RESUMO
Chronic and excessive glucocorticoid (GC) exposure can cause Cushing's syndrome, resulting in fat accumulation in selected body areas. Particularly in the brown adipose tissue (BAT), GC acts negatively, resulting in whitening of the tissue. We hypothesized that dysregulation of microRNAs by GC could be an additional mechanism to explain its negative actions in BAT. Male Wistar rats were divided into two groups: (1) Control sham and (2) GC group that was administered dexamethasone 6.25 mg/200 µL via osmotic pump implantation over 28 days. After this period, the animals were euthanized and BAT tissue was properly stored. Human fat cells treated with dexamethasone were used to translate the experimental results found in animals to human biology. GC-treated rat BAT presented with large lipid droplets, severely impaired thermogenic activation, and reduced glucose uptake measured by 18F-FDG PET/CT. GC exposure induced a reduction in the mitochondrial OXPHOS system and oxygen consumption. MicroRNA profiling of BAT revealed five top-regulated microRNAs and among them miR-21-5p was the most significantly upregulated in GC-treated rats compared to the control group. Although upregulation of miR-21-5p in the tissue, differentiated primary brown adipocytes from GC-treated rats had decreased miR-21-5p levels compared to the control group. To translate these results to the clinic, human brown adipocytes were treated with dexamethasone and miR-21-5p inhibitor. In human brown cells, inhibition of miR-21-5p increased brown adipocyte differentiation and prevented GC-induced glucose uptake, resulting in a lower glycolysis rate. In conclusion, high-dose GC therapy significantly impacts brown adipose tissue function, with a notable association between glucose uptake and miR-21-5p.
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In this study, we constructed engineered exosomes carrying the long non-coding RNA (lncRNA) SVIL-AS1 (SVIL-AS1 Exos), and explored its role and mechanism in lung cancer. After the construction of SVIL-AS1 Exos, their physicochemical characteristics were identified. Then, their function and effect in three different cell lines, A549, HeLa, and HepG2, were detected using western blot, the quantitative reverse transcriptase polymerase chain reaction, flow cytometry, 5-ethynyl-2'-deoxyuridine, and Cell Counting Kit-8 experiments. Finally, a mouse xenograft model was constructed to analyze tumor growth and explore the in vivo utility of SVIL-AS1 Exos using hematoxylin and eosin staining, immunohistochemistry, and the TdT-mediated dUTP nick end labeling assay. The results demonstrated that SVIL-AS1 Exos preferentially targeted A549 lung cancer cells over HeLa and HepG2 cells. SVIL-AS1 Exos promoted apoptosis and inhibited A549 cell proliferation by elevating expression of the lncRNA, SVIL-AS1. In vivo, SVIL-AS1 Exos effectively inhibited the growth of lung cancer A549 cells. Furthermore, SVIL-AS1 Exos suppressed the expression of miR-21-5p and upregulated the expression of caspase-9, indicating that SVIL-AS1 may regulate the development of lung cancer through the miR-21-5p/caspase-9 pathway. In conclusion, the engineered SVIL-AS1 Exos targeted lung cancer cells to inhibit the expression of miR-21-5p, upregulate the expression of caspase-9, and inhibit the development of lung cancer.
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BACKGROUND: MiR-21-5p is a highly expressed microRNA that plays an important role in various cancer-promoting processes, including anchorage-independent growth, invasion, migration metastasis, and drug resistance in lung cancer. Studies indicate that miR-21-5p may contribute to these processes by promoting epithelial-mesenchymal transition (EMT). Ras homolog gene family member B (RhoB), a gene downregulated by miR-21-5p, has also been linked to EMT in lung cancer. However, the role of the miR-21-5p/RhoB axis in EMT regulation in lung adenocarcinoma remains unclear. In this study, we aimed to investigate the regulatory role of the miR-21-5p/RhoB axis in EMT and related in vitro functional characteristics such as migration, invasion, cisplatin resistance, and the formation of tumor spheroids. METHODS AND RESULTS: A549 cells were transfected with the miR-21-5p inhibitor, RhoB siRNA, and their corresponding negative controls. Wound healing, transwell invasion, Methyl thiazole tetrazolium (MTT), and sphere formation assays were also performed to evaluate the migration, invasion, cisplatin resistance, and anchorage-independent growth of A549 cells. RT-qPCR was used to determine the mRNA expression levels of EMT markers. MiR-21-5p knockdown inhibited migration, invasion, cisplatin resistance, and sphere formation while upregulating E-cadherin and downregulating Slug. Furthermore, RhoB silencing restored EMT and related in vitro functional characteristics in A549 cells. CONCLUSIONS: Knockdown of miR-21-5p inhibits EMT and related in vitro functional characteristics by upregulating RhoB, suggesting that miR-21-5p may promote EMT through downregulation of RhoB.
