -
Aging Dec 2023Response to oncogenic factors like UV, GADD45 family in skin participates in scavenging ROS, DNA repair and cell cycle control. Because of this, the previous study of...
Response to oncogenic factors like UV, GADD45 family in skin participates in scavenging ROS, DNA repair and cell cycle control. Because of this, the previous study of the chronic UVB injury model has found that hsa-miR-300 can conduct intercellular transport by exosomes and target regulation of GADD45B. Whether the hsa-miR-300-GADD45B still regulates tumor development by cell cycle pathway is unclear. Through transcriptomic analysis of primary (n=39) and metastatic (n=102) melanoma, it was confirmed that in metastatic samples, some of the 97 down-regulated genes participate in maintaining skin homeostasis while 42 up-regulated genes were enriched in cancer-related functions. Furthermore, CDKN1A, CDKN2A, CXCR4 and RAD51 in the melanoma pathway, were also differentially expressed between normal skin and melanoma. CDKN1A and CDKN2A were also found to be involved in TP53-dependent cell cycle regulation. In conclusion, it was speculated that CDKN1A, CDKN2A, TP53, GADD45B and hsa-miR-300 may have regulatory relationships. It was demonstrated that there is a bidirectional regulation between hsa-miR-300 and TP53. In addition, miR-300 can regulate CDKN1A by GADD45B/TP53 and promote melanoma growth by accelerating the cell cycle transition from G1/S to G2 phase.
Topics: Humans; Melanoma; Cell Cycle; MicroRNAs; Cell Division; Cell Cycle Checkpoints; GADD45 Proteins; Antigens, Differentiation
PubMed: 38070141
DOI: 10.18632/aging.205276 -
Analytical Cellular Pathology... 2022Osteosarcoma is the most common primary malignant bone tumor in children and adolescents with poor prognosis. MicroRNA-181a-5p (miR-181a-5p) is involved in the...
Osteosarcoma is the most common primary malignant bone tumor in children and adolescents with poor prognosis. MicroRNA-181a-5p (miR-181a-5p) is involved in the progression of various tumors; however, its role and underlying mechanism in osteosarcoma remains unclear. In this study, we found that miR-181a-5p was upregulated in human osteosarcoma cells and tissues. miR-181a-5p mimic significantly promoted, while miR-181a-5p inhibitor blocked the proliferation, colony formation, migration, invasion, and cell cycle progression of osteosarcoma cells. Mechanistically, miR-181a-5p bound to the 3'-untranslational region of phosphatase and tensin homolog (PTEN) and reduced its protein expression, thereby activating protein kinase B (PKB/AKT) pathway. Either PTEN overexpression or AKT inhibition notably blocked the tumor-promoting effects of miR-181a-5p. Moreover, we observed that miR-181a-5p mimic further inhibited growth of human osteosarcoma cells in the presence of adriamycin or cisplatin. Overall, miR-181a-5p promotes osteosarcoma progression via PTEN/AKT pathway and it is a promising therapeutic target to treat osteosarcoma.
Topics: Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Humans; MicroRNAs; Osteosarcoma; PTEN Phosphohydrolase; Proto-Oncogene Proteins c-akt
PubMed: 35310933
DOI: 10.1155/2022/3421600 -
International Journal of Molecular... May 2023microRNAs (miRNAs) play an important role in the pathology of glioblastoma (GBM), which is the most malignant and most common primary malignant brain tumor. miRNAs can...
