RESUMEN

BACKGROUND: The objective of this study was to determine the role and regulatory mechanism of miR-380 in cholangiocarcinoma. METHODS: The TargetScan database and a dual-luciferase reporter assay system were used to determine if LIS1 was a target gene of miR-380. The Cell Counting Kit 8 assay, flow cytometry, and Transwell assay were used to detect the effects of miR-380 and LIS1 on the proliferation, S-phase ratio, and invasiveness of HCCC-9810/HuCCT1/QBC939 cells. Western blotting was used to determine the effect of miR-380 on MMP-2/p-AKT. Immunohistochemistry detected the regulatory effect of miR-380 on the expression of MMP-2/p-AKT/LIS1. RESULTS: Expression of miR-380 in cholangiocarcinoma was decreased but expression of LIS1 was increased. LIS1 was confirmed to be a target gene of miR-380. Transfection with miR-380 mimics inhibited the proliferation, S-phase arrest, and invasion of HCCC-9810/HuCCT1/QBC939 cells, and LIS1 reversed these inhibitory effects. miR-380 inhibitor promoted proliferation, S-phase ratio, and invasiveness of HCCC-9810/HuCCT1/QBC939 cells. si-LIS1 salvaged the promotive effect of miR-380 inhibitor. Overexpression of miR-380 inhibited expression of MMP-2/p-AKT/LIS1, but miR-380 inhibitor promoted their expression. CONCLUSION: An imbalance of miR-380 expression is closely related to cholangiocarcinoma, and overexpression of miR-380 inhibits the expression of MMP-2/p-AKT by directly targeting LIS1.

RESUMEN

OBJECTIVE: αS1-Casein is more closely associated with milk allergic reaction than other milk protein components. microRNA (miRNA) is a class of small non-coding RNAs that modulate multiple biological progresses by the target gene. However, the post-transcriptional regulation of αS1-casein expression by miRNA in ruminants remains unclear. This study aims to explore the regulatory roles of miR-380-3p on αS1-casein synthesis in goat mammary epithelial cells (GMEC). METHODS: αS1-Casein gene and miR-380-3p expression was measured in dairy goat mammary gland by quantitative real-time polymerase chain reaction (qRT-PCR). miR-380-3p overexpression and knockdown were performed by miR-380-3p mimic or inhibitor in GMEC. The effect of miR-380-3p on αS1-casein synthesis was detected by qRT-PCR, western blot, luciferase and chromatin immunoprecipitation assays in GMEC. RESULTS: Compared with middle-lactation period, αS1-casein gene expression is increased, while miR-380-3p expression is decreased during peak-lactation of dairy goats. miR-380-3p reduces αS1-casein abundance by targeting the 3'-untranslated region (3'UTR) of αS1-casein mRNA in GMEC. miR-380-3p enhances ß-casein expression and signal transducer and activator of transcription 5a (STAT5a) activity. Moreover, miR-380-3p promotes ß-casein abundance through target gene αS1-casein, and activates ß-casein transcription by enhancing the binding of STAT5 to ß-casein gene promoter region. CONCLUSION: miR-380-3p decreases αS1-casein expression and increases ß-casein expression by targeting αS1-casein in GMEC, which supplies a novel strategy for reducing milk allergic potential and building up milk quality in ruminants.

RESUMEN

N(6)-methyladenosine (m6A)-modified microRNAs (miRNAs) are relevant to cancer progression. Also, although the involvement of miR-380-3p in regulating cancer progression in bladder cancer and neuroblastoma has been preliminarily explored, its role in other types of cancer, such as pancreatic cancer (PC), has not been studied. Thus, this study aimed to investigate the role of miR-380-3p in regulating PC progression. Here, through performing Real-Time qPCR, we evidenced that miR-380-3p was significantly upregulated in the clinical pancreatic cancer tissues and cells compared to their normal counterparts. Interestingly, miR-380-3p was enriched with m6A modifications, and elimination of m6A modifications by deleting METTL3 and METTL14 synergistically suppressed miR-380-3p expressions in PC cells. Next, the gain and loss-of-function experiments verified that knockdown of miR-380-3p suppressed cell proliferation, epithelial-mesenchymal transition (EMT), and tumorigenesis in PC cells in vitro and in vivo, whereas miR-380-3p overexpression had opposite effects. Furthermore, the underlying mechanisms were uncovered, and our data suggested that miR-380-3p targeted the 3' untranslated regions (3'UTRs) of PTEN for its inhibition and degradation, resulting in the activation of the downstream Akt signal pathway. Moreover, the rescuing experiments validated that both PTEN overexpression and Akt pathway inhibitor LY294002 abrogated the promoting effects of miR-380-3p overexpression on cancer aggressiveness in PC cells. Collectively, this study firstly investigated the role of the m6A-associated miR-380-3p/PTEN/Akt pathway in regulating PC progression, which provided novel therapeutic and diagnostic biomarkers for this cancer.

