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Background: The prognosis and survival of individuals with cetuximab-resistant colorectal cancer (CRC) remain severely impacted by therapy for this disease. The study investigated the underlying mechanisms of Pseudomonas aeruginosa-mannose sensitive hemagglutinin (PA-MSHA), a type of therapeutic biological product approved in China, for cetuximab-resistant CRC. Methods: Cell proliferation, apoptosis, migration and invasion were detected by cell counting kit-8 (CCK-8) assay, flow cytometry, wound healing assay and transwell assay. Massively parallel sequencing of cetuximab-resistant CRC cells with PA-MSHA treatment was used to screen the differential expression profile of miRNAs. The directly target gene of miR-7-5p was revealed by dual luciferase assay. Apoptosis and invasion related proteins were detected by Western blot. Results: PA-MSHA could successfully stop the migrating and invading of cetuximab-resistant CRC cells while also inducing apoptosis. Tumor-bearing experiments in nude mice showed that PA-MSHA slowed tumor growth and lengthened mouse life. The sequencing data showed that miR-7-5p was considerably upregulated after PA-MSHA treatment. As anticipated, miR-7-5p overexpression improved PA-MSHA's anticancer properties both in vitro and in vivo. The target gene of miR-7-5p was confirmed to be Akt3 by dual luciferase assay, and Akt3 silencing undid the inhibition of PA-MSHA efficacy caused by miR-7-5p downregulation. Additionally, PA-MSHA therapy significantly reduced the activation of Wnt-ß-catenin pathway, and Akt3 expression was positively linked with several important Wnt-ß-catenin pathway genes, including Wnt and CTNNB1. Finally, we discovered that patients with CRC who had developed cetuximab resistance or disease progression had remarkably decreased serum miR-7-5p levels. Conclusions: PA-MSHA controlled the miR-7-5p/Akt3/Wnt-ß-catenin pathway to provide substantial efficacy against cetuximab-resistant CRC.
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Cancer patients, prone to severe COVID-19, face immune challenges due to their disease and treatments. Identifying biomarkers, particularly extracellular vesicle (EV)-derived microRNAs (miRNAs), is vital for comprehending their response to COVID-19 vaccination. Therefore, this study aimed to investigate specific EV-miRNAs in the plasma of cancer patients under active treatment who received the COVID-19 booster vaccine. The selected miRNAs (EV-hsa-miR-7-5p, EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, EV-hsa-miR-145- 5p, and EV-hsa-miR-223-3p) are involved in regulating SARS-CoV-2 spike protein and cytokine release, making them potential biomarkers for vaccination response. The study involved 54 cancer patients. Plasma and serum samples were collected at pre-boost vaccination, and at 3 and 6 months post-boost vaccination. Anti-spike antibody levels were measured. Additionally, RNA was extracted from EVs isolated from plasma and the expression levels of miRNAs were assessed. The results showed a significantly positive antibody response after COVID-19 boost vaccination. The expression levels of EV-hsa-miR-7-5p, EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, and EV-hsa-miR-223-3p increased significantly after 6 months of COVID-19 booster vaccination. Interestingly, an increased expression of certain EV-hsa-miRNAs was positively correlated. Bioinformatic analysis revealed that these correlated miRNAs play a critical role in regulating the targets present in antiviral responses and cytokine production. These findings suggest that EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, and EV-hsa-miR-223-3p may be crucial in immune response induced by mRNA vaccines.
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Basic research to expand treatment options for multiple myeloma is greatly needed due to the refractory nature of the disease. Histone deacetylase (HDAC) inhibitors, which are epigenetic regulators, are attractive but have limited applications. MicroRNAs (miRNAs), which are also epigenetic regulators, are important molecules that may lead to future therapeutic breakthroughs. In this study, we comprehensively searched for miRNAs that are altered by HDAC inhibitors in myeloma cells. We identified miR-7-5p (miR-7) as a miRNA downregulated by HDAC inhibitors. Transfection of myeloma cell lines with miR-7 suppressed cell proliferation, induced apoptosis, and enhanced the effects of the HDAC inhibitor panobinostat. Expression of miR-7 was downregulated by c-Myc inhibition, but upregulated by bortezomib. Comprehensive examination of miR-7 targets revealed four candidates: SLC6A9, LRRC59, EXOSC2, and PSME3. Among these, we focused on PSME3, an oncogene involved in proteasome capacity in myeloma cells. PSME3 knockdown increases myeloma cell death and panobinostat sensitivity. In conclusion, miR-7, which is downregulated by HDAC inhibitors, is a tumor suppressor that targets PSME3. This miR-7 downregulation may be involved in HDAC inhibitor resistance. In addition, combinations of anti-myeloma drugs that complement changes in miRNA expression should be considered.
