RESUMEN
Myocarditis (MC) is a myocardial inflammatory disease that threats human life. Pitavastatin (Pit) is a unique lipophilic statin with potent effects on lowering plasma total cholesterol and triacylglycerols. It has been reported to have pleiotropic effects, such as reducing inflammation and oxidative stress. However, the regulatory mechanism of Pit in MC remains a mystery. Two MC models were established in vitro (lipopolysaccharides-(LPS)-stimulated H9c2 cells) and in vivo (intraperitoneal injection of LPS in mice). The levels of microRNA-106b-5p (miR-106b-5p) and mitogen-activated protein kinase kinase kinase 2 (MAP3K2) were detected. ELISA was used to analyze in vivo cell inflammatory factors and myocardial injury markers, kits were used to detect the expression of antioxidant enzymes, cell counting kit-8 (CCK-8) was used to detect cell proliferation, and flow cytometry was used to detect apoptosis. Hematoxylin and eosin (HE) staining was used to detect the pathological changes of myocardial tissue in mice, and TUNEL staining was used to detect in vivo tissue cell apoptosis. The regulatory mechanism of Pit on miR-106b-5p/MAP3K2 was verified by a series of functional rescue experiments. The results demonstrated that in LPS-induced H9c2 cells, antioxidant enzymes decreased and pro-inflammatory factors and cardiac injury markers increased (p<0.05). However, these phenomenons were attenuated by Pit pretreatment. LPS decreased miR-106b-5p and elevated MAP3K2 in H9c2 cells, while Pit could recover their expression patterns (p<0.05). MAP3K2 was confirmed as a target gene of miR-106b-5p. Upregulating miR-106b-5p or downregulating MAP3K2 could further promote the protective effect of Pit, and vice versa (p<0.05). In addition, in the LPS-induced MC mouse model, histological examination showed that Pit significantly improved the myocardial tissue damage in MC mice, while downregulating miR-106b-5p or upregulating MAP3K2 could suppress the ameliorative effect of Pit (p<0.05). In conclusion, our study demonstrated that Pit ameliorates myocardial injury by suppressing myocardial inflammation and oxidative stress by modulating the miR-106b-5p/MAP3K2 axis.
Asunto(s)
Lipopolisacáridos , MicroARNs , Miocarditis , Estrés Oxidativo , Animales , MicroARNs/metabolismo , MicroARNs/genética , Estrés Oxidativo/efectos de los fármacos , Miocarditis/tratamiento farmacológico , Miocarditis/metabolismo , Miocarditis/patología , Masculino , Ratones , Línea Celular , Lipopolisacáridos/toxicidad , Quinolinas/farmacología , MAP Quinasa Quinasa Quinasa 2/metabolismo , Ratas , Apoptosis/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Miocardio/patología , Miocardio/metabolismo , Ratones Endogámicos BALB C , Inflamación/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patologíaRESUMEN
In this study, Holstein dairy cows raised in Ningxia were selected as the research object. Mammary epithelial cells (BMECs) were extracted from the milk of eight Holstein cows with significantly different milk fat expression rates and transcribed for sequencing. Bioinformatics analysis was used to analyse the correlation of fat milk percentage, and the critical miR-2285f regulating milk fat was screened out. The target gene binding sites were predicted, and 293T cells and mammary epithelial cells were used as miRNA and target gene models for functional verification in vitro. The tissue difference of miR-2285f Holstein cows was quantitatively analysed by transfecting miR-2285f mimic and inhibitor. Assay (dual luciferase reporter gene assay) and quantitative real-time PCR (quantitative real-time PCR, qRT-PCR), triglyceride (TAG) detection, oil red O detection of lipid droplets, Western Blot assay, Edu and Flow cytometry, The molecular regulatory effects of miR-2285f and target gene MAP2K2 on milk fat metabolism of Holstein dairy cows were studied. The wild-type vector and mutant vector of map2k2-3'utr were constructed, and double luciferase reporting experiments were conducted to verify that MAP2K2 was one of the target genes of miR-2285f. According to qRT-PCR and Western Blot analysis, miR-2285f mainly regulates the expression of MAP2K2 protein in BMECs at the translation level. Bta-miR-2285f can promote cell proliferation and slow cell apoptosis by regulating MAP2K2. Bta-miR-2285f can promote triglyceride (TAG) and lipid droplet accumulation in mammary epithelial cells by targeting MAP2K2. Bta-miR-2285f can regulate protein levels of fat milk marker gene PPARG by targeting MAP2K2. In conclusion, miR-2285f can target the expression of the MAP2K2 gene, promote the proliferation of dairy mammary epithelial cells, inhibit cell apoptosis and regulate the milk fat metabolism in dairy mammary epithelial cells. The results of this study revealed the function of miR-2285f in regulating the differential expression of fat milk in Holstein dairy cows at the cellular level. They provided a theoretical and experimental basis for analysing the regulation network of milk fat synthesis of Holstein dairy cows and the molecular breeding of dairy cows.