Assuntos
Adenocarcinoma de Pulmão , Movimento Celular , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares , MicroRNAs , Proteína rhoB de Ligação ao GTP , Humanos , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína rhoB de Ligação ao GTP/genética , Proteína rhoB de Ligação ao GTP/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Movimento Celular/genética , Células A549 , Resistencia a Medicamentos Antineoplásicos/genética , Cisplatino/farmacologia , Regulação para Cima/genética , Proliferação de Células/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Invasividade Neoplásica/genética , Caderinas/genética , Caderinas/metabolismoRESUMO
Bladder cancer (BC) is one of the most prevalent malignant tumors worldwide, and the incidence is especially higher in males. Extensive evidence has demonstrated the pivotal role of circular RNAs (circRNAs) in BC progression. However, the exact regulatory mechanism of circRNAs in BC remains incompletely elucidated and warrants further exploration. This study screened a novel circRNA-circPGM5 from thousands of circRNAs by high-throughput sequencing. We found that circPGM5, originating from the PGM5 gene, was significantly lower expressed in BC tissues. Quantitative real-time PCR (qRT-PCR) verified that circPGM5 showed relatively low expression in 50 pairs of BC tissues and EJ and T24 cells. Notably, circPGM5 expression was correlated with stage, grade, and lymphatic metastasis of BC. Through RNA-FISH assay, we confirmed that circPGM5 predominantly localized in the cytoplasm. Functionally, overexpression of circPGM5 inhibited the proliferation, migration, and invasion of BC cells in vitro. Remarkably, circPGM5 demonstrated markedly significant tumor growth and metastasis suppression in vivo. Mechanistically, we discovered that circPGM5 upregulated the mitogen-activated protein kinase 10 (MAPK10) expression by influencing the oncogenic miR-21-5p activity through miR-21-5p absorption. This modulation of MAPK10 impacted the phosphorylation of the tumor suppressor Foxo3a in BC. In conclusion, our findings uncovered the tumor-suppressing role of circPGM5 in BC via the miR-21-5p/MAPK10/Foxo3a axis.
Assuntos
Proliferação de Células , Proteína Forkhead Box O3 , MicroRNAs , RNA Circular , Neoplasias da Bexiga Urinária , Humanos , MicroRNAs/metabolismo , MicroRNAs/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Proteína Forkhead Box O3/metabolismo , Proteína Forkhead Box O3/genética , RNA Circular/genética , RNA Circular/metabolismo , Fosforilação , Linhagem Celular Tumoral , Masculino , Animais , Camundongos , Camundongos Nus , Regulação Neoplásica da Expressão Gênica , Progressão da Doença , Feminino , Movimento Celular , Pessoa de Meia-Idade , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND AND AIMS: Exosome-based therapies are gaining increasing attention, with growing evidence suggesting a link between alterations in mesentery adipose tissue (MAT) and intestinal disease in Crohn's disease (CD). However, the specific mechanism by which mesenchymal stem cells (MSCs)-Exos may alleviate colitis through targeting MAT remains not fully understood. METHODS: Human umbilical cord MSCs (HucMSCs) were cultured to isolate the corresponding exosomes (HucMSCs-Exos), which were confirmed by their morphology, size distribution, and expression of markers. In vivo, 2,4,6-trinitrobenzenesulfonic acid solution (TNBS) and dextran sodium sulfate (DSS) -induced mouse colitis models were used to detect the therapeutic effects of HucMSCs-Exos. ELISA, qRT-PCR, western blotting, and immunofluorescence determined the expression of key molecules. Luciferase reporter assay was used to confirm the relationship between miR-21-5p and SPRY2. RESULTS: Exosomes treatment through mesenteric injection demonstrated therapeutic effects on mesenteric inflammation and colitis. These therapeutic benefits were contingent on macrophages, significantly facilitating the M2 polarization of mesenteric macrophages. The expression data from GSE159814 and GSE211008 revealed that exosomal miR-21-5p was enriched in HucMSCs-Exos and could be delivered to macrophages. Additionally, the results indicated that miR-21-5p could directly target the 3'UTR of SPRY2 and activate the phosphorylation of ERK to modify macrophage phenotypes. Mechanistically, exosomal miR-21-5p derived from HucMSCs could promote macrophage M2 polarization via the SPRY2/ERK axis. CONCLUSION: Mesenteric injection of HucMSCs-Exos significantly alleviates mesenteric inflammation and colitis by promoting mesenteric macrophage M2 polarization, making it a promising approach to treat colitis and suggesting therapeutic potential role of exosomal miR-21-5p in CD.
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The toxicity of silica nanoparticles (SiNPs) to lung is known. We previously demonstrated that exposure to SiNPs promoted pulmonary impairments, but the precise pathogenesis remains elucidated. Ferroptosis has now been identified as a unique form of oxidative cell death, but whether it participated in SiNPs-induced lung injury remains unclear. In this work, we established a rat model with sub-chronic inhalation exposure of SiNPs via intratracheal instillation, and conducted histopathological examination, iron detection, and ferroptosis-related lipid peroxidation and protein assays. Moreover, we evaluated the effect of SiNPs on epithelial ferroptosis, possible mechanisms using in vitro-cultured human bronchial epithelial cells (16HBE), and also assessed the ensuing impact on fibroblast activation for fibrogenesis. Consequently, fibrotic lesions occurred in the rat lungs, concomitantly by enhanced lipid peroxidation, iron overload, and ferroptosis. Consistently, the in vitro data showed SiNPs triggered oxidative stress and caused the accumulation of lipid peroxides, resulting in ferroptosis. Importantly, the mechanistic investigation revealed miR-21-5p as a key player in the epithelial ferroptotic process induced by SiNPs via targeting GCLM for GSH depletion. Of note, ferrostatin-1 could greatly suppress ferroptosis and alleviate epithelial injury and ensuing fibroblast activation by SiNPs. In conclusion, our findings first revealed SiNPs triggered epithelial ferroptosis through miR-21-5p/GCLM signaling and thereby promoted fibroblast activation for fibrotic lesions, and highlighted the therapeutic potential of inhibiting ferroptosis against lung impairments upon SiNPs exposure.