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Apoptosis , Bortezomib , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Inhibidores de Histona Desacetilasas , MicroARNs , Mieloma Múltiple , Panobinostat , MicroARNs/genética , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Panobinostat/farmacología , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Bortezomib/farmacología , Ácidos Hidroxámicos/farmacología , Regulación hacia Abajo/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
BACKGROUND: The presence of distant metastasis at the time of initial diagnosis is a prevalent issue in non-small cell lung cancer (NSCLC), affecting around 30-40 % of the patients. Acidic tumor microenvironment (TME) provides favorable conditions that increase the invasiveness and aggressiveness of NSCLC. The activity of the glycolytic enzyme lactate dehydrogenase (LDHA) increases intracellular lactate accumulation, which creates an acidic TME. However, it is not yet known whether LDHA is involved in enhancing the metastatic potential of NSCLC and if LDHA is a druggable therapeutic target for NSCLC. PURPOSE: We aimed to investigate the molecular mechanisms underlying the enhanced NSCLC metastasis in acidic TME, and to explore whether sulforaphane (SFN), an active compound in Raphani Semen, can serve as a LDHA inhibitor to inhibit NSCLC metastasis in the acidic TME. METHODS: To mimic the acidic TME, NSCLC cells were cultured in acidic medium (pH 6.6), normal medium (pH 7.4) served as control. Western blotting, bioinformatic analysis, luciferase assay and rescue experiments were used to explore the mechanism and investigate the anti-metastatic effect of SFN both in vitro and in vivo. RESULTS: Acidic environment increases the expression of LDHA which in turn increases the production of lactic acid that contributes to the acidity of TME. Interestingly, elevated LDHA expression results from increased c-Myc expression, which transactivates LDHA. c-Myc expression is directly regulated by miR-7-5p. In vitro study shows that overexpression of miR-7-5p reverses the acidic pH-enhanced c-Myc and LDHA expressions and also abolishes the enhanced NSCLC cell migration. More importantly, SFN significantly inhibits NSCLC growth and metastasis by reducing lactate production via the miR-7-5p/c-Myc/LDHA axis. Besides, it also regulates the expressions of monocarboxylate transporter 1 (MCT1) and MCT4 that transport lactate across cell membrane. CONCLUSIONS: The miR-7-5p/c-Myc/LDHA axis is involved in the enhanced NSCLC metastasis in the acidic TME. SFN, a novel LDHA inhibitor, reduces lactate production by targeting the miR-7-5p/c-Myc/LDHA axis, and hence inhibits NSCLC metastasis. Our findings not only delineate a novel mechanism, but also support the clinical translation of SFN as a novel therapeutic agent for treating metastatic NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Isotiocianatos , L-Lactato Deshidrogenasa , Neoplasias Pulmonares , MicroARNs , Proteínas Proto-Oncogénicas c-myc , Sulfóxidos , Microambiente Tumoral , Isotiocianatos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Microambiente Tumoral/efectos de los fármacos , Sulfóxidos/farmacología , MicroARNs/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Proteínas Proto-Oncogénicas c-myc/metabolismo , Línea Celular Tumoral , L-Lactato Deshidrogenasa/metabolismo , Ratones Desnudos , Ratones , Concentración de Iones de Hidrógeno , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Movimiento Celular/efectos de los fármacosRESUMEN
Subgroup J avian leukosis virus (ALV-J) is a major pathogen in poultry, causing substantial economic losses to the poultry industry worldwide. Exosomal small RNAs derived from virus-infected cells or biological fluids can serve as viral transmission vectors. However, the role and mechanism of exosomal miRNA in ALV-J infection are unclear. In this study, we demonstrated that exosomal microRNA-7-25207 (miR-7-25207) could increase the titers of ALV-J. Exosomes isolated from ALV-J-infected DF-1 cells (Exo-ALV-J) contained partial viral proteins from ALV-J and could transmit the infection to uninfected DF-1 cells, leading to productive infection. Additionally, the RNA expression profile of exosomes was altered following ALV-J infection. miRNA analysis revealed that the expression of exosomal miR-7-25207 increased. Overexpression of miR-7-25207 significantly increased the titers of ALV-J in transfected cells. Furthermore, miR-7-25207 directly suppressed the expression of Akt and PRC1. Akt, in turn, directly inhibited CyclinQ1 expression, while PRC1 directly interfered with YAF2 expression. In conclusion, ALV-J infection activates the expression of miR-7-25207, which is subsequently delivered via exosomes to uninfected cells, increasing ALV-J titers by targeting Akt-CyclinQ1 and PRC1-YAF2 dual pathways. These findings suggest that exosomal miR-7-25207 may serve as a potential biomarker for clinical parameters in ALV-J infection.