Asunto(s)
Células Epiteliales , Glándulas Mamarias Animales , MicroARNs , Leche , Animales , Bovinos , MicroARNs/metabolismo , MicroARNs/genética , Femenino , Leche/química , Glándulas Mamarias Animales/metabolismo , Células Epiteliales/metabolismo , MAP Quinasa Quinasa Quinasa 2/metabolismo , MAP Quinasa Quinasa Quinasa 2/genética , Metabolismo de los Lípidos , Triglicéridos/metabolismo , Apoptosis , Humanos , Regulación de la Expresión Génica , Proliferación CelularRESUMEN
MicroRNAs (miRNAs) can positively regulate gene expression through an unconventional RNA activation mechanism involving direct targeting 3' untranslated regions (UTRs). Our prior study found miR-93-5p activates mitogen-activated protein kinase kinase kinase 2 (MAP3K2) in hepatocellular carcinoma (HCC) via its 3'UTR. However, the underlying mechanism remains elusive. Here, we identified two candidate AU-rich element (ARE) motifs (ARE1 and ARE2) adjacent to the miR-93-5p binding site located within the MAP3K2 3'UTR using AREsite2. Luciferase reporter and translation assays validated that only ARE2 participated in MAP3K2 activation. Integrative analysis revealed that human antigen R (HuR), an ARE2-associated RNA-binding protein (RBP), physically and functionally interacted with the MAP3K2 3'UTR. Consequently, an HuR-ARE2 complex was shown to facilitate miR-93-5p-mediated upregulation of MAP3K2 expression. Furthermore, bioinformatics analysis and studies of HCC cells and specimens highlighted an oncogenic role for HuR and positive HuR-MAP3K2 expression correlation. HuR is also an enhancing factor in the positive feedback circuit comprising miR-93-5p, MAP3K2, and c-Jun demonstrated in our prior study. The newly identified HuR-ARE2 involvement enriches the mechanism of miR-93-5p-driven MAP3K2 activation and suggests new therapeutic strategies warranted for exploration in HCC.
Asunto(s)
Regiones no Traducidas 3' , Carcinoma Hepatocelular , Proteína 1 Similar a ELAV , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas , MAP Quinasa Quinasa Quinasa 2 , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Regiones no Traducidas 3'/genética , MAP Quinasa Quinasa Quinasa 2/metabolismo , MAP Quinasa Quinasa Quinasa 2/genética , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Línea Celular Tumoral , Biosíntesis de ProteínasRESUMEN
Aberrant activation of Ras/Raf/mitogen-activated protein kinase (MAPK) signaling is frequently linked to metastatic prostate cancer (PCa); therefore, the characterization of modulators of this pathway is critical for defining therapeutic vulnerabilities for metastatic PCa. The lysine methyltransferase SET and MYND domain 3 (SMYD3) methylates MAPK kinase kinase 2 (MAP3K2) in some cancers, causing enhanced activation of MAPK signaling. In PCa, SMYD3 is frequently overexpressed and associated with disease severity; however, its molecular function in promoting tumorigenesis has not been defined. We demonstrate that SMYD3 critically regulates tumor-associated phenotypes via its methyltransferase activity in PCa cells and mouse xenograft models. SMYD3-dependent methylation of MAP3K2 promotes epithelial-mesenchymal transition associated behaviors by altering the abundance of the intermediate filament vimentin. Furthermore, activation of the SMYD3-MAP3K2 signaling axis supports a positive feedback loop continually promoting high levels of SMYD3. Our data provide insight into signaling pathways involved in metastatic PCa and enhance understanding of mechanistic functions for SMYD3 to reveal potential therapeutic opportunities for PCa.
