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1.
Mol Med Rep ; 20(5): 4459-4466, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31702042

RESUMO

Uncoupling protein 2 (UCP2) plays a positive role in sepsis. However, the role of UCP2 in experimental sepsis in astrocytes remains unknown. The present study was designed to determine whether UCP2 has a protective effect in an experimental sepsis model in astrocytes asnd to clarify the mechanisms responsible for its neuroprotective effects after sepsis. An experimental astrocyte model mimicking sepsis­induced brain injury was established using lipopolysaccharide (LPS) and interferon (IFN)­Î³. Additionally, UCP2 knockdown in astrocytes was achieved by adenovirus transfection. Tumor necrosis factor (TNF)­α and interleukin (IL)­1ß activity, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels were assessed. The mitochondrial ultrastructure was evaluated, and the expression of UCP2 was determined by western blotting. LPS with IFN­Î³ co­stimulation increased the mRNA and protein expression levels of UCP2 in astrocytes, damaged the mitochondrial structure, and accelerated the release of TNF­α and IL­1ß, resulting in a decrease in the MMP, and the excessive generation of ROS. Moreover, sepsis also caused a reduction in ATP production. The knockdown of UCP2 exacerbated astrocyte injury and mitochondrial impairment. In conclusion, both the function and morphology of mitochondria were damaged in an experimental model of sepsis in astrocytes, and knockdown of UCP2 using shRNA exacerbated this impairment, suggesting that UCP2 has a positive effect on astrocytes as determined in an experimental sepsis model.

2.
Oxid Med Cell Longev ; 2019: 2758262, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31182990

RESUMO

Uncoupling protein 2 (UCP2) has a cardioprotective role under septic conditions, but the underlying mechanism remains unclear. This study aimed at investigating the effects of UCP2 on the oxidative stress and apoptosis of cardiomyocytes induced by lipopolysaccharide (LPS). First, LPS increased UCP2 expression in cardiomyocytes in a time-dependent manner. LPS increased the production of lactate dehydrogenase (LDH), reactive oxygen species (ROS), and malondialdehyde (MDA) and decreased the level of superoxide dismutase (SOD). However, UCP2 knockdown increased the LPS-induced cardiac injury and oxidative stress. In addition, LPS damaged the mitochondrial ultrastructure and led to the disruption of mitochondrial membrane potential (MMP), as well as the release of mitochondrial cytochrome c. UCP2 knockdown aggravated mitochondrial injury and the release of mitochondrial cytochrome c. LPS increased the protein levels of Bax and cleaved-caspase-3, decreased the protein level of Bcl-2, and upregulated the protein level of mitogen-activated protein kinase. However, upon UCP2 knockdown, the protein levels of Bax and cleaved-caspase-3 increased even further, and the protein level of Bcl-2 was further decreased. The protein level of phosphorylated p38 was also further enhanced. Thus, UCP2 protects against LPS-induced oxidative stress and apoptosis in cardiomyocytes.

3.
Cell Death Differ ; 26(12): 2758-2773, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31092884

RESUMO

Yap is the key component of Hippo pathway which plays crucial roles in tumorigenesis. Inhibition of Yap activity could promote apoptosis, suppress proliferation, and restrain metastasis of cancer cells. However, how Yap is regulated is not fully understood. Here, we reported Yap being negatively regulated by its circular RNA (circYap) through the suppression of the assembly of Yap translation initiation machinery. Overexpression of circYap in cancer cells significantly decreased Yap protein but did not affect its mRNA levels. As a consequence, it remarkably suppressed proliferation, migration and colony formation of the cells. We found that circYap could bind with Yap mRNA and the translation initiation associated proteins, eIF4G and PABP. The complex containing overexpressed circYap abolished the interaction of PABP on the poly(A) tail with eIF4G on the 5'-cap of the Yap mRNA, which functionally led to the suppression of Yap translation initiation. Individually blocking the binding sites of circYap on Yap mRNA or respectively mutating the binding sites for PABP and eIF4G derepressed Yap translation. Significantly, breast cancer tissue from patients in the study manifested dysregulation of circYap expression. Collectively, our study uncovered a novel molecular mechanism in the regulation of Yap and implicated a new function of circular RNA, supporting the pursuit of circYap as a potential tool for future cancer intervention.