Asunto(s)

Adenosina , MicroARNs , Neoplasias Pancreáticas , Adenosina/análogos & derivados , Adenosina/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Humanos , Metiltransferasas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regulación hacia Arriba

RESUMEN

The objective of this study is to investigate lncRNA FLG-AS1-mediated miR-380-3p/SOCS6 axis in inflammation, oxidative stress, and apoptosis of retinal epithelial cells in diabetic retinopathy (DR). Fasting blood was collected from 60 DR patients and 60 healthy controls. The Pearson correlation was used to analyze the correlation between the expression levels of FLG-AS1 and miR-380-3p in DR patients. qRT-PCR and/or Western blotting were used to detect the expression of FLG-AS1, miR-380-3p, and SOCS6. After gain of function of FLG-AS1 or SOCS6 or loss of function of miR-380-3p, high glucose (HG)-treated human retinal pigment epithelial ARPE-19 cells were subjected to TUNEL assessment of apoptosis. ELISA was performed to detect the expression levels of IL-1ß, IL-6, and TNF-α in cell culture supernatant. DCFH-DA was used to detect the level of ROS in the cells. MDA and SOD assay kits were used to measure the activity of MDA and SOD in the cells. Dual-luciferase reporter assay was performed to verify the binding between miR-380-3p and FLG-AS1 or between miR-380-3p and SOCS6. Streptozotocin injections were used to induce diabetes in rats which were injected with FLG-AS1 overexpression lentiviral vectors in the eye. Twenty weeks later, retinal tissue was isolated and stained with hematoxylin-eosin or TUNEL. Compared to that in healthy controls, FLG-AS1 expression decreased 2.5-fold and miR-380-3p expression increased 2.6-fold in the serum of DR patients. The expression levels of FLG-AS1 and miR-380-3p were negatively correlated in DR patients (r = -0.3772, P = 0.003). Overexpression of FLG-AS1 reduced inflammation, oxidative stress, and apoptosis of HG-treated ARPE-19 cells and alleviated retinal injury in diabetic rats. FLG-AS1 promoted the expression of SOCS6 by targeting miR-380-3p. Inhibition of miR-380-3p or overexpression of SOCS6 reduced inflammation, oxidative stress, and apoptosis of HG-treated ARPE-19 cells. FLG-AS1 mitigates DR by regulating retinal epithelial cell inflammation, oxidative stress, and apoptosis via the miR-380-3p/SOCS6 axis.

Asunto(s)

Diabetes Mellitus Experimental , Retinopatía Diabética , MicroARNs , ARN Largo no Codificante , Animales , Apoptosis , Proliferación Celular , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Eosina Amarillenta-(YS)/metabolismo , Células Epiteliales/metabolismo , Glucosa/metabolismo , Hematoxilina/metabolismo , Humanos , Inflamación/metabolismo , Interleucina-6/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Estrés Oxidativo , ARN Largo no Codificante/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Pigmentos Retinianos/metabolismo , Estreptozocina/metabolismo , Superóxido Dismutasa/metabolismo , Proteínas Supresoras de la Señalización de Citocinas , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Neuroblastoma is a deadly childhood cancer arising in the developing sympathetic nervous system. High-risk patients are currently treated with intensive chemotherapy, which is curative in only 50% of children and leaves some surviving patients with life-long side effects. microRNAs (miRNAs) are critical regulators of neural crest development and are deregulated during neuroblastoma tumorigenesis, making miRNA-based drugs an attractive therapeutic avenue. A functional screen of >1,200 miRNA mimics was conducted in neuroblastoma cell lines to discover miRNAs that sensitized cells to low doses (30% inhibitory concentration [IC30]) of doxorubicin and vincristine chemotherapy used in the treatment of the disease. Three miRNAs, miR-99b-5p, miR-380-3p, and miR-485-3p, had potent chemosensitizing activity with doxorubicin in multiple models of high-risk neuroblastoma. These miRNAs underwent genomic loss in a subset of neuroblastoma patients, and low expression predicted poor survival outcome. In vitro functional assays revealed each of these miRNAs enhanced the anti-proliferative and pro-apoptotic effects of doxorubicin. We used RNA sequencing (RNA-seq) to show that miR-99b-5p represses neuroblastoma dependency genes LIN28B and PHOX2B both in vitro and in patient-derived xenograft (PDX) tumors. Luciferase reporter assays demonstrate that PHOX2B is a direct target of miR-99b-5p. We anticipate that restoring the function of the tumor-suppressive miRNAs discovered here may be a valuable therapeutic strategy for the treatment of neuroblastoma patients.

