RESUMO
Nuclear localization of the metabolic enzyme PKM2 is widely observed in various cancer types. We identify nuclear PKM2 as a non-canonical RNA-binding protein (RBP) that specifically interacts with folded RNA G-quadruplex (rG4) structures in precursor mRNAs (pre-mRNAs). PKM2 occupancy at rG4s prevents the binding of repressive RBPs, such as HNRNPF, and promotes the expression of rG4-containing pre-mRNAs (the "rG4ome"). We observe an upregulation of the rG4ome during epithelial-to-mesenchymal transition and a negative correlation of rG4 abundance with patient survival in different cancer types. By preventing the nuclear accumulation of PKM2, we could repress the rG4ome in triple-negative breast cancer cells and reduce migration and invasion of cancer cells in vitro and in xenograft mouse models. Our data suggest that the balance of folded and unfolded rG4s controlled by RBPs impacts gene expression during tumor progression.
RESUMO
p53 is an intensely studied tumor-suppressive transcription factor. Recent studies suggest that the RNA-binding protein (RBP) ZMAT3 is important in mediating the tumor-suppressive effects of p53. Here, we globally identify ZMAT3-regulated RNAs and their binding sites at nucleotide resolution in intact colorectal cancer (CRC) cells. ZMAT3 binds to thousands of mRNA precursors, mainly at intronic uridine-rich sequences and affects their splicing. The strongest alternatively spliced ZMAT3 target was CD44, a cell adhesion gene and stem cell marker that controls tumorigenesis. Silencing ZMAT3 increased inclusion of CD44 variant exons, resulting in significant up-regulation of oncogenic CD44 isoforms (CD44v) and increased CRC cell growth that was rescued by concurrent knockdown of CD44v Silencing p53 phenocopied the loss of ZMAT3 with respect to CD44 alternative splicing, suggesting that ZMAT3-mediated regulation of CD44 splicing is vital for p53 function. Collectively, our findings uncover a p53-ZMAT3-CD44 axis in growth suppression in CRC cells.
Assuntos
Processamento Alternativo/genética , Receptores de Hialuronatos/genética , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Carcinogênese/genética , Neoplasias Colorretais/genética , Técnicas de Silenciamento de Genes , Inativação Gênica , Células HCT116 , Células HEK293 , Humanos , Receptores de Hialuronatos/metabolismo , Ligação Proteica/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators Pax5 and Arid3a as well as Igf2bp3 mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.
Assuntos
Reprogramação Celular/genética , Proteínas de Ligação a DNA/metabolismo , Redes Reguladoras de Genes , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/genética , Camundongos , MicroRNAs/metabolismo , Modelos Animais , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
In mammals, gene silencing by the RNA-induced silencing complex (RISC) is a well-understood cytoplasmic posttranscriptional gene regulatory mechanism. Here, we show that embryonic stem cells (ESCs) contain high levels of nuclear AGO proteins and that in ESCs nuclear AGO protein activity allows for the onset of differentiation. In the nucleus, AGO proteins interact with core RISC components, including the TNRC6 proteins and the CCR4-NOT deadenylase complex. In contrast to cytoplasmic miRNA-mediated gene silencing that mainly operates on cis-acting elements in mRNA 3' untranslated (UTR) sequences, in the nucleus AGO binding in the coding sequence and potentially introns also contributed to post-transcriptional gene silencing. Thus, nuclear localization of AGO proteins in specific cell types leads to a previously unappreciated expansion of the miRNA-regulated transcriptome.