4.
Oncogene ; 37(44): 5829-5842, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29973691

RESUMO

Circular RNAs are a large group of noncoding RNAs that are widely expressed in mammalian cells. Genome-wide analyses have revealed abundant and evolutionarily conserved circular RNAs across species, which suggest specific physiological roles of these species. Using a microarray approach, we detected increased expression of a circular RNA circ-Dnmt1 in eight breast cancer cell lines and in patients with breast carcinoma. Silencing circ-Dnmt1 inhibited cell proliferation and survival. Ectopic circ-Dnmt1 increased the proliferative and survival capacities of breast cancer cells by stimulating cellular autophagy. We found that circ-Dnmt1-mediated autophagy was essential in inhibiting cellular senescence and increasing tumor xenograft growth. We further found that ectopically expressed circ-Dnmt1 could interact with both p53 and AUF1, promoting the nuclear translocation of both proteins. Nuclear translocation of p53 induced cellular autophagy while AUF1 nuclear translocation reduced Dnmt1 mRNA instability, resulting in increased Dnmt1 translation. From here, functional Dnmt1 could then translocate into the nucleus, inhibiting p53 transcription. Computational algorithms revealed that both p53 and AUF1 could bind to different regions of circ-Dnmt1 RNA. Our results showed that the highly expressed circular RNA circ-Dnmt1 could bind to and regulate oncogenic proteins in breast cancer cells. Thus circ-Dnmt1 appears to be an oncogenic circular RNA with potential for further preclinical research.

5.
Cell Death Differ ; 25(12): 2195-2208, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29795334

RESUMO

TP53 mutations occur in many different types of cancers that produce mutant p53 proteins. The mutant p53 proteins have lost wild-type p53 activity and gained new functions that contribute to malignant tumor progression. Different p53 mutations create distinct profiles in loss of wild-type p53 activity and gain of functions. Targeting the consequences generated by the great number of p53 mutations would be extremely complex. Therefore, in this study we used a workaround and took advantage of the fact that mutant p53 cannot bind H2AX. Using this, we developed a new approach to repress the acquisition of mutant p53 functions. We show here that the delivery of a circular RNA circ-Ccnb1 inhibited the function of three p53 mutations. By microarray analysis and real-time PCR, we detected decreased circ-Ccnb1 expression levels in patients bearing breast carcinoma. Ectopic delivery of circ-Ccnb1 inhibited tumor growth and extended mouse viability. Using proteomics, we found that circ-Ccnb1 precipitated p53 in p53 wild-type cells, but instead precipitated Bclaf1 in p53 mutant cells. Further experiments showed that H2AX serves as a bridge, linking the interaction of circ-Ccnb1 and wild-type p53, thus allowing Bclaf1 to bind Bcl2 resulting in cell survival. In the p53 mutant cells, circ-Ccnb1 formed a complex with H2AX and Bclaf1, resulting in the induction of cell death. We found that this occurred in three p53 mutations. These results shed light on the possible development of new approaches to inhibit the malignancy of p53 mutations.