Asunto(s)

MicroARNs , Neuroblastoma , Niño , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética

RESUMEN

Nonalcoholic fatty liver disease (NAFLD) is characterized by high morbidity. Although long noncoding RNAs (lncRNAs) are known to have a role in NAFLD pathogenesis, the identified lncRNA types are limited. In this study, NAFLD models were established in vitro and in vivo using free fatty acid-treated LO2 cells and high-fat diet-fed mice, respectively. Microarray data were downloaded from the Gene Expression Omnibus database, and AC012668 was selected for further analysis. Cell viability and apoptosis were measured using Cell Counting Kit 8 and flow cytometry assays. RNA expression was detected using reverse transcription-quantitative polymerase chain reaction. Triglyceride (TG) content and lipid deposition were detected using enzyme-linked immunosorbent assay and Oil-Red O staining. Western blotting was used to visualize protein expression. Starbase and TargetScan were used to predict the target miRNA and gene, and the predictions were verified through RNA pull-down and luciferase reporter assays. AC012668 expression levels were significantly suppressed in NAFLD models, whereas AC012668 overexpression inhibited lipogenesis-related gene (SCD1, SREBP1, FAS) expression and TG/lipid accumulation in vitro. Subsequently, miR-380-5p was predicted and verified to target AC012668, and its expression was notably increased in the NAFLD cell model. Moreover, transfection of miR-380-5p antagonized the effects of AC012668 on lipid formation and accumulation. LRP2 was confirmed to be the target gene of miR-380-5p and was downregulated in the NAFLD cell model. Silencing LRP2 reversed the effects of the miR-380-5p inhibitor on lipid formation and accumulation. AC012668 inhibited NAFLD progression via the miR-380-5p/LRP2 axis. These findings may provide a novel strategy against NAFLD.

Asunto(s)

Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , MicroARNs/genética , Enfermedad del Hígado Graso no Alcohólico , ARN Largo no Codificante/genética , Animales , Línea Celular , Humanos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , ARN Largo no Codificante/metabolismo

RESUMEN

BACKGROUND: This study aimed to explore the role of miR-380-5p in cerebral ischemia/reperfusion (CIR) injury-induced neuronal cell death and the potential signaling pathway involved. METHODOLOGY: Human neuroblastoma cell line SH-SY5Y cells were used in this study. Oxygen and glucose deprivation/reperfusion (OGD/R) model was used to mimic ischemia/reperfusion injury. CCK-8 assay and flow cytometry were used to examine cell survival. Quantitative real time PCR (RT-qPCR) assay and Western blotting were used to measure the change of RNA and protein expression, respectively. TargetScan and Luciferase assay was used to confirm the target of miR-380-5p. Malondialdehyde (MDA) superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) were measured using commercial kits. RESULTS: miR-380-5p was downregulated in SH-SY5Y cells after OGD/R. Cell viability was increased by miR-380-5p, while cell apoptosis was reduced by miR-380-5p mimics. MDA was reduced by miR-380-5p mimics, while SOD and GSHPx were increased by miR-380-5p. Results of TargetScan and luciferase assay have showed that BACH1 is the direct target of miR-380-5p. Expression of NRF2 was upregulated after OGD/R, but was not affected by miR-380-5p. mRNA expression of HO-1 and NQO1 and ARE activity were increased by miR-380-5p. Overexpression of BACH1 reversed the antioxidant and neuroprotective effects of miR-380-5p. CONCLUSION: miR-380-5p inhibited cell death induced by CIR injury through target BACH1 which also facilitated the activation of NRF2, indicating the antioxidant and neuroprotective effects of miR-380-5p.