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INTRODUCTION: Chronic low-grade inflammation and oxidative stress are pivotal contributors to the metabolic complications associated with obesity. Selenoprotein P (SELENOP) and glutathione peroxidase 1 (GPx1) are selenoproteins involved in the reduction of reactive oxygen species and pro-inflammatory cytokines levels. Nutritional epigenomics revealed the interaction of microRNAs and nutrients with an important impact on metabolic pathways involved in obesity. However, the knowledge regarding the influence of microRNA on selenium biomarkers and its impact on metabolic pathways related to obesity remains scarce. Thus, the aim of this study was to investigate the association of plasma miR-7-5p expression with selenium and inflammatory biomarkers in women with overweight/obesity. MATERIAL AND METHODS: Anthropometric evaluations were performed and blood samples were collected for the analysis of fasting glucose, insulin, inflammatory and selenium biomarkers, and miR-7-5p expression in 54 women with overweight/obesity. Gene expression of SELENOP and GPX1 were evaluated in peripheral mononuclear blood cells. RESULTS: This study observed a negative correlation between SELENOP levels and miR-7-5p (rho = -0.350; p = 0.018). Additionally, it was observed that body fat (OR = 0.737; p = 0.011), age (OR = 1.214; p = 0.007), and miR-7-5p (OR = 0.990; p = 0.015) emerged as significant predictors of SELENOP levels. CONCLUSIONS: In conclusion, we observed a significant inverse association between miR-7-5p expression and SELENOP concentration in overweight/obese women, suggesting that age and percentage of body fat are also associated. TRIAL REGISTRATION NUMBER: Brazilian Registry of Clinical Trials (ReBEC) number RBR-2nfy5q.
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BACKGROUND: Primary liver cancer is the third leading cause of cancer related deaths worldwide, and the disease is associated with high incidence rate of thrombosis. Studies indicate that Tissue Factor Pathway Inhibitor (TFPI) plays a role in cancer development. We aimed to study its expression, clinical role and regulation by micro RNAs (miRNAs) in hepatocellular carcinoma (HCC). METHODS: Publically available datasets were used for clinical analysis of TFPI and miRNAs expression by web analysis tools. miRNA mimics targeting TFPIα 3'untranslated region (UTR) were selected from target prediction programs and verified by luciferase reporter assay. In vitro effects of miRNAs overexpression in HCC cell lines on TFPI expression and cell proliferation and apoptosis were analysed. RESULTS: TFPI expression was significantly increased in HCC tumours compared to normal tissue. Low TFPI tumour expression was associated with better survival probability. Four candidate miRNAs were selected from the target prediction programs. miR-7-5p and miR-1236-3p were validated in HepG2 and Huh7 cells to reduce TFPI mRNA and protein levels following overexpression. Furthermore, miR-7-5p and miR-1236-3p reduced TFPIα-3'UTR-controlled luciferase activity. The two validated miRNAs inhibited proliferation of HepG2 cells, and had clinical significance in HCC. CONCLUSIONS: TFPI was increased in HCC tumours compared to normal tissue and high TFPI expression was associated with an unfavorable outcome in HCC patients. miR-7-5p and miR-1236-3p were identified as novel regulators of TFPI in vitro.
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Carcinoma Hepatocelular , Lipoproteínas , Neoplasias Hepáticas , MicroARNs , Femenino , Humanos , Masculino , Persona de Mediana Edad , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/mortalidad , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Lipoproteínas/genética , Lipoproteínas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , MicroARNs/genética , MicroARNs/metabolismo , PronósticoRESUMEN
The circular RNA (circRNA) Cdr1as is conserved across mammals and highly expressed in neurons, where it directly interacts with microRNA miR-7. However, the biological function of this interaction is unknown. Here, using primary cortical murine neurons, we demonstrate that stimulating neurons by sustained depolarization rapidly induces two-fold transcriptional upregulation of Cdr1as and strong post-transcriptional stabilization of miR-7. Cdr1as loss causes doubling of glutamate release from stimulated synapses and increased frequency and duration of local neuronal bursts. Moreover, the periodicity of neuronal networks increases, and synchronicity is impaired. Strikingly, these effects are reverted by sustained expression of miR-7, which also clears Cdr1as molecules from neuronal projections. Consistently, without Cdr1as, transcriptomic changes caused by miR-7 overexpression are stronger (including miR-7-targets downregulation) and enriched in secretion/synaptic plasticity pathways. Altogether, our results suggest that in cortical neurons Cdr1as buffers miR-7 activity to control glutamatergic excitatory transmission and neuronal connectivity important for long-lasting synaptic adaptations.
