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PURPOSE: We aim to investigate the potential role and underlying mechanisms of linc00174 on pyroptosis in the pathogenesis of DR. METHODS: Expression patterns of linc00174, miR-26a-5p and PTEN in human retinal microvascular endothelial cells (hRMECs) were detected by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. Biological functions of linc00174 on cell proliferation and pyroptosis were evaluated by CCK-8, flow cytometry, caspase-1 activity assays, respectively. Luciferase reporter assay was employed to verify the interaction between miR-26a-5p and linc00174/PTEN. Streptozotocin (STZ)-induced DR in mice was further constructed to verify the potential role of linc00174 in vivo. Hematoxylin and eosin (H&E) and immunohistochemical staining were performed to assess the pathological changes and caspase-1 expression in retinal tissues. RESULTS: Up-regulated linc00174 and PTEN and down-regulated miR-26a-5p were uncovered in hRMECs treated with high glucose (HG). Mechanistically, linc00174 served as a sponge of miR-26a-5p to facilitate PTEN expression. Functionally, knockdown of linc00174 inhibited HG-induced pyroptosis of hRMECs via targeting miR-26a-5p. Moreover, linc00174/miR-26a-5p axis participated in HG-induced pyroptosis via PTEN/Akt signaling cascade. Further, silencing of linc00174 attenuated pyroptosis via regulating miR-26a-5p/PETN axis in DR mice. CONCLUSIONS: Collectively, our study reveals that linc10074 deteriorates the pathogenesis of DR via miR-26a-5p/PTEN/Akt signalling cascade, which may shed light on the discovery of potential therapeutic agents for DR treatment.
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Diabetes Mellitus , Retinopatia Diabética , MicroRNAs , Animais , Caspases/metabolismo , Proliferação de Células , Diabetes Mellitus/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Amarelo de Eosina-(YS)/metabolismo , Glucose/metabolismo , Hematoxilina/metabolismo , Humanos , Camundongos , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piroptose , Sincalida/metabolismo , EstreptozocinaRESUMO
The present study was to identify abnormal methylation genes implicated in esophageal squamous cell carcinoma (ESCC). Genomic methylation alterations in ESCC tissues were analyzed using laser-microdissection and whole-genome bisulfite sequencing. CXCL14 promoter was frequently hypermethylated in ESCC tissues. The correlation of CXCL14 hypermethylation status and the mRNA and protein expression levels were validated using nested methylation-specific PCR (nMS-PCR), RNAscope in situ hybridization (RISH) and Western blot. RISH results showed completely negative CXCL14 expression in 34.3% (34/99) ESCC, compared with those in the basal layer cells of normal epithelia. Low expression of CXCL14 was more present in patients with lower differentiation. The anticancer role of CXCL14 has been commonly associated with immune regulation in the literature. Here, we observed by functional analysis that CXCL14 can also act as a tumor suppressor in ESCC cells. 5-Aza-dC treatment suppressed CXCL14 methylation and up-regulated the expression of CXCL14. Ectopic expression of CXCL14 suppressed the proliferation, invasion, tumor growth, and lung metastasis of ESCC cells. Both ectopic expression and induction of CXCL14 with 5-Aza-dC inhibited the activity of SRC, MEK1/2 and STAT3 in ESCC cells, while activated EGFR. Importantly, a combination of CXCL14 expression and SRC or EGFR inhibitor dramatically repressed the proliferation of ESCC cells and the growth of xenografts. Our findings revealed a direct tumor suppressor role of CXCL14, but not through the immune system. The data suggest that for ESCC patients with low level CXCL14, increasing CXCL14 expression combined with inhibition of SRC or EGFR might be a promising therapeutic strategy.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Metilação de DNA , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , FenótipoRESUMO
Community detection and structural hole spanner (the node bridging different communities) identification, revealing the mesoscopic and microscopic structural properties of complex networks, have drawn much attention in recent years. As the determinant of mesoscopic structure, communities and structural hole spanners discover the clustering and hierarchy of networks, which has a key impact on transmission phenomena such as epidemic transmission, information diffusion, etc. However, most existing studies address the two tasks independently, which ignores the structural correlation between mesoscale and microscale and suffers from high computational costs. In this article, we propose an algorithm for simultaneously detecting communities and structural hole spanners via hyperbolic embedding (SDHE). Specifically, we first embed networks into a hyperbolic plane, in which, the angular distribution of the nodes reveals community structures of the embedded network. Then, we analyze the critical gap to detect communities and the angular region where structural hole spanners may exist. Finally, we identify structural hole spanners via two-step connectivity. Experimental results on synthetic networks and real networks demonstrate the effectiveness of our proposed algorithm compared with several state-of-the-art methods.