RESUMEN

Bladder cancer is one of the most commonly diagnosed and fatal malignancies of the urinary tract. Noncoding RNAs have been reported to be new biomarkers and effective treatment targets for bladder cancer. In the present study, we identified a novel bladder cancer-related circRNA-miRNA-mRNA network, the circ_0004463/miR-380-3p/FOXO1 axis. circ_0004463 is significantly downregulated, whereas miR-380-3p is upregulated in bladder carcinoma tissue samples and cells. circ_0004463 acts as a tumor suppressor by inhibiting bladder cancer cell proliferation. Genes that negatively correlated with miR-380-3p and genes that miR-380-3p might target are enriched in mitochondrial respiration chain-related pathways. miR-380-3p promotes the proliferation of bladder cancer cells and mitochondrial respiration by acting as an oncogenic miRNA. circ_0004463 competes with FOXO1 for miR-380-3p binding to counteract miR-380-3p-mediated repression of FOXO1. Circ_0004463 overexpression inhibits cancer cell proliferation and mitochondrial respiration in bladder cancer cell lines, while miR-380-3p overexpression dramatically reverses the roles of circ_0004463 overexpression. In conclusion, the circ_0004463/miR-380-3p/FOXO1 axis could regulate mitochondrial respiration and bladder cancer cell apoptosis via FOXO1 signaling.

Asunto(s)

Apoptosis/genética , Proteína Forkhead Box O1/metabolismo , MicroARNs/metabolismo , Mitocondrias/metabolismo , ARN Circular/metabolismo , Transducción de Señal/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Respiración de la Célula/genética , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , MicroARNs/genética , Oncogenes , Fenotipo , ARN Circular/genética , Transfección , Regulación hacia Arriba/genética , Neoplasias de la Vejiga Urinaria/patología

RESUMEN

BACKGROUND: Spinal cord injury (SCI) is a severe central nervous system trauma. The present study aimed to evaluate the effect of HIF-1α on inflammation in spinal cord injury (SCI) to uncover the molecular mechanisms of anti-inflammation. RESULTS: HIF-1α was reduced in SCI model rats and HIF-1α activation reduced TNF-α, IL-1ß, IL-6 and IL-18 levels in SCI model rats. Meanwhile, Circ 0001723 expression was down-regulated and miR-380-3p expression was up-regulated in SCI model rats. In vitro model, down-regulation of Circ 0001723 promoted TNF-α, IL-1ß, IL-6 and IL-18 levels, compared with control negative group. However, over-expression of Circ 0001723 reduced TNF-α, IL-1ß, IL-6 and IL-18 levels in vitro model. Down-regulation of Circ 0001723 suppressed HIF-1α protein expressions and induced NLRP3 and Caspase-1 protein expressions in vitro model by up-regulation of miR-380-3p. Next, inactivation of HIF-1α reduced the pro-inflammation effects of Circ 0001723 in vitro model. Then, si-NLRP3 also inhibited the pro-inflammation effects of Circ 0001723 in vitro model via promotion of autophagy. CONCLUSIONS: We concluded that HIF-1α reduced inflammation in spinal cord injury via miR-380-3p/ NLRP3 by Circ 0001723.

Asunto(s)

Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/metabolismo , MicroARNs/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , ARN Circular/genética , Traumatismos de la Médula Espinal/metabolismo , Animales , Citocinas/sangre , Regulación de la Expresión Génica , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

BACKGROUND: Chronic obstructive pulmonary disease (COPD) not only causes respiratory damage, but also affects circulatory function, which can be life-threatening in severe cases. Therefore, it is very essential to reveal the molecular mechanism of its pathogenesis. METHODS: Human Bronchial Epithelial cells were exposed to 20% cigarette smoke extract (CSE) condition to simulate the cells in COPD patients. GEO database was applied to analyze the expression of miR-380 in COPD patients. The expression level of miR-380 in CSE model was determined with qRT-PCR. Cells proliferation, apoptosis, and inflammation response-related factors were detected with cell counting kit 8, flow cytometry, and Western blot, respectively. A correlation between miR-380 and Cholinergic Receptor Nicotinic Alpha 4 subunit (CHRNA4) was predicted with bioinformatics software, and confirmed by dual luciferase assay. Rescue assay was applied to explore further relationship between miR-380 and CHRNA4. RESULTS: miR-380 showed a tendency of high expression in COPD patients and CSE models. Overexpression of miR-380 promoted the inhibitory effect of cells proliferation, and promotion effects of cells apoptosis and inflammation response, which were caused by CSE. CHRNA4, which was lower expressed in COPD patients, was affirmed as a target of miR-380 and negatively modulated by miR-380. Rescue assay indicated that exhausting of CHRNA4 attenuated the moderating effects of miR-380 inhibitor on cells damage induced by CSE. CONCLUSIONS: Depletion of miR-380 alleviated cells damage caused by CSE through targeting CHRNA4, suggesting that miR-380/CHRNA4 may serve as novel therapeutic targets for COPD treatment.