microRNAs (miRNAs) play an important role in the pathology of glioblastoma (GBM), which is the most malignant and most common primary malignant brain tumor. miRNAs can target multiple genes simultaneously and are considered as potential therapeutic agents or targets. This study aimed to determine the role of miR-3174 in the pathobiology of GBM using both in vitro and in vivo approaches. This is the first study deciphering the role of miR-3174 in GBM. We studied the expression of miR-3174 and found it to be downregulated in a panel of GBM cell lines, GSCs and tissues relative to astrocytes and normal brain tissue. This finding led us to hypothesize that miR-3174 has a tumor-suppressive role in GBM. Exogenous expression of miR-3174 inhibited GBM cell growth and invasion, and hampered the neurosphere formation ability of GSCs. miR-3174 downregulated the expression of multiple tumor-promoting genes including CD44, MDM2, RHOA, PLAU and CDK6. Further, overexpression of miR-3174 reduced tumor volume in nude mice with intracranial xenografts. Immuno-histochemical study of brain sections with intracranial tumor xenografts revealed the pro-apoptotic and anti-proliferative activity of miR-3174. In conclusion, we demonstrated that miR-3174 has a tumor-suppressive role in GBM and could be exploited for therapeutic purposes.
Topics: Animals; Mice; Humans; MicroRNAs; Glioblastoma; Mice, Nude; Genes, Tumor Suppressor; Brain; Cell Proliferation; Brain Neoplasms; Cell Line, Tumor; Gene Expression Regulation, Neoplastic
PubMed: 37298284
DOI: 10.3390/ijms24119326 -
Molecular Metabolism Apr 2020It is well established that the liver-specific miR-122, a bona fide tumor suppressor, plays a critical role in lipid homeostasis. However, its role, if any, in amino...
OBJECTIVE
It is well established that the liver-specific miR-122, a bona fide tumor suppressor, plays a critical role in lipid homeostasis. However, its role, if any, in amino acid metabolism has not been explored. Since glutamine (Gln) is a critical energy and anaplerotic source for mammalian cells, we assessed Gln metabolism in control wild type (WT) mice and miR-122 knockout (KO) mice by stable isotope resolved metabolomics (SIRM) studies.
METHODS
Six-to eight-week-old WT and KO mice and 12- to 15-month-old liver tumor-bearing mice were injected with [U-C,N]-L-Gln, and polar metabolites from the liver tissues were analyzed by nuclear magnetic resonance (NMR) imaging and ion chromatography-mass spectrometry (IC-MS). Gln-metabolism was also assessed in a Gln-dependent hepatocellular carcinoma (HCC) cell line (EC4). Expressions of glutaminases (Gls and Gls2) were analyzed in mouse livers and human primary HCC samples.
RESULTS
The results showed that loss of miR-122 promoted glutaminolysis but suppressed gluconeogenesis in mouse livers as evident from the buildup of C- and/or N-Glu and decrease in glucose-6-phosphate (G6P) levels, respectively, in KO livers. Enhanced glutaminolysis is consistent with the upregulation of expressions of Gls (kidney-type glutaminase) and Slc1a5, a neutral amino acid transporter in KO livers. Both Gls and Slc1a5 were confirmed as direct miR-122 targets by the respective 3'-UTR-driven luciferase assays. Importantly, expressions of Gls and Slc1a5 as well as glutaminase activity were suppressed in a Gln-dependent HCC (EC4) cell line transfected with miR-122 mimic that resulted in decreased C-Gln, C-á-ketoglutarate, C-isocitrate, and C-citrate levels. In contrast, C-phosphoenolpyruvate and C-G6P levels were elevated in cells expressing ectopic miR-122, suggesting enhanced gluconeogenesis. Finally, The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database analysis showed that expression of GLS is negatively correlated with miR-122 in primary human HCCs, and the upregulation of GLS RNA is associated with higher tumor grade. More importantly, patients with higher expressions of GLS or SLC1A5 in tumors exhibited poor survival compared with those expressing lower levels of these proteins.
CONCLUSIONS
Collectively, these results show that miR-122 modulates Gln metabolism both in vitro and in vivo, implicating the therapeutic potential of miR-122 in HCCs that exhibit relatively high GLS levels.