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BACKGROUND: There is no clear conclusion on the relationship between thyroid disease and obesity and lifestyle factors such as smoking and drinking. In this study, we analysed the association of body mass index (BMI), smoking and drinking with subclinical hypothyroidism (SHO) and thyroid nodules (TNs) with the results of a cross-sectional survey of urban residents in central China and discussed the potential mechanism linking these predictive factors and the two diseases. METHODS: This study included 1279 participants who were recruited from a Chinese community in 2011 and 2012. A questionnaire, laboratory examination and ultrasound diagnosis were conducted on these participants. Binary logistic regression analysis was used to analyse these factors. RESULTS: Overweight (BMI ≥ 25 kg/m2) was closely related to SHO and TNs in univariate and multivariate logistic regression analyses. Smoking had a protective effect on SHO and TNs, while drinking had a protective effect on TNs in univariate logistic regression and multivariate logistic regression with some covariates, but there was no significant difference between smoking and drinking and the two kinds of thyroid diseases in multivariate logistic regression analysis with all the covariates. In subgroup analysis, BMI ≥ 25 kg/m2 was significantly associated with SHO in people with positive thyroid antibodies (odds ratio (OR) = 2.221, 95 % confidence interval (CI): 1.168-4.184, P = 0.015) and smokers (OR = 2.179, 95 % CI: 1.041-4.561, P = 0.039). BMI ≥ 25 kg/m2 was significantly associated with TNs in people over 60 years old (OR = 2.069, 95 % CI: 1.149-3.724, P = 0.015) and drinkers (OR = 3.065, 95 % CI: 1.413-6.648, P = 0.005). Drinking alcohol had a protective effect on TNs in smokers (OR = 0.456, 95 % CI: 0.240-0.865, P = 0.016) and people with BMI ≥ 25 kg/m2 (OR = 0.467, 95 % CI: 0.236-0.925, P = 0.029). No significant association was found between smoking and the two thyroid diseases in different subgroups. CONCLUSIONS: Obesity is a risk factor for both TNs and SHO, especially in elderly individuals and people with positive thyroid autoantibodies. Obesity and metabolic syndrome may be more associated with TNs than SHO. Smoking may have a protective effect on thyroid disease, while drinking may have a protective effect only on TNs.
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Consumo de Bebidas Alcoólicas/efeitos adversos , Índice de Massa Corporal , Obesidade/complicações , Fumar/efeitos adversos , Doenças da Glândula Tireoide/epidemiologia , Adulto , China/epidemiologia , Estudos Transversais , Feminino , Seguimentos , Humanos , Estilo de Vida , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Risco , Doenças da Glândula Tireoide/etiologia , Doenças da Glândula Tireoide/patologiaRESUMO
Diabetic retinopathy (DR) has been regarded as a sight-threatening vascular complication of diabetes mellitus. Accumulating evidence has identified the involvement of long non-coding RNAs (lncRNAs) in DR pathogenesis. We aim to investigate the role and underlying mechanism of linc00174 in the DR process. Samples of human vitreous humour from proliferative DR and non-diabetic individuals were collected to examine the levels of linc00174. Human retinal microvascular endothelial cells (HRMECs) exposed with high glucose (HG) were employed to simulate the pathological statues of DR. Short hairpin RNA specifically targeting linc00174 was applied. CCK-8, transwell, and matrigel tube formation were performed to evaluate cell proliferation, migration, and angiogenesis. Bioinformatics analysis and luciferase reporter assay were conducted to verify the linc00174/miR-150-5p/vascular endothelial growth factor A (VEGFA) regulatory network. Western blotting was employed to determine the expression of VEGFA. Linc00174 was significantly elevated in patients with DR, as well as HG-stimulated HRMECs, of which knockdown repressed HG-induced proliferation, migration, and angiogenesis. miR-150-5p was identified as a downstream effector to be involved in linc00174-mediated protective effects. miR-150-5p directly bound to the 3' untranslated region of VEGFA. The linc00174/miR-150-5p/VEGFA axis was confirmed in retinal vascular dysfunction. The linc00174 deteriorates diabetic retinal microangiopathy via regulating miR-150-5p/VEGFA pathway, indicating a novel therapeutic target for DR treatment.