Asunto(s)

Bronquios/patología , Células Epiteliales/metabolismo , MicroARNs/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Receptores Nicotínicos/metabolismo , Secuencia de Bases , Fumar Cigarrillos , Regulación hacia Abajo/genética , Células Epiteliales/patología , Técnicas de Silenciamiento del Gen , Humanos , MicroARNs/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , Receptores Nicotínicos/genética

RESUMEN

BACKGROUND: Spinal cord injury (SCI) is a severe central nervous system trauma. The present study aimed to evaluate the effect of HIF-1α on inflammation in spinal cord injury (SCI) to uncover the molecular mechanisms of anti-inflammation. RESULTS: HIF-1α was reduced in SCI model rats and HIF-1α activation reduced TNF-α, IL-1ß, IL-6 and IL-18 levels in SCI model rats. Meanwhile, Circ 0001723 expression was down-regulated and miR-380-3p expression was up-regulated in SCI model rats. In vitro model, down-regulation of Circ 0001723 promoted TNF-α, IL-1ß, IL-6 and IL-18 levels, compared with control negative group. However, over-expression of Circ 0001723 reduced TNF-α, IL-1ß, IL-6 and IL-18 levels in vitro model. Down-regulation of Circ 0001723 suppressed HIF-1α protein expressions and induced NLRP3 and Caspase-1 protein expressions in vitro model by up-regulation of miR-380-3p. Next, inactivation of HIF-1α reduced the pro-inflammation effects of Circ 0001723 in vitro model. Then, si-NLRP3 also inhibited the pro-inflammation effects of Circ 0001723 in vitro model via promotion of autophagy. CONCLUSIONS: We concluded that HIF-1α reduced inflammation in spinal cord injury via miR-380-3p/ NLRP3 by Circ 0001723.

Asunto(s)

Animales , Masculino , Ratas , Traumatismos de la Médula Espinal/metabolismo , MicroARNs/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , ARN Circular/genética , Inflamación/metabolismo , Regulación de la Expresión Génica , Citocinas/sangre , Ratas Sprague-Dawley

RESUMEN

Laboratorial and epidemiological research has established a relationship between paraquat (PQ) exposure and a risk for Parkinson's disease. Previously, we have investigated the effects of nuclear factor erythroid 2 related factor 2 (Nrf2) and microRNAs in PQ-induced neurotoxicity, addressing the function of miR-380-3p, a microRNA dysregulated by PQ, as well as Nrf2 deficiency. Nrf2 is known to mediate the expression of a variety of genes, including noncoding genes. By chromatin immunoprecipitation, we identified the relationship between Nrf2 and miR-380-3p in transcriptional regulation. qRT-PCR, Western blots, and dual-luciferase reporter gene assay showed that miR-380-3p blocked the translation of the transcription factor specificity protein-3 (Sp3) in the absence of degradation of Sp3 mRNA. Results based on cell counting analysis, annexin v-fluorescein isothiocyanate/propidium iodide double-staining assay, and propidium iodide staining showed that overexpression of miR-380-3p inhibited cell proliferation, increased the apoptotic rate, induced cell cycle arrest, and intensified the toxicity of PQ in mouse neuroblastoma (N2a [Neuro2a]) cells. Knockdown of Sp3 inhibited cell proliferation and eclipsed the alterations induced by miR-380-3p in cell proliferation. Two mediators of apoptosis and cell cycle identified in previous studies as Sp3-regulated, namely cyclin-dependent kinase inhibitor 1 (p21) and calmodulin (CaM), were dysregulated by PQ, but not Sp3 deficiency. In conclusion, Nrf2-regulated miR-380-3p inhibited cell proliferation and enhanced the PQ-induced toxicity in N2a cells potentially by blocking the translation Sp3 mRNA. We conclude that CaM and p21 were involved in PQ-induced toxicity.