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Células Epiteliais , Ferroptose , Pulmão , MicroRNAs , Nanopartículas , Transdução de Sinais , Dióxido de Silício , Ferroptose/efeitos dos fármacos , Animais , Humanos , MicroRNAs/metabolismo , MicroRNAs/genética , Ratos , Nanopartículas/química , Transdução de Sinais/efeitos dos fármacos , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Masculino , Glutamato-Cisteína Ligase/metabolismo , Glutamato-Cisteína Ligase/genética , Linhagem Celular , Ratos Sprague-Dawley , Estresse Oxidativo/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Glutationa/metabolismo , Cicloexilaminas/farmacologia , FenilenodiaminasRESUMO
Sepsis is a systemic inflammatory response syndrome resulting from the invasion of the human body by bacteria and other pathogenic microorganisms. One of its most prevalent complications is acute lung injury, which places a significant medical burden on numerous countries and regions due to its high morbidity and mortality rates. MicroRNA (miRNA) plays a critical role in the body's inflammatory response and immune regulation. Recent studies have focused on miR-21-5p in the context of acute lung injury, but its role appears to vary in different models of this condition. In the LPS-induced acute injury model of A549 cells, there is differential expression, but the specific mechanism remains unclear. Therefore, our aim is to investigate the changes in the expression of miR-21-5p and SLC16A10 in a type II alveolar epithelial cell injury model induced by LPS and explore the therapeutic effects of their targeted regulation. A549 cells were directly stimulated with 10 µg/ml of LPS to construct a model of LPS-induced cell injury. Cells were collected at different time points and the expression of interleukin 1 beta (IL-1ß), tumor necrosis factor-α (TNF-α) and miR-21-5p were measured by RT-qPCR and western blot. Then miR-21-5p mimic transfection was used to up-regulate the expression of miR-21-5p in A549 cells and the expression of IL-1ß and TNF-α in each group of cells was measured by RT-qPCR and western blot. The miRDB, TargetScan, miRWalk, Starbase, Tarbase and miR Tarbase databases were used to predict the miR-21-5p target genes and simultaneously, the DisGeNet database was used to search the sepsis-related gene groups. The intersection of the two groups was taken as the core gene. Luciferase reporter assay further verified SLC16A10 as the core gene with miR-21-5p. The expression of miR-21-5p and SLC16A10 were regulated by transfection or inhibitors in A549 cells with or without LPS stimulation. And then the expression of IL-1ß and TNF-α in A549 cells was tested by RT-qPCR and western blot in different groups, clarifying the role of miR-21-5p-SLC16A10 axis in LPS-induced inflammatory injury in A549 cells. (1) IL-1ß and TNF-α mRNA and protein expression significantly increased at 6, 12, and 24 h after LPS stimulation as well as the miR-21-5p expression compared with the control group (P < 0.05). (2) After overexpression of miR-21-5p in A549 cells, the expression of IL-1ß and TNF-α was significantly reduced after LPS stimulation, suggesting that miR-21-5p has a protection against LPS-induced injury. (3) The core gene set, comprising 51 target genes of miR-21-5p intersecting with the 1448 sepsis-related genes, was identified. This set includes SLC16A10, TNPO1, STAT3, PIK3R1, and FASLG. Following a literature review, SLC16A10 was selected as the ultimate target gene. Dual luciferase assay results confirmed that SLC16A10 is indeed a target gene of miR-21-5p. (4) Knocking down SLC16A10 expression by siRNA significantly reduced the expression of IL-1ß and TNF-α in A549 cells after LPS treatment (P < 0.05). (5) miR-21-5p inhibitor increased the expression levels of IL-1ß and TNF-α in A549 cells after LPS stimulation (P < 0.05). In comparison to cells solely transfected with miR-21-5p inhibitor, co-transfection of miR-21-5p inhibitor and si-SLC6A10 significantly reduced the expression of IL-1ß and TNF-α (P < 0.05). MiR-21-5p plays a protective role in LPS-induced acute inflammatory injury of A549 cells. By targeting SLC16A10, it effectively mitigates the inflammatory response in A549 cells induced by LPS. Furthermore, SLC16A10 holds promise as a potential target for the treatment of acute lung injury.
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Lesão Pulmonar Aguda , Células Epiteliais Alveolares , Sistemas de Transporte de Aminoácidos Neutros , MicroRNAs , Humanos , Células A549 , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Regulação da Expressão Gênica , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Lipopolissacarídeos/toxicidade , MicroRNAs/genética , MicroRNAs/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismoRESUMO
The potential function and mechanism of circRNAs in regulating malignant performances of Osteosarcoma (OS) cells have not been well investigated. The expression level of CircLMO7, miR-21-5p and ARHGAP24 were detected by RT-qPCR. The relationship between miR-21-5p and circ-LMO7, as well as between miR-21-5p and ARHGAP24, was predicted and examined through bioinformatics analysis and luciferase reporter gene experiments. Moreover, OS cell growth, invasion, migration, and apoptosis were detected using the cell counting kit-8 (CCK-8), transwell and flow cytometry assays, respectively. ARHGAP24 protein level was measured using western blotting. In present study, we choose to investigate the role and mechanism of circ-LOM7 on OS cell proliferation, migration and invasion. circ-LOM7 was found to be down-regulated in OS tissues and cell lines. Enforced expression of circ-LOM7 suppressed the growth, invasion, and migration of OS cells. In contrast, decreasing circ-LMO7 expression had opposite effects. Furthermore, miR-21-5p was predicted to be sponged by circ-LMO7, and had an opposite role of circ-LMO7 in OS. Moreover, ARHGAP24 served as miR-21-5p's downstream target. Mechanistically, circ-LMO7 was packed in exosomes and acted as a cancer-suppresser on OS by sponging miR-21-5p and upregulating the expression of ARHGAP24. The exosomal circ-LMO7 expression was significantly decreased in OS cell exosomes, and co-culture experiments showed that exosomal circ-LMO7 suppressed the proliferation ability of OS cells. Circ-LMO7 exerts as a tumor suppressor in OS, and the circ-LMO7/miR-21-5P/ARHGAP24 axis is involved in OS progression.