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Ácido Glutámico , MicroARNs , Neuronas , Transmisión Sináptica , MicroARNs/genética , MicroARNs/metabolismo , Animales , Neuronas/metabolismo , Ratones , Ácido Glutámico/metabolismo , Transmisión Sináptica/genética , Plasticidad Neuronal/genética , ARN Circular/genética , ARN Circular/metabolismo , Sinapsis/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Regulación de la Expresión Génica , Células CultivadasRESUMEN
Despite advances in treatment strategies, colorectal cancer (CRC) continues to cause significant morbidity and mortality, with mounting evidence a close link between immune system dysfunctions issued. Interleukin-2 receptor gamma (IL-2RG) plays a pivotal role as a common subunit receptor in the IL-2 family cytokines and activates the JAK-STAT pathway. This study delves into the role of Interleukin-2 receptor gamma (IL-2RG) within the tumor microenvironment and investigates potential microRNAs (miRNAs) that directly inhibit IL-2RG, aiming to discern their impact on CRC clinical outcomes. Bioinformatics analysis revealed a significant upregulation of IL-2RG mRNA in TCGA-COAD samples and showed strong correlations with the infiltration of various lymphocytes. Single-cell analysis corroborated these findings, highlighting IL-2RG expression in critical immune cell subsets. To explore miRNA involvement in IL-2RG dysregulation, mRNA was isolated from the tumor tissues and lymphocytes of 258 CRC patients and 30 healthy controls, and IL-2RG was cloned into the pcDNA3.1/CT-GFP-TOPO vector. Human embryonic kidney cell lines (HEK-293T) were transfected with this construct. Our research involved a comprehensive analysis of miRPathDB, miRWalk, and Targetscan databases to identify the miRNAs associated with the 3' UTR of human IL-2RG. The human microRNA (miRNA) molecules, hsa-miR-7-5p and hsa-miR-26b-5p, have been identified as potent suppressors of IL-2RG expression in CRC patients. Specifically, the downregulation of hsa-miR-7-5p and hsa-miR-26b-5p has been shown to result in the upregulation of IL-2RG mRNA expression in these patients. Prognostic evaluation of IL-2RG, hsa-miR-7-5p, and hsa-miR-26b-5p, using TCGA-COAD data and patient samples, established that higher IL-2RG expression and lower expression of both miRNAs were associated with poorer outcomes. Additionally, this study identified several long non-coding RNAs (LncRNAs), such as ZFAS1, SOX21-AS1, SNHG11, SNHG16, SNHG1, DLX6-AS1, GAS5, SNHG6, and MALAT1, which may act as competing endogenous RNA molecules for IL2RG by sequestering shared hsa-miR-7-5p and hsa-miR-26b-5p. In summary, this investigation underscores the potential utility of IL-2RG, hsa-miR-7-5p, and hsa-miR-26b-5p as serum and tissue biomarkers for predicting CRC patient prognosis while also offering promise as targets for immunotherapy in CRC management.
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Neoplasias Colorrectales , Regulación Neoplásica de la Expresión Génica , Subunidad gamma Común de Receptores de Interleucina , MicroARNs , Femenino , Humanos , Masculino , Persona de Mediana Edad , Secuencia de Bases , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Células HEK293 , Inmunoterapia , Subunidad gamma Común de Receptores de Interleucina/genética , MicroARNs/genética , MicroARNs/metabolismo , PronósticoRESUMEN
BACKGROUND: We recently reported that upregulation of Musashi 2 (MSI2) protein in the rare neuromuscular disease myotonic dystrophy type 1 contributes to the hyperactivation of the muscle catabolic processes autophagy and UPS through a reduction in miR-7 levels. Because oleic acid (OA) is a known allosteric regulator of MSI2 activity in the biogenesis of miR-7, here we sought to evaluate endogenous levels of this fatty acid and its therapeutic potential in rescuing cell differentiation phenotypes in vitro. In this work, four muscle cell lines derived from DM1 patients were treated with OA for 24 h, and autophagy and muscle differentiation parameters were analyzed. RESULTS: We demonstrate a reduction of OA levels in different cell models of the disease. OA supplementation rescued disease-related phenotypes such as fusion index, myotube diameter, and repressed autophagy. This involved inhibiting MSI2 regulation of direct molecular target miR-7 since OA isoschizomer, elaidic acid (EA) could not cause the same rescues. Reduction of OA levels seems to stem from impaired biogenesis since levels of the enzyme stearoyl-CoA desaturase 1 (SCD1), responsible for converting stearic acid to oleic acid, are decreased in DM1 and correlate with OA amounts. CONCLUSIONS: For the first time in DM1, we describe a fatty acid metabolism impairment that originated, at least in part, from a decrease in SCD1. Because OA allosterically inhibits MSI2 binding to molecular targets, reduced OA levels synergize with the overexpression of MSI2 and contribute to the MSI2 > miR-7 > autophagy axis that we proposed to explain the muscle atrophy phenotype.