Assuntos
Proteínas Argonautas/fisiologia , Inativação Gênica/fisiologia , MicroRNAs/fisiologia , Animais , Proteínas Argonautas/genética , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular , Citoplasma , Células-Tronco Embrionárias/metabolismo , Humanos , Mamíferos , Camundongos , MicroRNAs/genética , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro , RNA Interferente Pequeno , Proteínas de Ligação a RNA , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Fatores de TranscriçãoRESUMO
Hyperlactatemia often occurs in critically ill patients during severe sepsis/septic shock and is a powerful predictor of mortality. Lactate is the end product of glycolysis. While hypoxia due to inadequate oxygen delivery may result in anaerobic glycolysis, sepsis also enhances glycolysis under hyperdynamic circulation with adequate oxygen delivery. However, the molecular mechanisms involved are not fully understood. Mitogen-activated protein kinase (MAPK) families regulate many aspects of the immune response during microbial infections. MAPK phosphatase (MKP)-1 serves as a feedback control mechanism for p38 and JNK MAPK activities via dephosphorylation. Here, we found that mice deficient in Mkp-1 exhibited substantially enhanced expression and phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) 3, a key enzyme that regulates glycolysis following systemic Escherichia coli infection. Enhanced PFKFB3 expression was observed in a variety of tissues and cell types, including hepatocytes, macrophages, and epithelial cells. In bone marrow-derived macrophages, Pfkfb3 was robustly induced by both E. coli and lipopolysaccharide, and Mkp-1 deficiency enhanced PFKFB3 expression with no effect on Pfkfb3 mRNA stability. PFKFB3 induction was correlated with lactate production in both WT and Mkp-1-/- bone marrow-derived macrophage following lipopolysaccharide stimulation. Furthermore, we determined that a PFKFB3 inhibitor markedly attenuated lactate production, highlighting the critical role of PFKFB3 in the glycolysis program. Finally, pharmacological inhibition of p38 MAPK, but not JNK, substantially attenuated PFKFB3 expression and lactate production. Taken together, our studies suggest a critical role of p38 MAPK and MKP-1 in the regulation of glycolysis during sepsis.
Assuntos
Fosfatase 1 de Especificidade Dupla , Glicólise , Sepse , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Camundongos , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 1 de Especificidade Dupla/metabolismo , Escherichia coli/metabolismo , Lactatos , Lipopolissacarídeos , Oxigênio , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Sepse/genética , Fosfofrutoquinase-2/metabolismoRESUMO
Mitogen-activated protein kinase phosphatase 1 (Mkp-1) KO mice produce elevated cytokines and exhibit increased mortality and bacterial burden following systemic Escherichia coli infection. To understand how Mkp-1 affects immune defense, we analyzed the RNA-Seq datasets previously generated from control and E. coli-infected Mkp-1+/+ and Mkp-1-/- mice. We found that E. coli infection markedly induced programmed death-ligand 1 (PD-L1) expression and that Mkp-1 deficiency further amplified PD-L1 expression. Administration of a PD-L1-neutralizing monoclonal antibody (mAb) to Mkp-1-/- mice increased the mortality of the animals following E. coli infection, although bacterial burden was decreased. In addition, the PD-L1-neutralizing mAb increased serum interferon (IFN)-γ and tumor necrosis factor alpha, as well as lung- and liver-inducible nitric oxide synthase levels, suggesting an enhanced inflammatory response. Interestingly, neutralization of IFN-α/ß receptor 1 blocked PD-L1 induction in Mkp-1-/- mice following E. coli infection. PD-L1 was potently induced in macrophages by E. coli and lipopolysaccharide in vitro, and Mkp-1 deficiency exacerbated PD-L1 induction with little effect on the half-life of PD-L1 mRNA. In contrast, inhibitors of Janus kinase 1/2 and tyrosine kinase 2, as well as the IFN-α/ß receptor 1-neutralizing mAb, markedly attenuated PD-L1 induction. These results suggest that the beneficial effect of type I IFNs in E. coli-infected Mkp-1-/- mice is, at least in part, mediated by Janus kinase/signal transducer and activator of transcription-driven PD-L1 induction. Our studies also support the notion that enhanced PD-L1 expression contributes to the bactericidal defect of Mkp-1-/- mice.