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Progressão da Doença , Exossomos , Proteínas Ativadoras de GTPase , MicroRNAs , Osteossarcoma , RNA Circular , Osteossarcoma/genética , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Exossomos/metabolismo , Exossomos/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Proliferação de Células , Camundongos , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Apoptose/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Regulação Neoplásica da Expressão Gênica , Masculino , FemininoRESUMO
INTRODUCTION: Cerebral ischemia (CI) induces a profound neuroinflammatory response, but the underlying molecular mechanism remains unclear. Exosomes from adipose-derived stem cells (ADSC-exos) have been found to play a crucial role in cell communication by transferring molecules including microRNAs (miRNAs), which have been shown to modulate the inflammatory response after CI and are viable molecular targets for altering brain function. The current study aimed to explore the contribution of ADSC-exosomal miR-21-5p to the neuroinflammation after CI. METHODS: The differentially expressed miR-21-5p in CI was screened based on literature search. The target mRNAs of miR-21-5p were predicted using online databases and verified by luciferase reporter assay. Then, BV2 cells were treated with hemin to simulate the inflammatory response after CI, and its animal model was induced using the MCAO method. Ischemia was evaluated in rats using 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining. ADSCs-exos were further isolated and identified by western blot analysis and transmission electron microscope. RESULTS: MiR-21-5p was significantly down-regulated in CI and alleviated neuropathic damage after CI by the PIK3R1/PI3K/AKT signaling axis. And miR-21-5p derived from ADSCs-exos alleviated neuroinflammation after CI via promoting microglial M2 polarization. CONCLUSION: We demonstrated that ADSC-exosomal miR-21-5p mitigated post-CI inflammatory response through the PIK3R1/PI3K/AKT signaling axis and could offer neuroprotection after CI through promoting polarization of M2 microglia.
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Modelos Animais de Doenças , Exossomos , MicroRNAs , Proteínas Proto-Oncogênicas c-akt , Ratos Sprague-Dawley , Transdução de Sinais , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Exossomos/metabolismo , Exossomos/transplante , Proteínas Proto-Oncogênicas c-akt/metabolismo , Masculino , Humanos , Linhagem Celular , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/genética , Doenças Neuroinflamatórias/metabolismo , Microglia/metabolismo , Microglia/patologia , Camundongos , Tecido Adiposo/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Pessoa de Meia-Idade , Regulação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
BACKGROUND: Endothelial cell (EC)-driven intraneural revascularization (INRV) and Schwann cells-derived exosomes (SCs-Exos) both play crucial roles in peripheral nerve injury (PNI). However, the interplay between them remains unclear. We aimed to elucidate the effects and underlying mechanisms of SCs-Exos on INRV following PNI. RESULTS: We found that GW4869 inhibited INRV, as well as that normoxic SCs-Exos (N-SCs-Exos) exhibited significant pro-INRV effects in vivo and in vitro that were potentiated by hypoxic SCs-Exos (H-SCs-Exos). Upregulation of glycolysis emerged as a pivotal factor for INRV after PNI, as evidenced by the observation that 3PO administration, a glycolytic inhibitor, inhibited the INRV process in vivo and in vitro. H-SCs-Exos more significantly enhanced extracellular acidification rate/oxygen consumption rate ratio, lactate production, and glycolytic gene expression while simultaneously suppressing acetyl-CoA production and pyruvate dehydrogenase E1 subunit alpha (PDH-E1α) expression than N-SCs-Exos both in vivo and in vitro. Furthermore, we determined that H-SCs-Exos were more enriched with miR-21-5p than N-SCs-Exos. Knockdown of miR-21-5p significantly attenuated the pro-glycolysis and pro-INRV effects of H-SCs-Exos. Mechanistically, miR-21-5p orchestrated EC metabolism in favor of glycolysis by targeting von Hippel-Lindau/hypoxia-inducible factor-1α and PDH-E1α, thereby enhancing hypoxia-inducible factor-1α-mediated glycolysis and inhibiting PDH-E1α-mediated oxidative phosphorylation. CONCLUSION: This study unveiled a novel intrinsic mechanism of pro-INRV after PNI, providing a promising therapeutic target for post-injury peripheral nerve regeneration and repair.