Topics: Animals; Cell Line, Tumor; Glutamine; Humans; Liver; Metabolomics; Mice; Mice, Knockout; Mice, Transgenic; MicroRNAs
PubMed: 32180557
DOI: 10.1016/j.molmet.2020.01.003 -
Iranian Journal of Allergy, Asthma, and... Jun 2021Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by inflammation of the articular tissue. This study aims to evaluate the expression of microRNA...
Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by inflammation of the articular tissue. This study aims to evaluate the expression of microRNA (miR)-146a-5p, miR-24-3p, and miR-125a-5p in the plasma of RA patients and compare them with those of healthy controls to obtain a specific expression profile for earlier diagnosis and assistance in treating patients. This study was performed on 50 RA patients and 50 healthy controls. Five microliters of blood were taken from each patient/control. Plasma RNA was extracted using the Trisol solution. cDNAs were synthesized; using moloney murine leukemia virus (MMLV) and deoxynucleoside triphosphate (dNTP). Real-time PCR was performed using SYBR green kit. The mean expression of miR-146a-5p, miR-24-3p, and miR-125a-5p in the RA group were 8.1±1.9, 6.5±1.2, and 6.8±2.2 and in the healthy group were 4.8±1.6, 3.6±2.2, and 3.4±1.7, respectively. Significant differences were also observed in the mean expression of these three miRNAs in four subgroups of RA patients with different disease activity based on disease activity score 28 (DAS28) (p<0.05). ROC curve analysis showed that miR-146a-5p (AUC=0.8, sensitivity= 96%, specificity=86%), miR-24-3p (AUC=0.7, Sensitivity=95%, Specificity=75%) and miR-125a-5p (AUC=0.71, sensitivity=93%, specificity=84%) could be used as suitable biomarkers for RA diagnosis. Increased expressions of miR-146a-5p, miR-24-3p, and miR-125a-5p in RA patients indicate that the miRNAs are involved in disease incidence and progression, and the measurement of their expression can play an essential role in the diagnosis and treatment of the disease.
Topics: Adult; Arthritis, Rheumatoid; Biomarkers; Case-Control Studies; Circulating MicroRNA; Female; Humans; Male; MicroRNAs; Middle Aged; Predictive Value of Tests; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation
PubMed: 34134454
DOI: 10.18502/ijaai.v20i3.6334 -
Nature Communications Jul 2021Osteoarthritis (OA), the most common aging-related joint disease, is caused by an imbalance between extracellular matrix synthesis and degradation. Here, we discover...
Osteoarthritis (OA), the most common aging-related joint disease, is caused by an imbalance between extracellular matrix synthesis and degradation. Here, we discover that both strands of microRNA-455 (miR-455), -5p and -3p, are up-regulated by Sox9, an essential transcription factor for cartilage differentiation and function. Both miR-455-5p and -3p are highly expressed in human chondrocytes from normal articular cartilage and in mouse primary chondrocytes. We generate miR-455 knockout mice, and find that cartilage degeneration mimicking OA and elevated expression of cartilage degeneration-related genes are observed at 6-months-old. Using a cell-based miRNA target screening system, we identify hypoxia-inducible factor-2α (HIF-2α), a catabolic factor for cartilage homeostasis, as a direct target of both miR-455-5p and -3p. In addition, overexpression of both miR-455-5p and -3p protect cartilage degeneration in a mouse OA model, demonstrating their potential therapeutic value. Furthermore, knockdown of HIF-2α in 6-month-old miR-455 knockout cartilage rescues the elevated expression of cartilage degeneration-related genes. These data demonstrate that both strands of a miRNA target the same gene to regulate articular cartilage homeostasis.
Topics: Animals; Cartilage; Cartilage, Articular; Chondrocytes; Extracellular Matrix; Gene Expression Regulation; Homeostasis; Humans; Hypoxia; Mice; Mice, Knockout; MicroRNAs; Osteoarthritis; SOX9 Transcription Factor; Transcription Factors
PubMed: 34230481
DOI: 10.1038/s41467-021-24460-7 -
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