Asunto(s)
Neoplasias de la Próstata , Masculino , Ratones , Animales , Humanos , Neoplasias de la Próstata/genética , Transducción de Señal , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Metiltransferasas/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , MAP Quinasa Quinasa Quinasa 2/genética , MAP Quinasa Quinasa Quinasa 2/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismoRESUMEN
Circular RNAs (circRNAs) have been identified as vital regulators in cardiovascular diseases, including acute myocardial infarction (AMI). In this study, the function and mechanism of circRNA heparan sulfate proteoglycan 2 (circHSPG2) in hypoxia-induced injury in AC16 cardiomyocytes were investigated. AC16 cells were stimulated with hypoxia to establish an AMI cell model in vitro. Real-time quantitative PCR and western blot assays were performed to quantify the expression levels of circHSPG2, microRNA-1184 (miR-1184), and mitogen-activated protein kinase kinase kinase 2 (MAP3K2). Counting Kit-8 (CCK-8) assay was used to measure cell viability. Flow cytometry was performed to detect cell cycle and apoptosis. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of inflammatory factors. Dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays were used to analyze the relationship between miR-1184 and circHSPG2 or MAP3K2. In AMI serum, circHSPG2 and MAP3K2 mRNA were highly expressed and miR-1184 was down-regulated. Hypoxia treatment elevated HIF1α expression and repressed cell growth and glycolysis. Moreover, hypoxia promoted cell apoptosis, inflammation, and oxidative stress in AC16 cells. Hypoxia-induced circHSPG2 expression in AC16 cells. CircHSPG2 knockdown alleviated hypoxia-induced AC16 cell injury. CircHSPG2 directly targeted miR-1184, and miR-1184 targeted and suppressed MAP3K2. Inhibition of miR-1184 or overexpression of MAP3K2 abolished the alleviated effect of circHSPG2 knockdown on hypoxia-induced AC16 cell injury. Overexpression of miR-1184 relieved hypoxia-induced impairment in AC16 cells by MAP3K2. CircHSPG2 could regulate MAP3K2 expression through miR-1184. CircHSPG2 knockdown protected AC16 cells from hypoxia-induced injury by regulating the miR-1184/MAP3K2 cascade.
Asunto(s)
MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Miocitos Cardíacos/metabolismo , Hipoxia de la Célula , Apoptosis/genética , Hipoxia , Estrés Oxidativo , Inflamación/metabolismo , MAP Quinasa Quinasa Quinasa 2/metabolismoRESUMEN
Aberrant activation of inflammation signaling triggered by tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), and interleukin-17 (IL-17) is associated with immunopathology. Here, we identify neural precursor cells expressed developmentally down-regulated gene 4-like (NEDD4L), a HECT type E3 ligase, as a common negative regulator of signaling induced by TNF-α, IL-1, and IL-17. NEDD4L modulates the degradation of mitogen-activated protein kinase kinase kinase 2 (MEKK2) via constitutively and directly binding to MEKK2 and promotes its poly-ubiquitination. In interleukin-17 receptor (IL-17R) signaling, Nedd4l knockdown or deficiency enhances IL-17-induced p38 and NF-κB activation and the production of proinflammatory cytokines and chemokines in a MEKK2-dependent manner. We further show that IL-17-induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF-κB activation but also for NEDD4L-mediated MEKK2 degradation and the subsequent shutdown of IL-17R signaling. Importantly, Nedd4l-deficient mice show increased susceptibility to IL-17-induced inflammation and aggravated symptoms of experimental autoimmune encephalomyelitis (EAE) in IL-17R signaling-dependent manner. These data suggest that NEDD4L acts as an inhibitor of IL-17R signaling, which ameliorates the pathogenesis of IL-17-mediated autoimmune diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental , MAP Quinasa Quinasa Quinasa 2 , Ubiquitina-Proteína Ligasas Nedd4 , Células-Madre Neurales , Animales , Ratones , Encefalomielitis Autoinmune Experimental/genética , Inflamación/metabolismo , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-17/genética , Células-Madre Neurales/metabolismo , FN-kappa B/metabolismo , Fosforilación , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , MAP Quinasa Quinasa Quinasa 2/metabolismoRESUMEN
OBJECTIVE: This study aimed to investigate the effects of micro ribonucleic acid (miR)-338-3p on the migration, invasion and proliferation of lung adenocarcinoma (LUAD) cells. METHODS: Bioinformatics analysis was employed to evaluate the function and expression of related genes in lung cancer. Human A549 and NCI-H1299 cells cultured to logarithmic growth stage were assigned to negative control (NC) mimic group, miR-338-3p mimic group (miR-mimic group), NC inhibitor group and miR-338-3p inhibitor group (miR-inhibitor group) treated with or without MAP3K2 overexpression (OE)-lentivirus, or TBHQ or FR180204. Transwell assay, cell colony formation assay, Western blotting and cell-cycle analysis were carried out. RESULTS: Bioinformatics results manifested that miR-338 and MAP3K2 were involved in LUAD. The expression levels of MAP3K2, p-ERK1/2, MMP-2, MMP-3, MMP-9, cyclin A2 and cyclin D1 were increased after addition of miR-338-3p inhibitor, consistent with the raised amount of LUAD cells in migration and invasion experiments and number of colonies formed, as well as the cell cycle, but miR-338-3p mimic reversed these results. Moreover, MAP3K2 overexpression elevated the level of p-ERK1/2. Meanwhile, after treatment with TBHQ or FR180204, the influence of miR-338-3p inhibitor or mimic was also verified. CONCLUSIONS: MiR-338-3p overexpression can modulate the ERK1/2 signaling pathway by targeting MAP3K2, thus inhibiting the migration, invasion and proliferation of human LUAD cells.