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Células Endoteliais , Exossomos , Glicólise , Traumatismos dos Nervos Periféricos , Células de Schwann , Células de Schwann/metabolismo , Exossomos/metabolismo , Animais , Células Endoteliais/metabolismo , Camundongos , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/terapia , Masculino , Ratos , MicroRNAs/metabolismo , MicroRNAs/genética , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Ratos Sprague-Dawley , Compostos de Anilina , Compostos de BenzilidenoRESUMO
Developing effective methods for alveolar bone defect regeneration is a significant challenge in orthopedics. Exosomes from human umbilical cord mesenchymal stem cells (HUMSC-Exos) have shown potential in bone repair but face limitations due to undefined application methods and mechanisms. To address this, HUMSC-Exos were encapsulated in polyvinyl alcohol (PVA) hydrogel (Exo@PVA) to create a novel material for alveolar bone repair. This combination enhanced the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) more effectively than Exos alone. Additionally, Exo@PVA significantly improved alveolar bone regeneration and defect repair in rats. The microRNA-21-5p (miR-21-5p) in Exo@PVA, identified through the GEO database and analyzed via in silico methods, played a crucial role. miR-21-5p promoted BMSC osteogenic differentiation by inhibiting WWP1-mediated KLF5 ubiquitination and enhanced HUVEC angiogenesis by targeting ATP2B4. These findings underscore the potential of an Exo-based approach with PVA hydrogel scaffolds for bone defect repair, operating through the miR-21-5p/WWP1/ATP2B4 signaling axis.
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Regeneração Óssea , Diferenciação Celular , Exossomos , Células Endoteliais da Veia Umbilical Humana , Células-Tronco Mesenquimais , MicroRNAs , Neovascularização Fisiológica , Osteogênese , Álcool de Polivinil , Cordão Umbilical , Humanos , Álcool de Polivinil/química , Osteogênese/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Regeneração Óssea/efeitos dos fármacos , Exossomos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Cordão Umbilical/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Ratos , Animais , Neovascularização Fisiológica/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Masculino , Hidrogéis/química , Hidrogéis/farmacologia , Ratos Sprague-Dawley , AngiogêneseRESUMO
Extracellular vesicles (EVs) have recently received increasing attention as essential mediators of communication between tumor cells and their microenvironments. Tumor-associated macrophages (TAMs) play a proangiogenic role in various tumors, especially head and neck squamous cell carcinoma (HNSCC), and angiogenesis is closely related to tumor growth and metastasis. This research focused on exploring the mechanisms by which EVs derived from TAMs modulate tumor angiogenesis in HNSCC. Our results indicated that TAMs infiltration correlated positively with microvascular density in HNSCC. Then we collected and identified EVs from TAMs. In the microfluidic chip, TAMs derived EVs significantly enhanced the angiogenic potential of pHUVECs and successfully induced the formation of perfusable blood vessels. qPCR and immunofluorescence analyses revealed that EVs from TAMs transferred miR-21-5p to endothelial cells (ECs). And targeting miR-21-5p of TAMs could effectively inhibit TAM-EVs induced angiogenesis. Western blot and tube formation assays showed that miR-21-5p from TAM-EVs downregulated LATS1 and VHL levels but upregulated YAP1 and HIF-1α levels, and the inhibitors of YAP1 and HIF-1α could both reduce the miR-21-5p enhanced angiogenesis in HUVECs. The in vivo experiments further proved that miR-21-5p carried by TAM-EVs promoted the process of tumor angiogenesis via YAP1/HIF-1α axis in HNSCC. Conclusively, TAM-derived EVs transferred miR-21-5p to ECs to target the mRNA of LATS1 and VHL, which inhibited YAP1 phosphorylation and subsequently enhanced YAP1-mediated HIF-1α transcription and reduced VHL-mediated HIF-1α ubiquitination, contributing to angiogenesis in HNSCC. These findings present a novel regulatory mechanism of tumor angiogenesis, and miR-21-5p/YAP1/HIF-1α might be a potential therapeutic target for HNSCC.
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Exossomos , Neoplasias de Cabeça e Pescoço , MicroRNAs , Carcinoma de Células Escamosas de Cabeça e Pescoço , Microambiente Tumoral , Humanos , Angiogênese , Células Endoteliais , Neoplasias de Cabeça e Pescoço/genética , MicroRNAs/genética , Proteínas Serina-Treonina Quinases , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Macrófagos Associados a Tumor , Exossomos/metabolismo , Animais , CamundongosRESUMO
OBJECTIVE: In this study, we investigated the impact of perioperative administration of Bifidobacterium triplex viable capsules on the serum levels of circulating miR-21-5p, miR-135-5p, and miR-155-5p in patients with colorectal cancer (CRC). The purpose of this study is to provide a foundation for future research on the use of Bifidobacterium triplex viable capsules to enhance postoperative recovery in patients with CRC. METHODS: A total of 60 patients with primary CRC admitted to the Department of General Surgery at Shanxi Bethune Hospital between June 2020 and December 2020 were selected and randomly divided into two groups: 20 cases in the control group and 40 cases in the experimental group. The experimental group was administered oral Bifidobacterium triplex viable capsules during the perioperative period, while the control group was administered oral placebo. Before and after the perioperative period, the expression levels of miR-21-5p, miR-135-5p, and miR-155-5p were compared in the serum of both groups of patients. Furthermore, we established the prognostic value of these three miRNAs in CRC patients. RESULTS: After surgery, the expression levels of miR-21-5p, miR-135-5p, and miR-155-5p decreased in both groups of patients (P < 0.05). Significantly greater differences were observed between miR-21-5p and miR-135-5p (P < 0.001). Expression levels of serum miR-21-5p (P = 0.020) and miR-135-5p (P = 0.023) decreased significantly more in the experimental group than in the control group. The levels of the above three miRNAs after surgery did not correlate with 3-year OS (HR = 4.21; 95% CI 0.37-47.48; log-rank P = 0.20) or 3-year DFS (HR = 1.57; 95% CI 0.32-7.66; log-rank P = 0.55) in two groups. CONCLUSION: Radical surgery reduces the levels of serum miR-21-5p, miR-135-5p, and miR-155-5p expression in patients with CRC. The use of Bifidobacterium triplex viable capsules assists in achieving quicker perioperative recovery from radical surgery in CRC patients, and this underlying mechanism may be associated with the regulation of serum miR-21-5p, miR-135-5p, and miR-155-5p expression levels.