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Distrofia Miotónica , Ácido Oléico , Ácido Oléico/farmacología , Distrofia Miotónica/tratamiento farmacológico , Distrofia Miotónica/metabolismo , Humanos , Diferenciación Celular/efectos de los fármacos , MicroARNs/metabolismo , Autofagia/efectos de los fármacos , Línea Celular , Proteínas de Unión al ARN/metabolismoRESUMEN
Spinal cord injury (SCI) is a complex problem in modern medicine. Fibroblast activation and fibroscarring after SCI impede nerve recovery. Non-coding RNA plays an important role in the progression of many diseases, but the study of its role in the progression of spinal fibrosis is still emerging. Here, we investigated the function of circular RNAs, specifically antisense to the cerebellar degeneration-related protein 1 (CDR1as), in spinal fibrosis and characterized its molecular mechanism and pathophysiology. The presence of CDR1as in the spinal cord was verified by sequencing and RNA expression assays. The effects of inhibition of CDR1as on scar formation, inflammation and nerve regeneration after spinal cord injury were investigated in vivo and in vitro. Further, gene expression of miR-7a-5p and protein expression of transforming Growth Factor Beta Receptor II (TGF-ßR2) were measured to evaluate their predicted interactions with CDR1as. The regulatory effects and activation pathways were subsequently verified by miR-7a-5p inhibitor and siCDR1as. These results indicate that CDR1as/miR-7a-5p/TGF-ßR2 interactions may exert scars and nerves functions and suggest potential therapeutic targets for treating spinal fibrotic diseases.
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Fibrosis , MicroARNs , ARN Circular , ARN Largo no Codificante , Transducción de Señal , Traumatismos de la Médula Espinal , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/fisiopatología , Animales , MicroARNs/genética , MicroARNs/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Proteínas Smad/metabolismo , Proteínas Smad/genética , Regeneración Nerviosa , Femenino , Masculino , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Ratones Endogámicos C57BL , Ratones , Recuperación de la FunciónRESUMEN
BACKGROUND: Circular RNAs (circRNAs) belong to a class of covalently closed single stranded RNAs that have been implicated in cancer progression. Former investigations showed that hsa-circ-0013561 is abnormally expressed in head and neck squamous cell carcinoma (HNSCC). Nevertheless, the role of hsa-circ-0013561 during the progress of HNSCC still unclear. METHODS: Present study applied FISH and qRT-PCR to examine hsa-circ-0013561 expression in HNSCC cells and tissue samples. Dual-luciferase reporter assay was employed to identify downstream targets of hsa-circ-0013561. Transwell migration, 5-ethynyl-2'-deoxyuridine incorporation, CCK8 and colony formation assays were utilized to test cell migration and proliferation. A mouse tumor xenograft model was utilized to determine the hsa-circ-0013561 roles in HNSCC progression and metastasis in vivo. RESULTS: We found that hsa-circ-0013561 was upregulated in HNSCC tissue samples. hsa-circ-0013561 downregulation inhibited HNSCC cell proliferation and migration to promote apoptosis and G1 cell cycle arrest. The dual-luciferase reporter assay revealed that miR-7-5p and PDK3 are hsa-circ-0013561 downstream targets. PDK3 overexpression or miR-7-5p suppression reversed the hsa-circ-0013561-induced silencing effects on HNSCC cell proliferation and migration. PDK3 overexpression reversed miR-7-5p-induced effects on HNSCC cell proliferation and migration. CONCLUSION: The findings suggest that hsa-circ-0013561 downregulation inhibits HNSCC metastasis and progression through PDK3 expression and miR-7-5p binding modulation.
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BACKGROUND: The pathogenesis of vascular dementia (VD) is complex, and currently, no effective treatments have been recommended. Floralozone is a colorless liquid first discovered in Lagotis Gaertn. Recently, its medicinal value has been increasingly recognized. Our previous study has demonstrated that Floralozone can improve cognitive dysfunction in rats with VD by regulating the transient receptor potential melastatin 2 (TRPM2) and N-methyl-D-aspartate receptor (NMDAR) signaling pathways. However, the mechanism by which Floralozone regulates TRPM2 and NMDAR to improve VD remains unclear. AMP-activated protein kinase (AMPK) is an energy regulator in vivo; however, its role of AMPK activation in stroke remains controversial. MiR-7a-5p has been identified to be closely related to neuronal function. PURPOSE: To explore whether Floralozone can regulate the miR-7a-5p level in vivo through AMPKα2 activation, affect the TRPM2 and NR2B expression levels, and improve VD symptoms. METHODS: The VD model was established by a modified bilateral occlusion of the common carotid arteries (2-VO) of Sprague-Dawley (SD) rats and AMPKα2 KO transgenic (AMPKα2-/-) mice. Primary hippocampal neurons were modeled using oxygen and glucose deprivation (OGD). Morris water maze (MWM) test, hematoxylin-eosin staining (HE staining), and TUNEL staining were used to investigate the effects of Floralozone on behavior and hippocampal morphology in rats. Minichromosome maintenance complex component 2(MCM2) positive cells were used to investigate the effect of Floralozone on neurogenesis. Immunofluorescence staining, qRT-PCR, and western blot analysis were used to investigate the effect of Floralozone on the expression levels of AMPKα2, miR-7a-5p, TRPM2, and NR2B. RESULTS: The SD rat experiment revealed that Floralozone improved spatial learning and memory, improved the morphology and structure of hippocampal neurons, reduced apoptosis of hippocampal neurons and promoted neurogenesis in VD rats. Floralozone could increase the miR-7a-5p expression level, activate AMPKα2 and NR2B expressions, and inhibit TRPM2 expression in hippocampal neurons of VD rats. The AMPKα2 KO transgenic (AMPKα2-/-) mice experiment demonstrated that Floralozone could regulate miR-7a-5p, TRPM2, and NR2B expression levels through AMPKα2 activation. The cell experiment revealed that the TRPM2 and NR2B expression levels were regulated by miR-7a-5p, whereas the AMPKα2 expression level was not. CONCLUSION: Floralozone could regulate miR-7a-5p expression level by activating the protein expression of AMPKα2, control the protein expression of TRPM2 and NR2B, improve the morphology and structure of hippocampus neurons, reduce the apoptosis of hippocampus neurons, promote neurogenesis and improve the cognitive dysfunction.