6.
Mol Ther ; 25(9): 2062-2074, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28676341

RESUMO

Delayed or impaired wound healing is a major health issue worldwide, especially in patients with diabetes and atherosclerosis. Here we show that expression of the circular RNA circ-Amotl1 accelerated healing process in a mouse excisional wound model. Further studies showed that ectopic circ-Amotl1 increased protein levels of Stat3 and Dnmt3a. The increased Dnmt3a then methylated the promoter of microRNA miR-17, decreasing miR-17-5p levels but increasing fibronectin expression. We found that Stat3, similar to Dnmt3a and fibronectin, was a target of miR-17-5p. Decreased miR-17-5p levels would increase expression of fibronectin, Dnmt3a, and Stat3. All of these led to increased cell adhesion, migration, proliferation, survival, and wound repair. Furthermore, we found that circ-Amotl1 not only increased Stat3 expression but also facilitated Stat3 nuclear translocation. Thus, the ectopic expressed circ-Amotl1 and Stat3 were mainly translocated to nucleus. In the presence of circ-Amotl1, Stat3 interacted with Dnmt3a promoter with increased affinity, facilitating Dnmt3a transcription. Ectopic application of circ-Amotl1 accelerating wound repair may shed light on skin wound healing clinically.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Transporte de RNA , RNA/genética , Fator de Transcrição STAT3/metabolismo , Cicatrização/genética , Animais , Sítios de Ligação , Movimento Celular , Proliferação de Células , DNA (Citosina-5-)-Metiltransferases/metabolismo , Fibroblastos/metabolismo , Proteínas de Membrana/genética , Camundongos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , RNA/química , RNA/metabolismo , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/genética , Transfecção
7.
Zhongguo Dang Dai Er Ke Za Zhi ; 18(2): 159-64, 2016 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-26903064

RESUMO

OBJECTIVE: To investigate the correlation between uncoupling protein 2 (UCP2) expression and myocardial mitochondria injury in rats with sepsis induced by lipopolysaccharide (LPS). METHODS: The rat model of sepsis was established through an intraperitoneal injection of LPS. Forty male Sprague-Dawley rats were randomly and equally divided into control group (an intraperitoneal injection of normal saline), sepsis 6 h group (LPS-6 h group), sepsis 12 h group (LPS-12 h group), sepsis 24 h group (LPS-24 h group), and sepsis 48 h group (LPS-48 h group). The serum and heart tissues were harvested at corresponding time points and myocardial mitochondria was extracted. The microplate reader was applied to measure creatine kinase (CK), creatine kinase-MB (CK-MB), and reactive oxygen species (ROS). Flow cytometry was applied to measure the degree of mitochondrial swelling and mitochondrial membrane potential (MMP). Western blot was used to measure the expression level of UCP2. Electron microscopy was applied to observe the morphological changes in heart tissues and myocardial mitochondria. RESULTS: Compared with the control group, the LPS groups had significantly increased serum levels of CK, CK-MB, and myocardial ROS, as well as a significantly increased degree of mitochondrial swelling (P<0.05), and these values reached their peaks at 24 hours after LPS injection. The LPS groups had a significant decrease in MMP (P<0.05), which reached the lowest level at 24 hours after LPS injection. Western blot showed that the LPS groups had a significant increase in the expression level of myocardial UCP2 compared with the control group (P<0.05), which reached its peak at 24 hours after LPS injection. The results of electron microscopy showed mitochondrial swelling, partial rupture of the mitochondrial membrane, and cavity formation in rats in the LPS groups. The most severe lesions occurred in the LPS-24 h group. In rats with LPS, the ROS level in the myocardial mitochondria and the degree of mitochondrial swelling were positively correlated with the expression level of UCP2 (r=0.796 and 0.893, respectively; P<0.05), while MMP was negatively correlated with the expression level of UCP2 (r=-0.903, P<0.05). CONCLUSIONS: In the rat model of sepsis, the myocardium and myocardial mitochondria have obvious injuries, and the expression level of UCP2 is closely correlated with mitochondrial injury. Therefore, UCP2 might play an important role in myocardial mitochondrial injury in sepsis.