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Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Regulação da Expressão Gênica no Desenvolvimento/genética , MicroRNAs/metabolismo , MicroRNAs/fisiologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia , Regiões 3' não Traduzidas , Idoso , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Retinopatia Diabética/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo MolecularRESUMO
Beauveria bassiana is a promising biocontrol agent due to its entomopathogenic activities and residue-free characteristics. However, its susceptibility to abiotic stresses and naturally low virulence limit the effective application of this fungus. To effectively obtain fungal strains with high biocontrol potential, fluorescence-activated cell sorting (FACS) was used to screen mutant libraries generated by atmospheric and room temperature plasma (ARTP). Among about 8000 mutants obtained by ARTP mutagenesis, six candidate mutants were selected according to the forward scatter (FSC) signal readings of FACS. B6, with a 37.4% higher FSC reading than wild-type (WT), showed a 32.6% increase in virulence. It also presented a 13.5% decrease in median germinating time (GT50) and a 12.1% increase in blastospore production. Comparative analysis between insect transcriptional responses to B6 and WT infection showed that the immune response coupled with protein digestion and absorption progress was highly activated in B6-infected Galleria mellonella larvae, while fatty acid synthesis was suppressed after 3â¯days of infection. Our results confirmed the feasibility of sorting B. bassiana with high biocontrol potential via the combination of ARTP and FACS and facilitated the understanding of insect-pathogen interactions, highlighting a new strategy for modifying entomopathogenic fungi to improve the efficiency of biological control.
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Beauveria , Mariposas , Animais , Citometria de Fluxo , Mutagênese , PlasmaRESUMO
The development of an ideal model plant located at a key phylogenetic node is critically important to advance functional and regulatory studies of key regulatory genes in the evolutionary developmental (evo-devo) biology field. In this study, we selected Chirita pumila in the family Gesneriaceae, a basal group in Lamiales, as a model plant to optimize its genetic transformation system established previously by us through investigating a series of factors and further conduct functional test of the CYC-like floral symmetry gene CpCYC. By transforming a RNAi:CpCYC vector, we successfully achieved the desired phenotypes of upright actinomorphic flowers, which suggest that CpCYC actually determines the establishment of floral zygomorphy and the horizontal orientation of flowers in C. pumila. We also confirmed the activities of CpCYC promoter in dorsal petals, dorsal/lateral staminodes, as well as the pedicel by transferring a CpCYC promoter:GUS vector into C. pumila. Furthermore, we testified the availability of a transient gene expression system using C. pumila mesophyll protoplasts. The improved transformation system together with the inherent biological features would make C. pumila an attractive new model in functional and regulatory studies for a broad range of evo-devo issues.