Assuntos
Antígeno B7-H1 , Fosfatase 1 de Especificidade Dupla , Infecções por Escherichia coli , Regulação da Expressão Gênica , Interferon Tipo I , Animais , Antígeno B7-H1/genética , Fosfatase 1 de Especificidade Dupla/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Infecções por Escherichia coli/imunologia , Regulação da Expressão Gênica/imunologia , Interferon Tipo I/genética , CamundongosRESUMO
Coronary microembolization (CME) is an intractable complication results from acute coronary syndrome. CME-induced myocardial apoptosis was associated with progressive cardiac contractile dysfunction. miR-29b-3p has been reported implicated in variety cardiovascular diseases, but its function in CME-induced myocardial injury is yet unknown. Herein, a rat model of CME was established by injecting microspheres into the left ventricle and found that the expression level of miR-29b-3p was markedly decreased in the CME rat heart tissues. By using echocardiography, CD31 immunohistochemistry staining, hematoxylin basic fuchsin picric acid (HBFP) staining, TUNEL staining, and western blotting analysis after CME, it was found that upregulating miR-29b-3p improved cardiac dysfunction, promoted angiogenesis, decreased myocardial microinfarct area, and inhibited myocardial apoptosis. Additionally, miR-29b-3p inhibition can reverse the protective benefits of miR-29b-3p overexpression. Mechanistically, the target genes of miR-29b-3p were identified as glycogen synthase kinase 3 (GSK-3ß) and Bcl-2 modifying factor (BMF) by bioinformatics analysis and luciferase reporter experiment. Overall, our findings imply that induction of miR-29b-3p, which negatively regulates GSK-3ß and BMF expression, attenuates CME-induced myocardial injury, suggesting a novel potential therapeutic target for cardioprotective after CME.
Assuntos
MicroRNAs , Ratos , Animais , Glicogênio Sintase Quinase 3 beta/genética , Regulação para Cima , MicroRNAs/genética , Apoptose/genética , Miocárdio/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genéticaRESUMO
Repressor element 1-silencing transcription factor (REST) plays a crucial role in the differentiation of neural progenitor cells (NPCs). C-terminal domain small phosphatases (CTDSPs) are REST effector proteins that reduce RNA polymerase II activity on genes required for neurogenesis. miR-26b regulates neurogenesis in zebrafish by targeting ctdsp2 mRNA, but the molecular events triggered by this microRNA (miR) remain unknown. Here, we show in a murine embryonic stem cell differentiation paradigm that inactivation of miR-26 family members disrupts the formation of neurons and astroglia and arrests neurogenesis at the neural progenitor level. Furthermore, we show that miR-26 directly targets Rest, thereby inducing the expression of a large set of REST complex-repressed neuronal genes, including miRs required for induction of the neuronal gene expression program. Our data identify the miR-26 family as the trigger of a self-amplifying system required for neural differentiation that acts upstream of REST-controlled miRs.
Assuntos
MicroRNAs , Animais , Diferenciação Celular/genética , Camundongos , MicroRNAs/genética , Neurogênese/genética , RNA Mensageiro/genética , Proteínas Repressoras , Fatores de Transcrição , Peixe-Zebra/genéticaRESUMO
We have previously shown that Mkp-1-deficient mice produce elevated TNF-α, IL-6, and IL-10 following systemic Escherichia coli infection, and they exhibited increased mortality, elevated bacterial burden, and profound metabolic alterations. To understand the function of Mkp-1 during bacterial infection, we performed RNA-sequencing analysis to compare the global gene expression between E. coli-infected wild-type and Mkp-1 -/- mice. A large number of IFN-stimulated genes were more robustly expressed in E. coli-infected Mkp-1 -/- mice than in wild-type mice. Multiplex analysis of the serum cytokine levels revealed profound increases in IFN-ß, IFN-γ, TNF-α, IL-1α and ß, IL-6, IL-10, IL-17A, IL-27, and GMSF levels in E. coli-infected Mkp-1 -/- mice relative to wild-type mice. Administration of a neutralizing Ab against the receptor for type I IFN to Mkp-1 -/- mice prior to E. coli infection augmented mortality and disease severity. Mkp-1 -/- bone marrow-derived macrophages (BMDM) produced higher levels of IFN-ß mRNA and protein than did wild-type BMDM upon treatment with LPS, E. coli, polyinosinic:polycytidylic acid, and herring sperm DNA. Augmented IFN-ß induction in Mkp-1 -/- BMDM was blocked by a p38 inhibitor but not by an JNK inhibitor. Enhanced Mkp-1 expression abolished IFN-ß induction by both LPS and E. coli but had little effect on the IFN-ß promoter activity in LPS-stimulated RAW264.7 cells. Mkp-1 deficiency did not have an overt effect on IRF3/7 phosphorylation or IKK activation but modestly enhanced IFN-ß mRNA stability in LPS-stimulated BMDM. Our results suggest that Mkp-1 regulates IFN-ß production primarily through a p38-mediated mechanism and that IFN-ß plays a beneficial role in E. coli-induced sepsis.