Asunto(s)
Adenocarcinoma del Pulmón , Neoplasias Pulmonares , MicroARNs , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , MAP Quinasa Quinasa Quinasa 2/genética , MAP Quinasa Quinasa Quinasa 2/metabolismo , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
Bone marrow mesenchymal stem cell (BMSC) chondrogenic differentiation contributes to the treatment of osteoarthritis (OA). Numerous studies have indicated that microRNAs (miRNAs) regulate the pathogenesis and development of multiple disorders, including OA. Nevertheless, the role of miR-20a-5p in OA remains obscure. Forty male C57BL/6 mice were divided into four groups and were surgically induced OA or underwent sham surgery in the presence or absence of miR-20a-5p. Flow cytometry was implemented to detect surface markers of BMSCs. Reverse transcription quantitative polymerase chain reaction revealed the upregulation of miR-20a-5p during BMSC chondrogenic differentiation. Western blotting displayed that miR-20a-5p inhibition decreased protein levels of cartilage matrix markers but enhanced those of catabolic and hypertrophic chondrocyte markers in BMSCs. Alcian blue staining, hematoxylineosin staining and micro-CT revealed that miR-20a-5p inhibition restrained chondrogenic differentiation and miR-20a-5p overexpression promoted the repair of damaged cartilage and subchondral bone, respectively. Luciferase reporter assay identified that mitogen activated protein kinase kinase kinase 2 (Map3k2) was a target of miR-20a-5p in BMSCs. Moreover, the expression of miR-20a-5p and Map3k2 was negatively correlated in murine cartilage tissues. Knocking down Map3k2 could rescue the suppressive influence of miR-20a-5p inhibition on chondrogenic differentiation of BMSCs. In conclusion, miR-20a-5p facilitates BMSC chondrogenic differentiation and contributes to cartilage repair in OA by suppressing Map3k2.
Asunto(s)
MAP Quinasa Quinasa Quinasa 2 , MicroARNs , Osteoartritis , Animales , Cartílago/metabolismo , Cartílago/patología , MAP Quinasa Quinasa Quinasa 2/metabolismo , Quinasas Quinasa Quinasa PAM , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Osteoartritis/metabolismoRESUMEN
BACKGROUND: Hepatitis B virus (HBV) is a top contributor to hepatoma. Circular RNAs (circRNAs) have been elucidated to have a close connection with HBV-induced hepatoma. This study aimed to explore the role of circRNA BTB domain and CNC homolog 1 (circBACH1) in HBV replication and hepatoma progression, as well as the potential mechanistic pathway. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to assess the expression of circBACH1, microRNA (miR)-200a-3p, and mitogen-activated protein kinase kinase kinase 2 (MAP3K2). HBV replication was determined by enzyme-linked immunosorbent assay (ELISA) and qRT-PCR assay. Cell viability and clonogenicity were detected via Cell Counting Kit-8 (CCK-8) assay and colony formation assay, respectively. Cell metastasis was examined by Transwell assay and wound healing assay. Annexing-V/PI staining was employed to monitor cell apoptosis using flow cytometry. Levels of MAP3K2, proliferation- and apoptosis-related proteins were analyzed by Western blotting. Target interaction between miR-200a-3p and circBACH1 or MAP3K2 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The role of circBACH1 in vivo was investigated by xenograft model assay. RESULTS: Expression of circBACH1 and MAP3K2 was increased, while miR-200a-3p expression was decreased in HCC tissues and HBV-transfected hepatoma cells. Depletion of circBACH1 or miR-200a-3p overexpression impeded HBV replication, proliferation, and metastasis in HBV-transfected hepatoma cells. CircBACH1 was able to regulate MAP3K2 expression by sponging miR-200a-3p. CircBACH1 regulated HBV replication and hepatoma progression through the miR-200a-3p/MAP3K2 pathway. Moreover, circBACH1 deficiency hampered tumor growth in vivo. CONCLUSION: CircBACH1 knockdown had inhibitory effects on HBV replication and hepatoma progression, at least partly by modulating the miR-200a-3p/MAP3K2 axis.