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Regulação da Expressão Gênica de Plantas/genética , Lamiales/genética , Transformação Genética/genética , Evolução Biológica , Flores/genética , Genes de Plantas/genética , Magnoliopsida/genética , Modelos Biológicos , Fenótipo , Filogenia , Proteínas de Plantas/genética , Fatores de Transcrição/metabolismoRESUMO
Biological control potential of entomopathogenic fungi depending on conidiation capacity, conidial stress tolerance and virulence can be improved through genetic engineering. To explore a possible role of trehalose biosynthesis pathway in improving fungal pest-control potential, we characterized biological functions of trehalose-6-phosphate phosphatase (BbTPP) in Beauveria bassiana, an insect mycopathogen that serves as a main source of fungal insecticides. Deletion of BbTPP resulted in abolished trehalose biosynthesis, reduced conidiation capacity, decreases in conidial thermotolerance and UV-B resistance, increased hyphal sensitivities to chemical stresses, and attenuated virulence. By contrast, over-expression of BbTPP led to increased trehalose accumulation, decreased T6P accumulation, and enhanced stress tolerance and virulence despite little impact on growth and conidiation under normal conditions. These results indicate that BbTPP serves as not only a key player in control of trehalose biosynthesis required for multiple cellular functions but also a potential candidate to be exploited for genetic improvement of fungal potential against insect pests.
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Estresse Fisiológico , Trealose , Animais , Proteínas Fúngicas , Deleção de Genes , Insetos , Monoéster Fosfórico Hidrolases , Esporos Fúngicos , VirulênciaRESUMO
The anti-Markovnikov hydroazidation of alkenes has been accomplished under visible-light irradiation by using [Ir(dF(CF3 )ppy)2 (dtbbpy)]PF6 as the photocatalyst and trimethylsilyl azide as the azidating agent. The reactions were greatly facilitated by water, the beneficial effect of which can be attributed to its participation in the reaction as the hydrogen donor, as indicated by deuterium isotope experiments. The reactions proceed under solvent free conditions in the presence of water. 4-Dimethylaminopyridine also exhibited a beneficial effect on the reactions. The present method enabled hydroazidation of several types of unactivated alkenes with good yields and high regioselectivity.
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The standard decoction of Chinese herbal decoction pieces is a standard reference substance to measure whether different dosage forms of Chinese medicine are basically consistent with those of clinical decoction,and provides new ideas and methods for effectively solving the problems of uneven quality in Chinese medicine dispensing granules. In this study,a systematic method for evaluating the quality of Scrophulariae Radix decoction was established from the perspective of " standard decoction",providing reference for the quality control of the Scrophulariae Radix dispensing granules. 15 batches of Scrophulariae Radix decoction pieces from different origins were collected,and 15 batches of standard decoctions were prepared according to the standardized process with water as solvent.Harpagide and harpagoside were used as quantitative detection indicators to determine the content,calculate the transfer rates and determine the extraction rate. The high performance liquid chromatography( HPLC) was used to establish a standard decoction fingerprint analysis method. The results showed that the transfer rates of harpagide and harpagoside in 15 batches of Scrophulariae Radix pieces standard decoction were( 70. 84±13. 39) % and( 48. 56±6. 40) % respectively; the extraction rate was( 57. 47±5. 89) %. Nine peaks were identified in the HPLC fingerprint,and the similarity was higher than 0. 97 between the fingerprints of 15 batches of standard decoction and the control fingerprint. In this study,the preparation process of standard decoction of Scrophulariae Radix pieces conformed to the traditional decoction preparation method. The sources of the samples were representative,and the established fingerprint method was stable and feasible,which can provide reference for the preparation and quality control of Scrophulariae Radix dispensing granules.
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Medicamentos de Ervas Chinesas/normas , Raízes de Plantas/química , Scrophularia/química , Cromatografia Líquida de Alta Pressão , Controle de QualidadeRESUMO
Gcn5 is a core histone acetyltransferase that catalyzes histone H3 acetylation on N-terminal lysine residues in yeasts and was reported to catalyze H3K9/K14 acetylation required for activating asexual development in Aspergillus. Here, we report a localization of Gcn5 ortholog in the nucleus and cytoplasm of Beauveria bassiana, a fungal insect pathogen. Deletion of gcn5 led to hypoacetylated H3 at K9/14/18/27 and 97% reduction in conidiation capacity as well as severe defects in colony growth and conidial thermotolerance. Two master conidiation genes, namely brlA and abaA, were transcriptionally repressed to undetectable level in Δgcn5, but sharply upregulated in wild-type, at the beginning time of conidiation. Based on chromatin immunoprecipitation, both DNA and acetylation levels of the distal and proximal fragments of the brlA promoter bound by acetylated H3K14 alone were upregulated in wild-type, but not in Δgcn5, at the mentioned time. In Δgcn5, normal cuticle infection was abolished while virulence through cuticle-bypassing infection was greatly attenuated, accompanied by drastically reduced activities of putative cuticle-degrading enzymes, retarded dimorphic transition and transcriptional repression of associated genes. These findings unveil a novel mechanism by which Gcn5 activates asexual development pathway by acetylating H3K14 and regulates the virulence-related cellular events in B. bassiana.