Assuntos
Fosfatase 1 de Especificidade Dupla/metabolismo , Infecções por Escherichia coli/metabolismo , Interferon beta/metabolismo , Animais , Células Cultivadas , Fosfatase 1 de Especificidade Dupla/deficiência , Fosfatase 1 de Especificidade Dupla/imunologia , Infecções por Escherichia coli/imunologia , Interferon beta/genética , Interferon beta/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células RAW 264.7 , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND/AIMS: Microvascular obstruction (MVO), an undesirable complication of percutaneous coronary intervention, is independently associated with adverse left ventricle remodeling and poor prognosis after acute myocardial infarction. Hypoxia and oxidative stress major roles in the pathophysiology of MVO. Pim1 serves an important protective role in the ischemic myocardium, but the underlying mechanisms remain poorly defined. Autophagy in early hypoxia or during moderate oxidative stress has been demonstrated to protect the myocardium. In this study, we investigated the association between the protective effect of Pim1 and autophagy after hypoxia and oxidative stress. METHODS: Ventricular myocytes from neonatal rat heart (NRVMs) were isolated. NRVMs were exposed to hypoxia and H2O2. Rapamycin and 3-methyladenine (3-MA) were used as an activator and inhibitor of autophagy, respectively. pHBAd-Pim1 was transfected into NRVMs. We assessed cardiomyocyte apoptosis by Annexin V-FITC/PI flow cytometry. Autophagy was evaluated by mRFP-GFP-LC3 adenovirus infection by confocal microscopy. Western blotting was used to quantify apoptosis or autophagy protein (caspase-3, LC3, P62, AMPK, mTOR, ATG5) concentrations. RESULTS: Autophagy and apoptosis in NRVMs significantly increased and peaked at 3 h and 6 h, respectively, after exposure to hypoxia and H2O2. The mTOR inhibitor rapamycin induced autophagy and decreased cardiomyocyte apoptosis, but the autophagy inhibitor 3-MA decreased autophagy and increased apoptosis at 3 h after exposure to hypoxia and H2O2. Pim1 levels in NRVMs increased at 3 h and decreased gradually after exposure to hypoxia and H2O2. Pim1 overexpression enhanced autophagy and decreased apoptosis. Pim1-induced promotion of autophagy is partly the result of activation of the AMPK/mTOR/ATG5 pathway after exposure to hypoxia and H2O2. CONCLUSION: Our results revealed that Pim1 overexpression prevented NRVMs from apoptosis via upregulating autophagy after exposure to hypoxia and oxidative stress, partly through activation of the AMPK/mTOR/ATG5 autophagy pathway.
Assuntos
Autofagia , Hipóxia Celular , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/metabolismo , Células Cultivadas , Peróxido de Hidrogênio/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-pim-1/genética , Ratos , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Myocardial apoptosis is considered to be the chief cause of progressive cardiac dysfunction induced by coronary microembolization (CME), and the Nrf2/HO-1 signaling pathway is involved in CME-induced myocardial apoptosis. Nicorandil (NIC) has multiple beneficial cardiovascular effects on myocardial injury. Therefore, this study was undertaken to analyze the role of NIC pretreatment in the inhibiting myocardial apoptosis after CME in rats. METHODS: Forty rats were divided into Sham group, CME group, CME plus NIC (NIC) group, and CME plus AAV9-Nrf2 (AAV9-Nrf2) group (nâ¯=â¯10 per group). CME-induced myocardial apoptosis model was established through injecting plastic microspheres (42⯵M) into the left ventricle except the Sham group. NIC group received nicorandil 3â¯mg/(kg.d) for 7 days before the operation. Cardiac function was assessed by echocardiography. The mRNA expression level of Nrf2 was detected by RT-PCR. The protein expression levels of Nrf2, HO-1, Bcl-2, Bax and cleaved caspase-3 were detected by Western blot. The size of the microinfarction area was measured by HBFP staining; myocardial apoptosis was analyzed by TUNEL staining. RESULTS: Compared with the sham group, the cardiac function and the expression level of Nrf2, HO-1 and Bcl-2were decreased, while myocardial apoptosis and the expression of Bax and cleaved caspase-3 were increased in the CME group. Compared with the CME group, cardiac function was significantly improved, the expression levels of Nrf2, HO-1, and Bcl-2 were increased, the expression of Bax and cleaved caspase-3 were decreased, and the myocardial apoptosis was attenuated in the NIC group and AAV9-Nrf2 group. CONCLUSION: NIC pretreatment effectively inhibit CME-induced myocardial apoptosis and improve cardiac function. The protective effects are mediated through the activation of the Nrf2/HO-1 signaling in cardiomyocytes.