Assuntos
Cardiomiopatias/metabolismo , Canais Iônicos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Sepse/metabolismo , Animais , Cardiomiopatias/genética , Modelos Animais de Doenças , Humanos , Canais Iônicos/genética , Lipopolissacarídeos/efeitos adversos , Masculino , Proteínas Mitocondriais/genética , Miocárdio/metabolismo , Ratos , Ratos Sprague-Dawley , Sepse/genética , Proteína Desacopladora 2
8.
Zhongguo Dang Dai Er Ke Za Zhi ; 17(8): 859-63, 2015 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-26287353

RESUMO

OBJECTIVE: To preliminarily investigate the long-term structural and functional injuries of mitochondria in rat brain caused by sepsis. METHODS: Wistar rats were randomly assigned into sepsis and control groups. A rat model of sepsis was prepared by an intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS) of gram-negative bacteria, and the survival assay was performed. Eight rats in the sepsis group were sacrificed at 12, 24, 48, or 72 hours after LPS injection, while rats in the control group were sacrificed after an intraperitoneal injection of an equal volume of normal saline. Mitochondria were extracted from rat brain tissue. Mitochondrial membrane potential (MMP) and mitochondrial swelling level were determined by flow cytometry, and the activities of electron transport chain complexes (I-V) were measured using enzyme assay kits. Hematoxylin-eosin (HE) staining and electron microscopy were used to observe morphological changes in brain tissue and mitochondria. RESULTS: The sepsis group had a significantly lower survival rate than the control group (P<0.01). The MMP and activities of electron transport chain complexes (I-V) in the sepsis group, which were significantly lower than those in the control group (P<0.05), were reduced to the lowest levels at 48 hours and partially recovered at 72 hours. The mitochondrial swelling level in the sepsis group, which was significantly higher than that in the control group (P<0.05), increased to the peak level at 48 hours and partially recovered at 72 hours. Hematoxylin and Eosin staining revealed substantial damages in the structure of brain tissue, and electron microscopy showed mitochondrial swelling, and vacuolization in a few mitochondria. CONCLUSIONS: In the rat model of LPS-induced sepsis, both structural and functional injuries are found in cerebral mitochondria, and achieve the peak levels probably at around 48 hours.


Assuntos
Encéfalo/fisiopatologia , Lipopolissacarídeos/toxicidade , Mitocôndrias/fisiologia , Sepse/fisiopatologia , Animais , Encéfalo/patologia , Encéfalo/ultraestrutura , Masculino , Potencial da Membrana Mitocondrial , Mitocôndrias/ultraestrutura , Ratos , Ratos Wistar , Sepse/induzido quimicamente , Sepse/mortalidade
9.
J Res Med Sci ; 20(2): 185-95, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25983774

RESUMO

Sepsis is a systemic inflammatory response to infection. Sepsis, which can lead to severe sepsis, septic shock, and multiple organ dysfunction syndrome, is an important cause of mortality. Pathogenesis is extremely complex. In recent years, cell hypoxia caused by mitochondrial dysfunction has become a hot research field. Sepsis damages the structure and function of mitochondria, conversely, mitochondrial dysfunction aggravated sepsis. The treatment of sepsis lacks effective specific drugs. The aim of this paper is to undertake a narrative review of the current experimental treatment for mitochondrial dysfunction in sepsis. The search was conducted in PubMed databases and Web of Science databases from 1950 to January 2014. A total of 1,090 references were retrieved by the search, of which 121 researches met all the inclusion criteria were included. Articles on the relationship between sepsis and mitochondria, and drugs used for mitochondrial dysfunction in sepsis were reviewed retrospectively. The drugs were divided into four categories: (1) Drug related to mitochondrial matrix and respiratory chain, (2) drugs of mitochondrial antioxidant and free radical scavengers, (3) drugs related to mitochondrial membrane stability, (4) hormone therapy for septic mitochondria. In animal experiments, many drugs show good results. However, clinical research lacks. In future studies, the urgent need is to develop promising drugs in clinical trials.