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Beauveria , Proteínas Fúngicas/genética , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Acetilação , Animais , Beauveria/genética , Beauveria/crescimento & desenvolvimento , Beauveria/patogenicidade , Proteínas Fúngicas/metabolismo , Insetos/microbiologia , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Esporos Fúngicos/metabolismo , Ativação Transcricional/genética , Virulência/genéticaRESUMO
Histone lysine acetylation orchestrates transcriptional activity essential for diverse cellular events across organisms, but it remains poorly understood how an acetylated lysine affects cellular functions in filamentous fungal pathogens. Here, we show the functions of a histone acetyltransferase that is phylogenetically close to Mst2 in fission yeast and specifically acetylates histone H3K14 in Beauveria bassiana, a fungal insect pathogen widely applied in arthropod pest management. Deletion of mst2 in B. bassiana resulted in moderate growth defects on rich and minimal media, delayed conidiation, and drastic reduction (75%) in conidiation capacity under normal culture conditions. The Δmst2 conidia suffered slower germination, decreased hydrophobicity, attenuated virulence, and reduced thermotolerance and UV-B resistance. The Δmst2 mutant also displayed increased sensitivities to DNA damaging, oxidative, cell wall perturbing, and osmotic stresses during conidial germination and colony growth at optimal 25 °C. Intriguingly, the phenotypic changes were accompanied with transcriptional repression of related gene sets, which are required for asexual development and conidial hydrophobicity or cascaded for CWI and HOG pathways, and encode the families of superoxide dismutases (SOD), catalases, heat-shock proteins, and trehalose or mannitol-metabolizing enzymes. Consequently, total SOD and catalase activities, trehalose and mannitol contents, and hydrophobicity were remarkably lowered in the hyphal cells or conidia of Δmst2. All of these changes were well restored by targeted mst2 complementation. Our results indicate that Mst2 enables to mediate global gene transcription and/or post-translation through H3K14 acetylation and plays an essential role in sustaining the biological control potential of B. bassiana against arthropod pests.
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Beauveria/enzimologia , Agentes de Controle Biológico , Proteínas Fúngicas/metabolismo , Histona Acetiltransferases/metabolismo , Transcrição Gênica , Acetilação , Animais , Beauveria/genética , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Histona Acetiltransferases/genética , Hifas/crescimento & desenvolvimento , Lepidópteros/microbiologia , Pressão Osmótica , Fenótipo , Esporos Fúngicos/crescimento & desenvolvimento , Estresse Fisiológico , VirulênciaRESUMO
Intracellular trehalose accumulation is relevant to fungal life and pathogenicity. Trehalose-6-phosphate synthase (TPS) is known to control the first step of trehalose synthesis, but functions of multiple TPS genes in some filamentous fungi are variable. Here, we examined the functions of two TPS genes (tpsA and tpsB) in Beauveria bassiana, a fungal insect pathogen widely applied in arthropod pest control. Intracellular TPS activity and trehalose content decreased by 71-75 and 72-80% in ΔtpsA, and 21-30 and 15-45% in ΔtpsB, respectively, and to undetectable levels in ΔtpsAΔtpsB, under normal and stressful conditions. The three mutants lost 33, 50, and 98% of conidiation capacity in standard cultures. Conidial quality indicated by viability, density, intracellular trehalose content, cell wall integrity, and hydrophobicity was more impaired in ΔtpsA than in ΔtpsB and mostly in ΔtpsAΔtpsB, which was also most sensitive to nutritional, chemical, and environmental stresses and least virulent to Galleria mellonella larvae. Almost all of phenotypic defects in ΔtpsAΔtpsB approached to the sums of those observed in ΔtpsA and ΔtpsB and were restored by targeted gene complementation. Altogether, TpsA and TpsB play complementary roles in sustaining trehalose synthesis, conidiation capacity, conidial quality, multiple stress tolerance, and virulence, highlighting a significance of both for the fungal adaptation to environment and host.