Assuntos
Apoptose , Heme Oxigenase (Desciclizante) , Infarto do Miocárdio , Miócitos Cardíacos , Fator 2 Relacionado a NF-E2 , Nicorandil , Animais , Ratos , Apoptose/efeitos dos fármacos , Cardiotônicos/administração & dosagem , Relação Dose-Resposta a Droga , Heme Oxigenase (Desciclizante)/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Nicorandil/administração & dosagem , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Fator 2 Relacionado a NF-E2/metabolismoRESUMO
Mitogen-activated protein kinase phosphatase (Mkp)-1 exerts its anti-inflammatory activities during Gram-negative sepsis by deactivating p38 and c-Jun N-terminal kinase (JNK). We have previously shown that Mkp-1+/+ mice, but not Mkp-1-/- mice, exhibit hypertriglyceridemia during severe sepsis. However, the regulation of hepatic lipid stores and the underlying mechanism of lipid dysregulation during sepsis remains an enigma. To understand the molecular mechanism underlying the sepsis-associated metabolic changes and the role of Mkp-1 in the process, we infected Mkp-1+/+ and Mkp-1-/- mice with Escherichia coli i.v., and assessed the effects of Mkp-1 deficiency on tissue lipid contents. We also examined the global gene expression profile in the livers via RNA-seq. We found that in the absence of E. coli infection, Mkp-1 deficiency decreased liver triglyceride levels. Upon E. coli infection, Mkp-1+/+ mice, but not Mkp-1-/- mice, developed hepatocyte ballooning and increased lipid deposition in the livers. E. coli infection caused profound changes in the gene expression profile of a large number of proteins that regulate lipid metabolism in wildtype mice, while these changes were substantially disrupted in Mkp-1-/- mice. Interestingly, in Mkp-1+/+ mice E. coli infection resulted in downregulation of genes that facilitate fatty acid synthesis but upregulation of Cd36 and Dgat2, whose protein products mediate fatty acid uptake and triglyceride synthesis, respectively. Taken together, our studies indicate that sepsis leads to a substantial change in triglyceride metabolic gene expression programs and Mkp-1 plays an important role in this process.
Assuntos
Fosfatase 1 de Especificidade Dupla/deficiência , Infecções por Escherichia coli/genética , Perfilação da Expressão Gênica/métodos , Metabolismo dos Lipídeos , Sepse/genética , Animais , Infecções por Escherichia coli/metabolismo , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Fígado/química , Redes e Vias Metabólicas , Camundongos , Sepse/metabolismo , Sepse/microbiologia , Análise de Sequência de RNA , Triglicerídeos/metabolismoRESUMO
BACKGROUND/AIMS: Myocardial apoptosis is heavily implicated in the myocardial injury caused by coronary microembolization (CME), and toll-like receptor 4 (TLR4) is considered to be involved in this apoptotic cascade. Therefore, the present study was designed to investigate the role of TLR4/NF-κB signaling pathway regulated by TAK-242, a selective TLR4 signal transduction inhibitor, in the myocardial apoptosis after CME in rats. METHODS: Forty-five rats were randomized (random number) into three groups: sham, CME and CME + TAK-242 (n = 15 per group).CME was induced by injecting polyethylene microspheres (42µm) into the left ventricular except the sham group. CME + TAK-242 group was treated with TAK-242 (2mg/kg) via the tail vein 30 minutes before CME modeling. Cardiac function was evaluated 6 hours after operation. Tissue biopsy was stained with HBFP to measure the size of micro-infarction area. TUNEL staining was used to detect myocardial apoptosis. Western blot and qPCR were used to evaluate the expression of TLR4, MyD88, NF-κB p65, p-IκBα and Cleaved caspase-3. RESULTS: Cardiac function in the CME group and CME + TAK-242 group were significantly decreased compared with the sham group (P < 0.05) and the micro-infarction area, the apoptotic index, the expression of TLR4, NF-κB p65, p-IκBα and Cleaved caspase-3 were increased significantly (P < 0.05). Cardiac function in the CME + TAK-242 group was significantly improved compared with the CME group (P < 0.05) and the micro-infarction area, the apoptotic index, the expression of TLR4, MyD88, NF-κB p65, p-IκBα and Cleaved caspase-3 were decreased significantly (P < 0.05). CONCLUSIONS: TAK-242 can effectively improve CME-induced cardiac dysfunction by regulating TLR4/NF-κB signaling pathway and then reducing the myocardial apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Doença das