10.
Brain Res ; 1620: 130-8, 2015 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-25998537

RESUMO

Sepsis-induced brain dysfunction (SIBD) is often the first manifestation of sepsis, and its pathogenesis is associated with mitochondrial dysfunction. In this study, we investigated the roles of the tyrosine kinase Src and protein tyrosine phosphatase 1B (PTP1B) in brain mitochondrial dysfunction using a rat model of lipopolysaccharide (LPS)-induced sepsis. We found that there was a gradual and significant increase of PTP1B levels in the rat brain after sepsis induction. In contrast, brain Src levels were reduced in parallel with the PTP1B increase. Sepsis led to significantly reduced tyrosine phosphorylation of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, II and III. Pretreatment of mitochondrial proteins with active PTP1B significantly inhibited complexes I and III activities in vitro, whereas Src enhanced complexes I, II, and III activities. PTP1B and Src were each co-immunoprecipitated with OXPHOS complexes I and III, suggesting direct interactions between both proteins and complexes I and III. Src also directly interacted with complex II. Furthermore, pretreatment of mitochondrial proteins with active PTP1B resulted in overproduction of reactive oxygen species and decreased mitochondrial membrane potential. Pretreatment with active Src produced the opposite effect. These results suggest that brain mitochondrial dysfunction following LPS-induced sepsis in rats is partly attributed to PTP1B and Src mediated decrease in mitochondrial protein tyrosine phosphorylation.


Assuntos
Encéfalo/metabolismo , Mitocôndrias/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Encefalopatia Associada a Sepse/fisiopatologia , Sepse/fisiopatologia , Quinases da Família src/metabolismo , Animais , Modelos Animais de Doenças , Lipopolissacarídeos , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Fosforilação/fisiologia , RNA Mensageiro/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Sepse/complicações , Encefalopatia Associada a Sepse/etiologia
11.
Int J Mol Med ; 35(6): 1525-36, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25873251

RESUMO

Uncoupling protein 2 (UCP2) regulates the production of mitochondrial reactive oxygen species (ROS) and cellular energy transduction under physiological or pathological conditions. In this study, we aimed to determine whether mitochondrial UCP2 plays a protective role in cardiomyocytes under septic conditions. In order to mimic the septic condition, rat embryonic cardiomyoblast-derived H9C2 cells were cultured in the presence of lipopolysaccharide (LPS) plus peptidoglycan G (PepG) and small interfering RNA (siRNA) against UCP2 (siUCP2) was used to suppress UCP2 expression. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR), western blot analysis, transmission electron microscopy (TEM), confocal microscopy and flow cytometry (FCM) were used to detect the mRNA levels, protein levels, mitochondrial morphology and mitochondrial membrane potential (MMP or ΔΨm) in qualitative and quantitative analyses, respectively. Indicators of cell damage [lactate dehydrogenase (LDH), creatine kinase (CK), interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the culture supernatant] and mitochondrial function [ROS, adenosine triphosphate (ATP) and mitochondrial DNA (mtDNA)] were detected. Sepsis enhanced the mRNA and protein expression of UCP2 in the H9C2 cells, damaged the mitochondrial ultrastructure, increased the forward scatter (FSC)/side scatter (SSC) ratio, increased the CK, LDH, TNF-α and IL-6 levels, and lead to the dissipation of MMP, as well as the overproduction of ROS; in addition, the induction of sepsis led to a decrease in ATP levels and the deletion of mtDNA. The silencing of UCP2 aggravated H9C2 cell damage and mitochondrial dysfunction. In conclusion, our data demonstrate that mitochondrial morphology and funtion are damaged in cardiomyocytes under septic conditions, while the silencing of UCP2 using siRNA aggravated this process, indicating that UCP2 may play a protective role in cardiomyocytes under septic conditions.


Assuntos
Canais Iônicos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , RNA Interferente Pequeno/farmacologia , Sepse/metabolismo , Animais , Linhagem Celular , Canais Iônicos/genética , Lipopolissacarídeos/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias Cardíacas/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos Cardíacos/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Sepse/induzido quimicamente , Sepse/genética , Sepse/patologia , Proteína Desacopladora 2
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