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Neoplasias Colorretais , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Prognóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/cirurgia , Regulação Neoplásica da Expressão GênicaRESUMO
The increased risk of fractures in patients with type 1 diabetes mellitus (T1DM) is nowadays well recognized. However, the exact mechanism of action of diabetic bone disease has not been fully elucidated. MicroRNAs (miRNAs) are gene regulators that operate post-transcriptionally and have been implicated in the development of various metabolic disorders including T1DM. Previous studies have implicated a role for miR-144-5p and miR-21-5p, which are involved in controlling oxidative stress by targeting Nrf2, in T1DM. To date, it is unclear whether miR-144-5p and miR-21-5p affect bone health in T1DM. Thus, this study aimed to investigate the influence of miR-144-5p and miR-21-5p knockdown in the development of bone disease in T1DM male mice. Therefore, T1DM was induced in 10-wk-old male mice using streptozotocin (STZ). One week later, after development of hyperglycemia, antagomir-144-5p and antagomir-21-5p or their non-targeting control were administered at 10 mg/kg BW once a week until the end of the experiment. At 14 wk of age, glucose levels, bone, and fat mass were analyzed. The results revealed that treating T1DM male mice with antagomir-144-5p and antagomir-21-5p did not protect against diabetes development or bone loss, despite the successful downregulation of the miRNAs and the normalization of Nrf2 mRNA levels in bone tissue. Histological and serological parameters of bone formation or resorption were not altered by the antagomir treatment. Finally, we measured the expression of miRNA-144-5p or miRNA-21-5p in the serum of 30 individuals with T1DM and compared them to non-diabetic controls, but did not find an altered expression of either miRNA. In conclusion, the knockdown of miR-144-5p and miR-21-5p does not affect STZ-induced diabetes development or loss of bone mass in male mice. However, it does normalize expression of the anti-oxidant factor Nrf2 in diabetic bone tissue.
RESUMO
OBJECTIVES: Trigeminal neuralgia (TN) is a common neuropathic pain. Voltage-gated potassium channel (Kv) has been confirmed to be involved in the occurrence and development of TN, but the specific mechanism is still unclear. MicroRNA may be involved in neuropathic pain by regulating the expression of Kv channels and neuronal excitability in trigeminal ganglion (TG). This study aims to explore the relationship between Kv1.1 and miR-21-5p in TG with a TN model, evaluate whether miR-21-5p has a regulatory effect on Kv1.1, and to provide a new target and experimental basis for the treatment of TN. METHODS: A total of 48 SD rats were randomly divided into 6 groups: 1) a sham group (n=12), the rats were only sutured at the surgical incision without nerve ligation; 2) a sham+agomir NC group (n=6), the sham rats were microinjected with agomir NC through stereotactic brain injection in the surgical side of TG; 3) a sham+miR-21-5p agomir group (n=6), the sham rats were microinjected with miR-21-5p agomir via stereotactic brain injection in the surgical side of TG; 4) a TN group (n=12), a TN rat model was constructed using the chronic constriction injury of the distal infraorbital nerve (dIoN-CCI) method with chromium intestinal thread; 5) a TN+antagonist NC group (n=6), TN rats were microinjected with antagonist NC through stereotactic brain injection method in the surgical side of TG; 6) a TN+miR-21-5p antagonist group (n=6), TN rats were microinjected with miR-21-5p antagonist through stereotactic brain injection in the surgical side of TG. The change of mechanical pain threshold in rats of each group after surgery was detected. The expressions of Kv1.1 and miR-21-5p in the operative TG of rats were detected by Western blotting and real-time reverse transcription polymerase chain reaction. Dual luciferase reporter genes were used to determine whether there was a target relationship between Kv1.1 and miR-21-5p and whether miR-21-5p directly affected the 3'-UTR terminal of KCNA1. The effect of brain stereotaxic injection was evaluated by immunofluorescence assay, and then the analogue of miR-21-5p (agomir) and agomir NC were injected into the TG of rats in the sham group by brain stereotaxic apparatus to overexpress miR-21-5p. The miR-21-5p inhibitor (antagomir) and antagomir NC were injected into TG of rats in the TN group to inhibit the expression of miR-21-5p. The behavioral changes of rats before and after administration were observed, and the expression changes of miR-21-5p and Kv1.1 in TG of rats after intervention were detected. RESULTS: Compared with the baseline pain threshold, the facial mechanical pain threshold of rats in the TN group was significantly decreased from the 5th to 15th day after the surgery (P<0.05), and the facial mechanical pain threshold of rats in the sham group was stable at the normal level, which proved that the dIoN-CCI model was successfully constructed. Compared with the sham group, the expression of Kv1.1 mRNA and protein in TG of the TN group was down-regulated (both P<0.05), and the expression of miR-21-5p was up-regulated (P<0.05). The results of dual luciferase report showed that the luciferase activity of rno-miR-21-5p mimics and KCNA1 WT transfected with 6 nmol/L or 20 nmol/L were significantly decreased compared with those transfected with mimic NC and wild-type KCNA1 WT, respectively (P<0.001). Compared with low dose rno-miR-21-5p mimics (6 nmol/L) co-transfection group, the relative activity of luciferase in the high dose rno-miR-21-5p mimics (20 nmol/L) cotransfection group was significantly decreased (P<0.001). The results of immunofluorescence showed that drugs were accurately injected into TG through stereotaxic brain. After the expression of miR-21-5p in the TN group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were increased. After overexpression of miR-21-5p in the sham group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were decreased. CONCLUSIONS: Both Kv1.1 and miR-21-5p are involved in TN and miR-21-5p can regulate Kv1.1 expression by binding to the 3'-UTR of KCNA1.