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Beauveria/enzimologia , Glucosiltransferases/metabolismo , Lepidópteros/microbiologia , Esporos Fúngicos/crescimento & desenvolvimento , Estresse Fisiológico , Trealose/biossíntese , Fatores de Virulência/metabolismo , Animais , Beauveria/genética , Beauveria/patogenicidade , Beauveria/fisiologia , Deleção de Genes , Teste de Complementação Genética , Glucosiltransferases/genética , Viabilidade Microbiana , Análise de Sobrevida , Virulência , Fatores de Virulência/genéticaRESUMO
PURPOSE: Facial skin fibroblasts imposed with cyclic stretch at 10% magnitude display considerable mechanotransduction properties and biochemical reactions in our previous study. However, it is poorly understood how these shared traits are fully parallel to the common features across all fibroblasts derived from different skin-based anatomical regions in response to cyclic stretch stimulation. Thus, the purpose of this study was to evaluate the effects of various cyclic stretches on fibroblasts derived from multiple anatomical skin sites of human bodies, and the optimal stretch magnitude was defined based on the changes to cell mechanical behavior. METHODS: Fibroblasts from skin areas of the scalp, anterior chest, suprapubic, axilla, and planta were cultured and characterized in vitro. Cyclic stretch at 0%, 5%, 10%, 15%, and 20% magnitudes was imposed at a loading frequency of 0.1 Hz for 48 hours, and thereafter, the mechanical behavior and biochemical reaction of the dermal fibroblasts were analyzed. RESULTS: Dermal fibroblasts from various anatomical sites preconditioned with varying cyclic stretch led to an evident increase in the cell proliferation ability, the expression of integrin ß1 and p130 Crk-associated substrate messenger RNA and protein, and the productions of type I collagen and transforming growth factor ß1, most importantly in a strain magnitude-dependent manner with the peak appearing in the range of 10% to 15% magnitude cyclic stretch. CONCLUSIONS: These findings may facilitate the subsequent studies on the conversion of normal skin fibroblasts into hypertrophic scar cells, which should be considered in an interpretation of the mechanisms of hypertrophic scarring and skin mechanics.
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Proliferação de Células/fisiologia , Cicatriz Hipertrófica/metabolismo , Fibroblastos/metabolismo , Mecanotransdução Celular/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Análise de Variância , Células Cultivadas , Cicatriz Hipertrófica/patologia , Colágeno/metabolismo , Fibroblastos/patologia , Humanos , Immunoblotting , Reação em Cadeia da Polimerase em Tempo Real/métodos , Pele/patologiaRESUMO
Thioredoxin (Trx) interacting protein (TXNIP) is a Trx-binding protein that inhibits the antioxidative function of Trx and is highly expressed in the serum and tissue samples from diabetes patients. This study was to explore whether TXNIP overexpression could cause INS-1 cell autophagy under normal glucose and lipid concentrations, and to analyze the role of autophagy in the apoptosis of INS-1 cells. The INS-1 cells cultured under normal conditions were divided into three groups: normal control, empty adenovirus vector (Ad-eGFP) and TXNIP overexpression (Ad-TXNIP-eGFP) groups. Forty-eight hours after transfection, the expression levels of TXNIP mRNA and protein were measured. Western blot was used to examine the protein expression levels of Beclin-1 and P62, as well as LC3-II/LC3-I ratio, which are associated with autophagy. IF/ICC was used to measure the autophagosome. In addition, the cleaved caspase-3/caspase-3 ratio, the apoptosis marker, was also measured, and the apoptotic rates were detected by flow cytometry (FCM). The results showed that the TXNIP mRNA and protein levels were significantly up-regulated in Ad-TXNIP-eGFP group, suggesting that TXNIP overexpression model was successfully established. In Ad-TXNIP-eGFP group, the protein levels of Beclin-1 and LC3-II/LC3-I ratio were increased, while the protein expression of P62 was decreased, compared with those in Ad-eGFP group. Red fluorescent intensity, representing autophagy level, was higher in Ad-TXNIP-eGFP group than that in Ad-eGFP group. These results suggested that TXNIP overexpression can significantly promote INS-1 cell autophagy. Meanwhile, cleaved caspase 3/caspase 3 ratio and the number of apoptotic cells were significantly increased in Ad-TXNIP-eGFP group. The inhibitor of autophagy, 3-MA, reduced TXNIP overexpression-induced apoptosis in INS-1 cells. Taken together, our data demonstrate that autophagy appears to be an important pathway in TXNIP overexpression-induced apoptosis in INS-1 cells.