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Proteínas Quinasas Activadas por AMP , Disfunción Cognitiva , Demencia Vascular , MicroARNs , Animales , Masculino , Ratones , Ratas , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/etiología , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/genética , Demencia Vascular/genética , Demencia Vascular/tratamiento farmacológico , Demencia Vascular/metabolismo , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/metabolismo , MicroARNs/genética , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Ratas Sprague-Dawley , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismoRESUMEN
BACKGROUND AND PURPOSE: The pathophysiological mechanisms underlying brain injury resulting from intracerebral hemorrhage (ICH) remain incompletely elucidated, and efficacious therapeutic interventions to enhance the prognosis of ICH patients are currently lacking. Previous research indicates that MicroRNA-7 (miR-7) can suppress the expression of Nod-like receptor protein 3 (NLRP3), thereby modulating neuroinflammation in Parkinson's disease pathogenesis. However, the potential regulatory effects miR-7 on NLRP3 inflammasome after ICH are yet to be established. This study aims to ascertain whether miR-7 mitigates secondary brain injury following experimental ICH by inhibiting NLRP3 and to investigate the underlying mechanisms. METHODS: An ICH model was established by stereotaxically injecting 100 µL of autologous blood into the right basal ganglia of Sprague-Dawley (SD) rats. Subsequently, these rats were allocated into three groups: sham, ICH + Vehicle, and ICH + miR-7, each comprising 18 animals. Twelve hours post-modeling, rats received intraventricular injections of 10 µL physiological saline, 10 µL phosphate, and 10 µL phosphate-buffered saline solution containing 0.5 nmol of miR-7 mimics, respectively. Neurological function was assessed on day three post-modeling, followed by euthanasia for brain tissue collection. Brain water content was determined using the dry-wet weight method. The expression of inflammatory cytokines in cerebral tissues surrounding the hematoma was analyzed through immunohistochemistry and Western blot assays. These cytokines were re-evaluated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Moreover, bioinformatics tools were employed to predict miR-7's binding to NLRP3. A wild-type luciferase reporter gene vector and a corresponding mutant vector were constructed, followed by transfection of miR-7 mimics into HEK293T cells to assess luciferase activity. RESULTS: Our study demonstrates that the administration of miR-7 mimics markedly reduced neurological function scores and attenuated brain edema in rats following ICH. A significant upregulation of NLRP3 expression in microglia/macrophage adjacent to the hematoma was observed, substantially reduced after the treatment with miR-7 mimics. Furthermore, this intervention ameliorated neurodegenerative changes and effectively decreased the protein and mRNA levels of pro-inflammatory cytokines, namely TNF-α, IL-1ß, IL-6, and Caspase1, in the cerebral tissues proximate to the hematomas. In addition, miR-7 mimics distinctly inhibited the luciferase activity associated with the wild-type reporter gene, an effect not mirrored in its mutant variant. CONCLUSIONS: The miR-7 suppressed NLRP3 expression in microglia/macrophage to reduce the production of inflammatory cytokines, leading to conducting certain neuroprotection post-ICH in rats.
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Lesiones Encefálicas , MicroARNs , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Humanos , Ratas , Lesiones Encefálicas/etiología , Hemorragia Cerebral/complicaciones , Citocinas/genética , Citocinas/metabolismo , Células HEK293 , Hematoma/complicaciones , Luciferasas/uso terapéutico , MicroARNs/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Fosfatos , Ratas Sprague-DawleyRESUMEN
Long noncoding RNA small nucleolar RNA host gene 1 (lncRNA SNHG1) plays a crucial role in the occurrence and progression of various tumors. This study investigates the function of lncRNA SNHG1 in hepatocellular carcinoma (HCC). We discovered that lncRNA SNHG1 is significantly upregulated in HCC and markedly enhances cell proliferation, migration, and invasion, while simultaneously suppressing apoptosis in HCC cells. Furthermore, lncRNA SNHG1 was found to downregulate miR-7-5p expression. Overexpression of lncRNA SNHG1 counteracted the suppression of HCC cell migration, proliferation, and invasion caused by miR-7-5p mimics, and reversed the miR-7-5p mimics' enhancement of apoptosis in HCC cells. Additionally, miR-7-5p was shown to negatively regulate IGF2BP2, with the silencing of IGF2BP2 diminishing the abilities of HCC cells to proliferate, migrate, and invade, and increasing their propensity for apoptosis. Overexpression of lncRNA SNHG1 negated these effects. Thus, lncRNA SNHG1 fosters HCC progression by upregulating IGF2BP2 expression through targeting miR-7-5p.