Assuntos
Canal de Potássio Kv1.1 , MicroRNAs , Neuralgia , Neuralgia do Trigêmeo , Animais , Ratos , Antagomirs , Regulação para Baixo , Luciferases , MicroRNAs/genética , Neuralgia/genética , Ratos Sprague-Dawley , RNA Mensageiro , Neuralgia do Trigêmeo/genética , Canal de Potássio Kv1.1/genéticaRESUMO
Follicular cysts are a common reproductive disorder in domestic animals that cause considerable economic losses to the farming industry. Effective prevention and treatment methods are lacking because neither the pathogenesis nor formation mechanisms of follicular cysts are well-understood. In this study, we first investigated the granulosa cells (GCs) of cystic follicles isolated from pigs. We observed a significant reduction in the expression of methyltransferase-like 3 (METTL3). Subsequent experiments revealed that METTL3 downregulation in GCs caused a decrease in m6A modification of pri-miR-21. This reduction further inhibited DGCR8 recognition and binding to pri-miR-21, dampening the synthesis of mature miR-21-5p. Additionally, the decrease in miR-21-5p promotes IL-1ß expression in GCs. Elevated IL-1ß activates the NFκB pathway, in turn upregulating apoptotic genes TNFa and BAX/BCL2. The subsequent apoptosis of GCs and inhibition of autophagy causes downregulation of CYP19A1 expression. These processes lower oestrogen secretion and contribute to follicular cyst formation. In conclusion, our findings provide a foundation for understanding and further exploring the mechanisms of follicular-cyst development in farm animals. This work has important implications for treating ovarian disorders in livestock and could potentially be extended to humans.
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Apoptose , Células da Granulosa , Metiltransferases , MicroRNAs , Animais , Feminino , Apoptose/genética , Células Cultivadas , Regulação para Baixo , Cisto Folicular/genética , Cisto Folicular/patologia , Cisto Folicular/metabolismo , Células da Granulosa/metabolismo , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Metiltransferases/metabolismo , Metiltransferases/genética , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/metabolismo , NF-kappa B/genética , Transdução de Sinais , Suínos , Proteínas de Ligação a RNA/metabolismoRESUMO
Bone marrow mesenchymal stem cell (BMSC)-derived exosomes (BMSC-Exos) have a variety of biological functions and are extensively involved in the regulation of inflammatory diseases, as well as tissue repair and regeneration. However, the mechanism of action of these compounds in dry eye disease (DED) in mice is still unclear. This study demonstrated that the Treg/Th17 ratio was strongly imbalanced in DED clinical samples. BMSC-Exos can modulate the Treg/Th17 balance, improve the integrity of the corneal epithelial layer, and ameliorate DED progression in mice. Mechanistically, BMSC-Exos dramatically decreased the levels of IL-17 and IL-22; increased the levels of IL-4, IL-10, and TGF-ß1; and increased tear secretion and the number of goblet cells in the conjunctiva in mice, thus alleviating the progression of DED. This effect is achieved by BMSC-Exos through the delivery of miR-21-5p to target and restrain TLR4, thereby restraining the MyD88/NF-κB pathway. Our study showed that the upregulation of miR-21-5p in BMSC-Exos may be a therapeutic target for DED. These findings support new ideas and a basis for treating DED, as well as for further study of the application value of exosomes in alleviating DED.