Asunto(s)
Carcinoma Hepatocelular , Hepatitis B , Neoplasias Hepáticas , MAP Quinasa Quinasa Quinasa 2 , MicroARNs , ARN Circular , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Proliferación Celular , Hepatitis B/genética , Hepatitis B/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Neoplasias Hepáticas/patología , MAP Quinasa Quinasa Quinasa 2/metabolismo , MicroARNs/genética , ARN Circular/genética , Replicación ViralRESUMEN
γδ T cells are a conserved population of lymphocytes that contributes to anti-tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL-2 or IL-15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA-181a as a key modulator of human γδ T cell differentiation. We observe that miR-181a is highly expressed in patients with prostate cancer and that this pattern associates with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR-181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR-181a overexpression restricts their levels of NKG2D and TNF-α. Upon in silico analysis, we identify two miR-181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR-181a as critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next-generation immunotherapies.
Asunto(s)
Diferenciación Celular , MicroARNs , Receptor Notch2 , Linfocitos T/citología , Humanos , Activación de Linfocitos , MAP Quinasa Quinasa Quinasa 2/metabolismo , Masculino , MicroARNs/genética , Neoplasias de la Próstata , Receptor Notch2/metabolismo , Transducción de SeñalRESUMEN
Previous studies have identified microRNA (miRNA/miR)36133p as a heat stress (HS)related miRNA in endothelial cells that can lead to apoptosis. However, the mechanism underlying the miR36133pmediated apoptosis of HSexposed endothelial cells remains unclear. In the present study, western blot analysis and reverse transcriptionquantitative PCR were used to determine protein and miRNA expression levels, respectively. Annexin Vfluorescein isothiocyanate/propidium iodide staining, caspase3 activity measurements and DNA fragmentation assays were performed to detect apoptosis. To evaluate whether mitogenactivated protein kinase kinase kinase 2 (MAP3K2) was a direct target of miR36133p, a luciferase reporter assay was performed. In addition, transient transfection was used to carry out loss and gainoffunction experiments. The results revealed that miR36133p expression was reduced in human umbilical vein endothelial cells (HUVECs) following HS, which led to apoptosis. Mechanistically, following HS, a decrease in miR36133p binding to the 3'untranslated region of MAP3K2 directly upregulated its expression, and the downstream p38 and caspase3 pathways, thereby leading to apoptosis. Taken together, the results of the present study demonstrated that HS suppressed miR36133p expression, which activated the MAP3K2/p38/caspase3 pathway, leading to the apoptosis of HUVECs. In conclusion, the miR36133p/MAP3K2/p38/caspase3 pathway may serve an indispensable role in regulating the progression of apoptosis, indicating a regulatory role of miR36133p in the pathophysiology of HSexposed endothelial cells.
Asunto(s)
Apoptosis/genética , Caspasa 3/metabolismo , Células Endoteliales/metabolismo , Respuesta al Choque Térmico , MAP Quinasa Quinasa Quinasa 2/metabolismo , MicroARNs/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Regiones no Traducidas 3' , Caspasa 3/genética , Muerte Celular , Fragmentación del ADN , Regulación hacia Abajo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , MAP Quinasa Quinasa Quinasa 2/genética , MicroARNs/genética , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Dysregulation of non-coding RNAs (ncRNAs) has been proved to play pivotal roles in epithelial-mesenchymal transition (EMT) and fibrosis. We have previously demonstrated the crucial function of long non-coding RNA (lncRNA) ATB in silica-induced pulmonary fibrosis-related EMT progression. However, the underlying molecular mechanism has not been fully elucidated. Here, we verified miR-29b-2-5p and miR-34c-3p as two vital downstream targets of lncRNA-ATB. As opposed to lncRNA-ATB, a significant reduction of both miR-29b-2-5p and miR-34c-3p was observed in lung epithelial cells treated with TGF-ß1 and a murine silicosis model. Overexpression miR-29b-2-5p or miR-34c-3p inhibited EMT process and abrogated the pro-fibrotic effects of lncRNA-ATB in vitro. Further, the ectopic expression of miR-29b-2-5p and miR-34c-3p with chemotherapy attenuated silica-induced pulmonary fibrosis in vivo. Mechanistically, TGF-ß1-induced lncRNA-ATB accelerated EMT as a sponge of miR-29b-2-5p and miR-34c-3p and shared miRNA response elements with MEKK2 and NOTCH2, thus relieving these two molecules from miRNA-mediated translational repression. Interestingly, the co-transfection of miR-29b-2-5p and miR-34c-3p showed a synergistic suppression effect on EMT in vitro. Furthermore, the co-expression of these two miRNAs by using adeno-associated virus (AAV) better alleviated silica-induced fibrogenesis than single miRNA. Approaches aiming at lncRNA-ATB and its downstream effectors may represent new effective therapeutic strategies in pulmonary fibrosis.