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This study is aimed at investigating the roles of Toll-like receptor 4 (TLR4) and microRNA-7 (miR-7) in colorectal cancer (CRC) development and progression. We assessed TLR4 and miR-7 expression in CRC cells and tissues using reverse transcription-quantitative polymerase chain reaction. The relationship between miR-7 and TLR4 was analyzed through dual luciferase reporter assays. MTT, wound healing, and cell invasion assays were conducted to examine the effects of TLR4 and miR-7 on CRC cell proliferation, migration, and invasion. Western blotting was used to explore the involvement of the TRAF6/NF-κB signaling pathway. miR-7 was underexpressed in CRC, while TLR4 levels were increased. miR-7 negatively regulated TLR4 expression and its knockdown enhanced CRC cell proliferation, migration, and invasion. TLR4 knockdown had the opposite effects. The TRAF6/NF-κB pathway was linked to TLR4's role in tumor progression. miR-7 might inhibit TRAF6/NF-κB target a signaling pathway of TLR4 and promote CRC occurrence. miR-7 may therefore be used as a sensitive biomarker in CRC patients.
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Neoplasias Colorrectales , MicroARNs , Receptor Toll-Like 4 , Humanos , Proliferación Celular , Neoplasias Colorrectales/genética , MicroARNs/genética , MicroARNs/metabolismo , FN-kappa B/genética , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Diffuse large B-cell lymphoma (DLBCL) is the leading cause of mortality from invasive hematological malignancies worldwide. MicroRNA-7-5p (miR-7-5p) has been shown to be a tumor suppressor in several types of tumors. However, its role in DLBCL is not fully understood. This study explored the role of miR-7-5p in the progression of DLBCL and pursued the underlying mechanism. Quantitative real-time PCR and transfection of miRNA mimic and inhibitors were used to assess the effects of miR-7-5p on autophagy and apoptosis in SU-DHL-4 and SU-DHL-10 cells. Dual-luciferase reporter assay was used to identify target genes of miR-7-5p. Immunofluorescence, flow cytometry, and western blotting (WB) were performed to explore the underlying mechanism and downstream pathways of miR-7-5p and AMBRA1 in DLBCL cells. MiR-7-5p was upregulated in DLBCL cells. Luciferase reporter assays implicated AMBRA1 as a downstream target of miR-7-5p in DLBCL. WB and flow cytometry showed that an increase in miR-7-5p level and a decrease in AMBRA1 expression led to a decrease in autophagy and apoptosis-related protein expression. Furthermore, miR-7-5p prevented c-MYC dephosphorylation through AMBRA1 downregulation. On the contrary, c-MYC increased the expression of miR-7-5p, thereby establishing positive feedback on miR-7-5p transcription. The addition of hydroxychloroquine, an autophagy inhibitor, reduced autophagy and increased apoptosis in DLBCL cells. In vivo experiments further proved that the increase of miR-7-5p played a regulatory role in the expression of downstream AMBRA1 and c-MYC. These results demonstrate that c-MYC-dependent MiR-7-5p suppressed autophagy and apoptosis by targeting AMBRA1 in DLBCL cells. MiR-7-5p also suppressed autophagy and apoptosis by targeting AMBRA1 in DLBCL cells. Therefore, these data suggest that targeting miR-7-5p may be a promising strategy in DLBCL therapy.
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Circular RNA (circRNA) ArfGAP with FG repeats 1 (circAGFG1) contributes to colorectal cancer (CRC) development. However, whether circAGFG1 regulates the resistance of CRC to oxaliplatin (L-OHP) remains unknown. CircAGFG1, microRNA-7-5p (miR-7-5p) and pyruvate kinase M2 (PKM2) RNA expression were quantified by quantitative real-time polymerase chain reaction. Protein expression was detected by western blot assay and immunohistochemistry assay. Glycolysis was analyzed through glucose uptake, lactate production and adenosine triphosphate (ATP) concentration assays. 50% inhibitory concentration of L-OHP was determined by cell counting kit-8 assay. Cell proliferation and apoptotic rate were analyzed by cell colony formation and flow cytometry analysis, respectively. Dual-luciferase reporter assay was used to identify the relationship among circAGFG1, miR-7- 5p and PKM2. The effect of circAGFG1 on L-OHP sensitivity in vivo was further evaluated by a xenograft model assay. CircAGFG1 and PKM2 expression were significantly increased, while miR-7-5p was decreased in L-OHP-resistant CRC tissues and cells. High circAGFG1 expression predicted a poor prognosis of CRC. CircAGFG1 knockdown or PKM2 depletion decreased glycolysis and cell proliferation and increased L-OHP sensitivity and cell apoptosis. PKM2 introduction rescued circAGFG1 silencing-induced effects in CRC cells. In terms of mechanism, circAGFG1 bound to miR-7-5p, which was identified to target PKM2. Also, circAGFG1 regulated PKM2 expression by interacting with miR-7-5p. Further, circAGFG1 knockdown improved the sensitivity of tumors to L-OHP in vivo. CircAGFG1 depletion inhibited L-OHP resistance by regulating the miR-7-5p/PKM2 pathway.