Coronárias/metabolismo , Embolia/metabolismo , Miocárdio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Caspase 3/metabolismo , Doença das Coronárias/tratamento farmacológico , Doença das Coronárias/patologia , Embolia/tratamento farmacológico , Embolia/patologia , Masculino , Fator 88 de Diferenciação Mieloide/metabolismo , Miocárdio/patologia , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND/AIMS: Coronary microembolization (CME) can lead to no-reflow or slow reflow, which is one of the important reasons for loss of clinical benefit from myocardial reperfusion therapy. MicroRNAs and autophagy are heavily implicated in the occurrence and development of almost all cardiovascular diseases. Therefore, the present study was designed to investigate the role of miR-30e-3p and autophagy in CME-induced myocardial injury rat model. METHODS: Sixty rats were randomly divided into six groups: sham, CME 1h,3h,6h,9h, and 12h (n = 10 per group). Our CME rat model was created by injecting polyethylene microspheres (42mm) into the left ventricle of the heart; the sham group was injected with same volume of normal saline. The cardiac function and serum cardiac troponin I (cTnI) level of each group was measured. HE staining and HBFP staining were used to evaluate the myocardial micro-infarction area of myocardium tissue samples. Then RT-qPCR and western blot were used to detect the expression of miR-30e-3p and, autophagy related protein LC3-II and p62, respectively. Transmission electron microscope (TEM) was used to identify autophagic vacuoles in tissue samples. RESULTS: The cardiac function of the CME 6h,9h, and 12h groups were significantly decreased compared to the sham group (P < 0.05) and the cTnI level in each group were also significantly increased (P < 0.05). The expression of miR-30e-3p in the CME 6h, 9h and 12h group were decreased significantly compared with the sham group (P < 0.05). Meanwhile, the expression of autophagy related protein LC3-II decreased significantly and p62 increased significantly in the CME 9h and 12h group (P < 0.05). TEM images showed typical autophagic vacuoles for each of the CME groups. CONCLUSIONS: Myocardial miR-30e-3p is down regulated after CME and is accompanied by inhibited autophagy and decreased cardiac function. Therefore, miR-30e-3p may be involved in CME-induced cardiac dysfunction by regulating myocardial autophagy.
Assuntos
Autofagia , Embolia/patologia , Traumatismos Cardíacos/etiologia , MicroRNAs/metabolismo , Animais , Vasos Coronários/lesões , Vasos Coronários/patologia , Modelos Animais de Doenças , Regulação para Baixo , Ecocardiografia , Embolia/complicações , Traumatismos Cardíacos/metabolismo , Traumatismos Cardíacos/patologia , Ventrículos do Coração/fisiopatologia , Masculino , MicroRNAs/genética , Microscopia Eletrônica de Transmissão , Microesferas , Proteínas Associadas aos Microtúbulos/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Polietileno/toxicidade , Ratos , Ratos Sprague-Dawley , Proteína Sequestossoma-1/metabolismo , Troponina I/sangue , Regulação para CimaRESUMO
BACKGROUND: Cardiomyocyte apoptosis is a common pathological manifestation that occurs in several heart diseases. This study aimed to explore the mechanism of microRNA-486 (miR-486) in cardiomyocyte apoptosis by interfering with the p53-activated BCL-2 associated mitochondrial pathway. METHODS: miR-486 mimics and inhibitors were transfected into the primary cardiomyocytes of suckling Sprague-Dawley rat pups, and H2O2 was used to induce apoptosis. Flow cytometry and TUNEL were both used to detect cardiomyocyte apoptosis, while the relative mRNA transcript and protein levels of miR-486, p53, Bbc3, BCL-2, and cleaved caspase-3 were detected using RT-PCR and western blot analysis, respectively. RESULTS: miR-486 overexpression significantly decreased the expressions of p53, Bbc3 and cleaved caspase-3 (P < 0.05), and BCL-2 expression was significantly increased (P < 0.05), which in turn caused a significant decrease in the rate of cardiomyocyte apoptosis (P < 0.05). In contrast, miR-486 silencing resulted in an elevated rate of cardiomyocyte apoptosis (P < 0.05). CONCLUSION: miR-486 may regulate cardiomyocyte apoptosis via p53-mediated BCL-2 associated mitochondrial apoptotic pathway. Therefore, up-regulating miR-486 expression in cardiomyocytes can effectively reduce the activation of the BCL-2 associated mitochondrial apoptotic pathway, consequently protecting cardiomyocytes.