Asunto(s)
Transición Epitelial-Mesenquimal , MicroARNs/metabolismo , Fibrosis Pulmonar/metabolismo , ARN Largo no Codificante/metabolismo , Células A549 , Animales , Línea Celular , Humanos , MAP Quinasa Quinasa Quinasa 2/genética , MAP Quinasa Quinasa Quinasa 2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patología , ARN Largo no Codificante/genética , Receptor Notch2/genética , Receptor Notch2/metabolismo , Elementos de Respuesta , Factor de Crecimiento Transformador beta/metabolismoAsunto(s)
Intestinos/metabolismo , MAP Quinasa Quinasa Quinasa 2/metabolismo , Nicho de Células Madre/fisiología , Células del Estroma/metabolismo , Animales , Diferenciación Celular/fisiología , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismoRESUMEN
Intestinal stromal cells are known to modulate the propagation and differentiation of intestinal stem cells1,2. However, the precise cellular and molecular mechanisms by which this diverse stromal cell population maintains tissue homeostasis and repair are poorly understood. Here we describe a subset of intestinal stromal cells, named MAP3K2-regulated intestinal stromal cells (MRISCs), and show that they are the primary cellular source of the WNT agonist R-spondin 1 following intestinal injury in mice. MRISCs, which are epigenetically and transcriptomically distinct from subsets of intestinal stromal cells that have previously been reported3-6, are strategically localized at the bases of colon crypts, and function to maintain LGR5+ intestinal stem cells and protect against acute intestinal damage through enhanced R-spondin 1 production. Mechanistically, this MAP3K2 specific function is mediated by a previously unknown reactive oxygen species (ROS)-MAP3K2-ERK5-KLF2 axis to enhance production of R-spondin 1. Our results identify MRISCs as a key component of an intestinal stem cell niche that specifically depends on MAP3K2 to augment WNT signalling for the regeneration of damaged intestine.
Asunto(s)
Mucosa Intestinal/citología , MAP Quinasa Quinasa Quinasa 2/metabolismo , Nicho de Células Madre , Células del Estroma/citología , Animales , Antígenos CD34 , Colitis/patología , Colitis/prevención & control , Epigénesis Genética , Femenino , Mucosa Intestinal/patología , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Especies Reactivas de Oxígeno/metabolismo , Tetraspanina 28 , Trombospondinas/biosíntesis , Trombospondinas/metabolismo , Antígenos Thy-1RESUMEN
The Hippo pathway is an evolutionarily conserved signaling pathway that controls organ size in animals via the regulation of cell proliferation and apoptosis. It consists of a kinase cascade, in which MST1/2 and MAP4Ks phosphorylate and activate LATS1/2, which in turn phosphorylate and inhibit YAP/TAZ activity. A variety of signals can modulate LATS1/2 kinase activity to regulate Hippo pathway. However, the full mechanistic details of kinase-mediated regulation of Hippo pathway signaling remain elusive. Here, we report that TNF activates LATS1/2 and inhibits YAP/TAZ activity through MEKK2/3. Furthermore, MEKK2/3 act in parallel to MST1/2 and MAP4Ks to regulate LATS1/2 and YAP/TAZ in response to various signals, such as serum and actin dynamics. Mechanistically, we show that MEKK2/3 interact with LATS1/2 and YAP/TAZ and phosphorylate them. In addition, Striatin-interacting phosphatase and kinase (STRIPAK) complex associates with MEKK3 via CCM2 and CCM3 to inactivate MEKK3 kinase activity. Upstream signals of Hippo pathway trigger the dissociation of MEKK3 from STRIPAK complex to release MEKK3 activity. Our work has uncovered a previous unrecognized regulation of Hippo pathway via MEKK2/3 and provides new insights into molecular mechanisms for the interplay between Hippo-YAP and NF-κB signaling and the pathogenesis of cerebral cavernous malformations.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , MAP Quinasa Quinasa Quinasa 2/metabolismo , MAP Quinasa Quinasa Quinasa 3/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proliferación Celular/fisiología , Células Cultivadas , Vía de Señalización Hippo , Humanos , Ratones , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVE: Acute myocardial infarction (AMI) is a serious cardiovascular disease with a high incidence worldwide and the main cause of sudden cardiac death. The aim of this article was to study the protective role of miR-335 in myocardial infarction (MI) and the underlying molecular mechanism. MATERIALS AND METHODS: Thirty Sprague Dawley (SD) rats were randomly divided into sham group, MI + NC group