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Síndromes do Olho Seco , Exossomos , Células-Tronco Mesenquimais , MicroRNAs , Fator 88 de Diferenciação Mieloide , NF-kappa B , Transdução de Sinais , Linfócitos T Reguladores , Células Th17 , Receptor 4 Toll-Like , Animais , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Exossomos/metabolismo , Exossomos/transplante , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Síndromes do Olho Seco/terapia , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Células Th17/metabolismo , Células Th17/imunologia , NF-kappa B/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/imunologia , Camundongos , Humanos , Camundongos Endogâmicos C57BL , Masculino , FemininoRESUMO
RESEARCH QUESTION: What is the effect of micro-RNA (miR)-21-5p-loaded bone marrow mesenchymal stem cell-derived exosomes (miR-21-Exo) on autoimmune premature ovarian insufficiency (POI)? DESIGN: The Cell Counting Kit 8 (CCK8) assay, fluorescence-activated cell sorting, western blotting, quantitative reverse transcriptase (qRT)-PCR and enzyme-linked immunosorbent assay (ELISA) verified the effect of miR-21-Exo on interferon-γ (IFN-γ)-induced KGN cells. qRT-PCR, western blotting and dual-luciferase reporter gene assays verified that miR-21-Exo mediated Msh homeobox 1 (MSX1) regulation of the Notch signalling pathway and that miR-21 interacted directly with MSX1. The effects of miR-21-Exo on the ovaries were verified by monitoring of the oestrous cycle, haematoxylin and eosin staining, follicle counts, ELISA, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), western blotting and qRT-PCR. RESULTS: The results showed that miR-21-Exo promoted IFN-γ-induced KGN cell proliferation and hormone synthesis, and inhibited apoptosis. Using dual-luciferase reporter gene assays, miR-21 and MSX1 were shown to have direct interactions. Moreover, the findings elucidated that miR-21-Exo inhibited cell apoptosis and promoted hormone synthesis by mediating MSX1 to regulate the Notch signalling pathway. miR-21-Exo restored the ovarian structure in a mouse model of autoimmune POI, promoted endocrine function and proliferation, and inhibited apoptosis and inflammation in vivo. CONCLUSIONS: This study demonstrates that miR-21-Exo regulates the MSX1-mediated Notch signalling pathway to inhibit granulosa cell apoptosis and improve hormone synthesis function, providing insight into a potential mechanism of molecular therapy for the treatment of autoimmune POI.
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Exossomos , Fator de Transcrição MSX1 , Células-Tronco Mesenquimais , MicroRNAs , Insuficiência Ovariana Primária , Feminino , MicroRNAs/metabolismo , MicroRNAs/genética , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/genética , Animais , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator de Transcrição MSX1/metabolismo , Fator de Transcrição MSX1/genética , Humanos , Ovário/metabolismo , Doenças Autoimunes/metabolismo , Apoptose , Proliferação de CélulasRESUMO
BACKGROUND: This study aims to investigate the roles of telocytes on the metastatic properties of breast cancer stem cells (CSCs), and to re-evaluate the effect of miR-21-5p expression on CSCs following the addition of telocytes. METHODS AND RESULTS: Telocytes from human bone marrow mononuclear cells were isolated/characterised. This was followed by the isolation/characterisation of CSCs from the MDA-MB-231. miR-21-5p was both overexpressed/inhibited in CSCs. Through co-culture studies, EMT transition and oncogenic properties of CSCs were investigated by analysing changes in ALDH1 and vimentin protein levels as well as changes in the ABCC11, SNAI1, LZTFL1, Oct 3/4, E- and N-cadherin gene expression levels. With the inhibition of miR-21-5p, significant increases in LZTFL and ABCC11 were observed with the addition of telocytes. The expression of the LZTFL gene, which decreased with the overexpression of miR-21-5p, increased in CSCs after co-culture with telocytes. While an increase expression of ABCC11, SNAI1, N-Cadherin, vimentin and ALDH was observed in CSCs after overexpression of miR-21-5p, significant decreases in these expressions were observed after co-culture with telocyte. CONCLUSIONS: In our study, by gene/protein level analysis we demonstrated that telocytes may have the potential to reduce cancer metastasis through miR-21-5p in breast cancer progression and reduce EMT transition.
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MicroRNAs , Neoplasias , Telócitos , Humanos , Vimentina/genética , Caderinas , Células-Tronco Neoplásicas , MicroRNAs/genéticaRESUMO
BACKGROUND: This research explores the significance of miR-215-5p and vasculogenic mimicry (VM) in forecasting the prognosis for hepatocellular carcinoma (HCC). METHODS: We analyzed HCC-associated miRNA expression profiles using data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). Samples included tissue and blood from 80 early-stage HCC patients and serum from 120 healthy individuals. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed to measure miR-215-5p and zinc finger E-box binding homeobox 2 (ZEB2) gene expressions. Hematoxylin and eosin (H&E) and CD34/Periodic Acid-Schiff (PAS) double staining assessed VM presence in HCC tissue sections. Bioinformatics tools predicted interactions between miR-215-5p and ZEB2, confirmed through luciferase reporter assays. We also examined the impact of miR-215-5p or ZEB2 overexpression on HCC cell invasion, migration, and VM formation using scratch, Transwell invasion assays, and Matrigel 3D cultures. RESULTS: Bioinformatics analysis indicated that miR-215-5p was under-expressed in HCC, particularly in cases with vascular invasion, which correlated with worse patient outcomes. In contrast, ZEB2, targeted by miR-215-5p, was overexpressed in HCC. RT-qPCR validated these expression patterns in HCC tissues. Among the HCC patients, 38 were VM positive and 42 VM negative. Logistic regression highlighted a negative correlation between miR-215-5p levels and VM positivity in HCC tissues and a positive correlation for ZEB2 with VM positivity and tumor vascular invasion. Lower miR-215-5p levels were linked to increased HCC recurrence and metastasis. Both bioinformatics analysis and luciferase assays demonstrated a direct interaction between miR-215-5p and ZEB2. Enhancing miR-215-5p levels reduced ZEB2 expression, consequently diminishing invasion, migration, and VM formation of the HCC cells in vitro. CONCLUSIONS: miR-215-5p expression inversely correlates with VM occurrence in HCC tissues, while ZEB2 expression shows a direct correlation. By targeting ZEB2, miR-215-5p may hinder VM in HCC tissues, helping to prevent vascular invasion and HCC recurrence. Thus, miR-215-5p emerges as a vital prognostic indicator for predicting vascular invasion and recurrence in HCC.