Assuntos
Apoptose , MicroRNAs/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Animais Lactentes , Apoptose/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica , Peróxido de Hidrogênio/toxicidade , MicroRNAs/genética , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Myocardial apoptosis is closely related to myocardial injury caused by coronary microembolization (CME).Nuclear factor erythroid 2-like (Nrf2) has been taken into account as an inhibitor of apoptosis in various tissues. Thus, this research aims to investigate which part Nrf2/HO-1 signaling pathway plays in myocardial apoptosis process following the effect of CME on rats. METHODS: Separate 40 rats then form them into a group of shame, a group of CME, a group of CME plus AAV-Nrf2(AAV-Nrf2 (CME) group) and a group of CME plus AAV-control (AAV-control (CME) group) stochastically and averagely. Rat CME was established by injecting into the left ventricular chamber, with or without pretreatment of adeno-associated virus Nrf2 (AAV-Nrf2). Echocardiological measurements, using Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) to stain, conducting Quantitative PCR in real time (RT-PCR) as well as Western blotting to evaluate the impacts of them functionally, morphologically and molecularly in CME. RESULTS: Nrf2 decreased in cardiomyocytes after CME. Upregulation of Nrf2 inside an organism through AAV connect to improving the function of heart as well as attenuating myocardial apoptosis, following the restrain of proapoptotic mRNAs and proteins like caspase-3, caspase-9 and bax expressing as well as the increase of antiapoptotic mRNA and proteins like HO-1 and bcl-2 expressing. CONCLUSION: Activation of Nrf2/HO-1 pathway can improve CME-induced cardiac dysfunction effectively and also reduce the myocardial apoptosis.
Assuntos
Apoptose , Estenose Coronária , Embolia , Heme Oxigenase (Desciclizante) , Miócitos Cardíacos , Fator 2 Relacionado a NF-E2 , Animais , Ratos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Estenose Coronária/metabolismo , Dependovirus , Modelos Animais de Doenças , Embolia/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Miócitos Cardíacos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Parvovirinae , Ratos Sprague-Dawley , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
BACKGROUND/AIMS: Phosphatase and tensin homolog deleted on chromosome ten (PTEN) has been recognized as a promoter of apoptosis in various tissues, and revealed to be up-regulated in circumstances of coronary microembolization (CME). However, whether this functional protein could be modified by pretreatment of atorvastatin in models of CME has not been disclosed yet. METHODS: Swine CME was induced by intra-coronary injection of inertia plastic microspheres (diameter 42 µm) into left anterior descending coronary, with or without pretreatment of atorvastatin or PTEN siRNA. Echocardiologic measurements, pathologic examination, TUNEL staining and western blotting were applied to assess their functional, morphological and molecular effects in CME. RESULTS: PTEN were aberrantly up-regulated in cardiomyocytes following CME, with both the mRNA and protein levels increased after CME modeling. Pretreatment with atorvastatin could attenuate the induction of PTEN. Furthermore, down-regulation of PTEN in vivo via siRNA was associated with an improved cardiac function, attenuated myocardial apoptosis, and concomitantly inhibited expressions of key proapoptotic proteins such as Bax, cleaved-caspase-3. Interestingly, atorvastatin could markedly attenuate PTEN expression and therefore partially reverse cardiac dysfunction and attenuate the apoptosis of the myocardium following CME. CONCLUSION: Modulation of PTEN was probably as a potential mechanism involved in the beneficial effects of pretreatment of atorvastatin to cardiac function and apoptosis in large animal models of CME.