and MI + agomiR-335 group. The expression of miR-335 in rat myocardium was detected by quantitative Real Time-Polymerase Chain Reaction (RT-PCR). Western blot was performed to detect the expression of TNF-α, IL-6, IL-1ß, Caspase-3, Cleaved Caspase-3 (C-Caspase-3) and MAP3K2 in rat myocardium. On the 7th day of the establishment of the rat MI model, a high-resolution small animal ultrasound system was utilized to detect the cardiac function of the rats, and the heart tissues and blood samples of the rats were collected. The corresponding kits were purchased to detect the contents of LDH, CK-MB, MDA and SOD in rat serum, and HE staining was employed to observe the morphology of rat myocardial tissue. RESULTS: The expression of miR-335 in myocardial infarcted zones and border zones of MI rats decreased significantly. The upregulation of miR-335 remarkably inhibited myocardial inflammation and apoptosis after MI, thus improving the cardiac function of MI rats. Compared with the sham group, the MAP3K2 expression in the MI + NC group was observably increased, while the overexpression of miR-335 could inhibit the expression of this protein. Through the Luciferase reporter gene experiment, we found that miR-335 could directly target MAP3K2. CONCLUSIONS: The expression of miR-335 decreased in myocardial tissue after MI, and the overexpression of miR-335 reduced myocardial damage by inhibiting oxidative stress, inflammation, and apoptosis via targeting MAP3K2, thereby improving the cardiac function of MI rats.
Asunto(s)
MAP Quinasa Quinasa Quinasa 2/metabolismo , MicroARNs/metabolismo , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Regulación hacia Arriba , Animales , Ecocardiografía , Femenino , MicroARNs/genética , Infarto del Miocardio/patología , Miocitos Cardíacos/patología , Ratas , Ratas Sprague-DawleyRESUMEN
Hepatocellular carcinoma (HCC) is a common disease with a significant mortality, and there is no effective treatment for advanced patients. Growing evidence indicates that circRNAs are closely related to HCC progression, may be used as biomarkers and targets for the diagnosis and treatment of HCC. Recent researches have shown that circPUM1 may play an oncogene role in a variety of human cancers, but its role in HCC development has not been reported. Our study found that circPUM1 could promote the proliferation, migration and invasion of HCC cells in vitro. In addition, in vivo studies showed that circPUM1 could increase the development of HCC tumours and regulate the expression of EMT-related proteins. Furthermore, we demonstrated that circPUM1 could promote the development of HCC by up-regulating the expression of MAP3K2 via sponging miR-1208. Our study suggested that circPUM1 may be a potential therapeutic target for HCC.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , MAP Quinasa Quinasa Quinasa 2/metabolismo , MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Western Blotting , Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Hepáticas/genética , MAP Quinasa Quinasa Quinasa 2/genética , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Neurofibromatosis type I (NF1) is characterized by prominent skeletal manifestations caused by NF1 loss. While inhibitors of the ERK activating kinases MEK1/2 are promising as a means to treat NF1, the broad blockade of the ERK pathway produced by this strategy is potentially associated with therapy limiting toxicities. Here, we have sought targets offering a more narrow inhibition of ERK activation downstream of NF1 loss in the skeleton, finding that MEKK2 is a novel component of a noncanonical ERK pathway in osteoblasts that mediates aberrant ERK activation after NF1 loss. Accordingly, despite mice with conditional deletion of Nf1 in mature osteoblasts (Nf1fl/fl;Dmp1-Cre) and Mekk2-/- each displaying skeletal defects, Nf1fl/fl;Mekk2-/-;Dmp1-Cre mice show an amelioration of NF1-associated phenotypes. We also provide proof-of-principle that FDA-approved inhibitors with activity against MEKK2 can ameliorate NF1 skeletal pathology. Thus, MEKK2 functions as a MAP3K in the ERK pathway in osteoblasts, offering a potential new therapeutic strategy for the treatment of NF1.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Imidazoles/farmacología , MAP Quinasa Quinasa Quinasa 2/metabolismo , Neurofibromatosis 1/etiología , Piridazinas/farmacología , Animales , Modelos Animales de Enfermedad , Activación Enzimática , Proteínas de la Matriz Extracelular/genética , Femenino , Humanos , MAP Quinasa Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa Quinasa 2/genética , Masculino , Ratones Transgénicos , Neurofibromatosis 1/tratamiento farmacológico , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Osteoblastos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Cráneo/citologíaRESUMEN