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Neoplasias Colorrectales , MicroARNs , Humanos , Oxaliplatino/farmacología , Línea Celular Tumoral , MicroARNs/farmacología , Apoptosis , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Proliferación CelularRESUMEN
Adoptive-cell-therapy (ACT) is important therapeutic approach against cancer. We previously showed that miR-7 deficiency endowed CD4+T cells with hyperactivation status in liver injury. However, whether CD4+T cells with miR-7 deficiency could elicit antitumor effect in ACT is still unclear. Naïve CD4+CD62Lhi T cells were purified from CD45.2 WT or CD45.2 miR-7def mice and transferred into syngeneic CD45.1WT mice bearing with lung tumor cells. The infiltration and function of T cells were measured by FCM and immunofluorescence assay. And naïve CD4+CD62Lhi T cells were purified from CD45.2 WT or CD45.2 miR-7def mice, then the cells were activated with CD3 antibody plus CD28 antibody in vitro for 24 h. Then, the cultured supernatant of LLC tumor cells or cytokines IFN-γ and IL-12 was added to establish Th1 polarization. Under these conditions, Th1 polarization-related molecules in these cells were analyzed by flow cytometry. Our data demonstrated a significant reduction in the growth and metastasis of lung cancer cells in the miR-7def CD4+T cell-transferred group, accompanied by a significant enhancement in the infiltration, proliferation, activation, and Th1 polarization of CD4+ T cells. Moreover, we observed the proliferation; activation of tumor-infiltrating CD8+ T cells was significantly increased in the local tumor of the CD45.2 miR-7def CD4+ T cell-transferred group, compared to the CD45.2 WT CD4+ T cell-transferred group. It is noteworthy that MAPK4, a target molecule of miR-7, was upregulated in CD4+ T cells from lung tumor tissues, resulting in an altered transduction of phosphorylation of NF-κB as well as AKT and ERK in vivo and in vitro. miR-7 deficiency promoted Th1-polarization of CD4+ T cells and elicited effective antitumor immune responses in ACT.
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Neoplasias Pulmonares , MicroARNs , Ratones , Animales , Linfocitos T CD8-positivos , Linfocitos T CD4-Positivos , Inmunoterapia Adoptiva , Neoplasias Pulmonares/terapia , MicroARNs/genética , Células TH1RESUMEN
INTRODUCTION: The epithelial immunomodulation and regeneration are intrinsic critical events against inflammatory bowel disease (IBD). MiR-7 is well documented as a promising regulator in the development of various diseases including inflammatory diseases. OBJECTIVES: This study aimed to assess the effect of miR-7 in intestinal epithelial cells (IECs) in IBD. METHODS: MiR-7def mice were given dextran sulfate sodium (DSS) to induce enteritis model. The infiltration of inflammatory cells was measured by FCM and immunofluorescence assay. 5'deletion assay and EMSA assays were performed to study the regulatory mechanism of miR-7 expression in IECs. The inflammatory signals and the targets of miR-7 were analyzed by RNA-seq and FISH assay. IECs were isolated from miR-7def, miR-7oe and WT mice to identify the immunomodulation and regeneration capacity. IEC-specific miR-7 silencing expression vector was designed and administered by the tail vein into murine DSS-induced enteritis model to evaluate the pathological lesions of IBD. RESULTS: We found miR-7 deficiency improved the pathological lesions of DSS-induced murine enteritis model, accompanied by elevated proliferation and enhanced transduction of NF-κB/AKT/ERK signals in colonic IECs, as well as decreased local infiltration of inflammatory cells. MiR-7 was dominantly upregulated in colonic IECs in colitis. Moreover, the transcription of pre-miR-7a-1, orchestrated by transcription factor C/EBPα, was a main resource of mature miR-7 in IECs. As for the mechanism, EGFR, a miR-7 target gene, was downregulated in colonic IECs in colitis model and Crohn's disease patients. Furthermore, miR-7 also controlled the proliferation and inflammatory-cytokine secretion of IECs in response to inflammatory-signals through EGFR/NF-κB/AKT/ERK pathway. Finally, IEC-specific miR-7 silencing promoted the proliferation and transduction of NF-κB pathway in IECs and alleviated the pathological damage of colitis. CONCLUSION: Our results present the unknown role of miR-7/EGFR axis in IEC immunomodulation and regeneration in IBD and might provide clues for the application of miRNA-based therapeutic strategies in colonic diseases.