Assuntos
Apoptose/efeitos dos fármacos , Atorvastatina/farmacologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Caspase 3/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Ecocardiografia , Embolia/etiologia , Embolia/metabolismo , Embolia/patologia , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Troponina I/sangue , Proteína X Associada a bcl-2/metabolismoRESUMO
In the global context of development transformation, digital transformation (DT) has emerged as a prevalent practice among international corporations, facilitating the simultaneous enhancement of economic growth. However, limited research on how digital transformation affects green production, especially at a micro level, poses risks by impeding the understanding of strategies for balancing economic growth and environmental sustainability. This study addresses a critical research gap by elucidating the influence of digitization on green total factor productivity (GTFP). We based our findings on a sample of 280 listed non-financial firms from 2007 to 2021 and computed core variables through text analysis and the super-SBM model. The data analysis using fixed effects models, correlation analysis, instrumental variable approach, and difference-in-differences method. The findings underscore the positive impact of DT on enhancing firms' green innovation, investment efficiency, and internal control, consequently significantly elevating the level of GTFP. Additionally, it was observed that normal DT contributes to the sustainable long-term improvement of a firm's GTFP, whereas excessive DT exhibits a positive impact on short-term GTFP. This study's insights into the positive impact of DT on GTFP serve to guide enterprises and policymakers in navigating a transition towards high-quality development, facilitating a balanced approach that fosters environmental conservation alongside economic growth in both emerging and global contexts.
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Since the 20th century, Red Culture has served as a significant informal institution guiding revolutionary trajectory and developmental course. However, integrating Red Culture into contemporary corporate management and leveraging its constructive influence within today's market-driven economy necessitates comprehensive exploration and thoughtful consideration. This study aims to explore the potential influence of Red Culture on contemporary innovation. Empirical findings reveal substantial and affirmative effects of Red Culture on corporate innovation. Specifically, a heightened Red Culture ambiance correlates with a marked increase in both innovation input and output within corporate. Further investigation underscores Red Culture's pivotal governance role in mitigating strategic manipulation of innovation and research and development practices, especially within the overarching framework of innovation-driven strategies. Moreover, Red Culture synergizes with formal innovation incentive mechanisms, jointly fostering corporate innovation. This study provides micro-level empirical evidence that elucidates the impact of Red Culture on corporate innovation. Additionally, it furnishes valuable policy insights for the practical implementation and enhancement of pertinent Red Culture initiative.
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OBJECTIVES: Recent studies have indicated that radiomics may have excellent performance and clinical application prospects in the differential diagnosis of benign and malignant vertebral compression fractures (VCFs). However, multimodal magnetic resonance imaging (MRI)-based radiomics model is rarely used in the differential diagnosis of benign and malignant VCFs, and is limited to lumbar. Herein, this study intends to develop and validate MRI radiomics models for differential diagnoses of benign and malignant VCFs in patients. METHODS: This cross-sectional study involved 151 adult patients diagnosed with VCF in The First Affiliated Hospital of Soochow University in 2016-2021. The study was conducted in three steps: (i) the original MRI images were segmented, and the region of interest (ROI) was marked out; (ii) among the extracted features, those features with Pearson's correlation coefficient lower than 0.9 and the top 15 with the highest variance and Lasso regression coefficient less than and more than 0 were selected; (iii) MRI images and combined data were studied by logistic regression, decision tree, random forest and extreme gradient boosting (XGBoost) models in training set and the test set (ratio of 8:2), respectively; and the models were further verified and evaluated for the differential diagnosis performance. The evaluated indexes included area under receiver (AUC) of operating characteristic curve, accuracy, sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and 95% confidence intervals (CIs). The AUCs were used to assess the predictive performance of different machine learning modes for benign and malignant VCFs. RESULTS: A total of 1144 radiomics features, and 14 clinical features were extracted. Finally, 12 radiomics features were included in the radiomics model, and 12 radiomics features with 14 clinical features were included in the combined model. In the radiomics model, the differential diagnosis performance in the logistic regression model with the AUC of 0.905 ± 0.026, accuracy of 0.817 ± 0.057, sensitivity of 0.831 ± 0.065, and negative predictive value of 0.813 ± 0.042, was superior to the other three. In the combined model, XGBoost model had the superior differential diagnosis performance with specificity (0.979 ± 0.026) and positive predictive value (0.971 ± 0.035). CONCLUSION: The multimodal MRI-based radiomics model performed well in the differential diagnosis of benign and malignant VCFs, which may provide a tool for clinicians to differentially diagnose VCFs.