OBJECTIVE: Myocardial infarction (MI) is caused by myocardial ischemia and hypoxia, which causes irreversible damage to the myocardium and seriously endangers human health. Exosomes are small, monolayer-structured extracellular vesicles that transport proteins, lipids, mRNAs, and miRNAs between cells. Mesenchymal stem cells (MSCs) can secrete a large number of exosomes and play a role in many pathophysiological processes. The purpose of this paper was to investigate the role of exosomal microRNA-338 (miR-338) in MI and its underlying mechanism of action. MATERIALS AND METHODS: We transfected rat bone marrow-derived MSCs with miR-338 mimic or negative control and extracted exosomes secreted by MSCs. Expression of miR-338 in MSCs, exosomes, and H9c2 cells co-cultured with exosomes was detected by PCR. Then, we treated H9c2 cells with H2O2. We transfected miR-338 inhibitor into H9c2 cells co-cultured with exosomes to further study the function of miR-338. Apoptosis of H9c2 cells were observed by Western blot, flow cytometry, and cell staining. We also established a MI rat model to study the function in vivo and injected exosomes in the myocardium. Seven days later, we used echocardiography to detect the heart function of rats. RESULTS: MiR-338 was upregulated in MSCs transfected with miR-338 mimic, exosomes, and H9c2 cells co-cultured with exosomes. When H9c2 cells were co-cultured with exosomes overexpressing miR-338, the expression of Bax was decreased while the expression of Bcl-2 was increased, and the apoptosis rate was also decreased as shown in flow cytometry, and the amount of caspase3 fluorescence was also decreased. Cardiac function was markedly improved after intramyocardial injection of exosomes overexpressing miR-338 in rats. It was demonstrated using computational tools, Western blot, and Luciferase reporter gene experiments that miR-338 could regulate JNK pathway via targeting MAP3K2. CONCLUSIONS: Exosomal miR-338 can inhibit cardiomyocyte apoptosis and improve cardiac function in rats with myocardial infarction by regulating MAP3K2/JNK signaling pathway.
Asunto(s)
Apoptosis/genética , Exosomas , Células Madre Mesenquimatosas , MicroARNs/genética , Infarto del Miocardio/terapia , Miocitos Cardíacos/metabolismo , Animales , Línea Celular , Femenino , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa Quinasa 2/metabolismo , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Ratas Sprague-DawleyRESUMEN
Cumulative evidence suggests that microRNAs (miRNAs) promote gene expression in cancers. However, the pathophysiologic relevance of miRNA-mediated RNA activation in hepatocellular carcinoma (HCC) remains to be established. Our previous miRNA expression profiling in seven-paired HCC specimens revealed miR-93-5p as an HCC-related miRNA. In this study, miR-93-5p expression was assessed in HCC tissues and cell lines by quantitative real-time PCR and fluorescence in situ hybridization. The correlation of miR-93-5p expression with survival and clinicopathological features of HCC was determined by statistical analysis. The function and potential mechanism of miR-93-5p in HCC were further investigated by a series of gain- or loss-of-function experiments in vitro and in vivo. We identified that miR-93-5p, overexpressed in HCC specimens and cell lines, leads to poor outcomes in HCC cases and promotes proliferation, migration, and invasion in HCC cell lines. Mechanistically, rather than decreasing target mRNA levels as expected, miR-93-5p binds to the 3'-untranslated region (UTR) of mitogen-activated protein kinase kinase kinase 2 (MAP3K2) to directly upregulate its expression and downstream p38 and c-Jun N-terminal kinase (JNK) pathway, thereby leading to cell cycle progression in HCC. Notably, we also demonstrated that c-Jun, a downstream effector of the JNK pathway, enhances miR-93-5p transcription by targeting its promoter region. Besides, downregulation of miR-93-5p significantly retarded tumor growth, while overexpression of miR-93-5p accelerated tumor growth in the HCC xenograft mouse model. Altogether, we revealed a miR-93-5p/MAP3K2/c-Jun positive feedback loop to promote HCC progression in vivo and in vitro, representing an RNA-activating role of miR-93-5p in HCC development.