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Apoptose , Autofagia , Proteínas de Transporte/metabolismo , Ilhotas Pancreáticas/citologia , Proteínas de Transporte/genética , Caspase 3/metabolismo , Linhagem Celular , Humanos , TransfecçãoRESUMO
OBJECTIVES: The objectives of this study were to investigate the functional effect of matrix metallopeptidase 14 (MMP14) on cell invasion in cervical cancer cells (HeLa line) and to study the underlying molecular mechanisms. METHODS: Expression vector of short hairpin RNA targeting MMP14 was treated in HeLa cells, and then, transfection efficiency was verified by a florescence microscope. Transwell assay was used to investigate cell invasion ability in HeLa cells. Quantitative polymerase chain reaction and Western blotting analysis were used to detect the expression of MMP14 and relative factors in messenger RNA and protein levels, respectively. RESULTS: Matrix metallopeptidase 14 short hairpin RNA expression vector transfection obviously decreased MMP14 expression in messenger RNA and protein levels. Down-regulation of MMP14 suppressed invasion ability of HeLa cells and reduced transforming growth factor ß1 and vascular endothelial growth factor B expressions. Furthermore, MMP14 knockdown decreased bone sialoprotein and enhanced forkhead box protein L2 expression in both RNA and protein levels. CONCLUSION: Matrix metallopeptidase 14 plays an important role in regulating invasion of HeLa cells. Matrix metallopeptidase 14 knockdown contributes to attenuating the malignant phenotype of cervical cancer cell.
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Movimento Celular , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 14 da Matriz/metabolismo , Osteopontina/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Apoptose , Western Blotting , Proliferação de Células , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/genética , Células HeLa , Humanos , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/genética , Invasividade Neoplásica , Osteopontina/genética , RNA Mensageiro/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/genética , Fator B de Crescimento do Endotélio Vascular/genéticaRESUMO
This study aimed to investigate the effects of harmine hydrochloride (HMH) on digestive tumor cells in vitro and its molecular mechanism. MTT assays showed that HMH inhibited the proliferation of some human cancer cell lines and had no obvious inhibitory effects on human LO2 cells. Flow cytometry assays showed that HMH trigged G2 phase arrest in MGC-803 cells and SMMC-7721 cells, while the expression of cyclin A, cyclin B, p21, Myt1, and p-cdc2 (Tyr15) was upregulated. Flow cytometry assays also showed that the percentages of apoptotic cells were increased, the mitochondrial transmembrane potential (ΔΨm) decreased, and the cleavage of caspase-9, caspase-3, and poly (Adenosine diphosphate ribose) polymerase (PARP) were observed, the expression of Bad increased, phospho-Bad (S112) decreased, pro-caspase-8 was cleaved, and Bid (22 kDa) was cleaved. The expression of p-ERK decreased in both cells. In conclusion, these results demonstrated that HMH upregulates the expression of p21, activates Myt1 and inhibits cdc2 by phospho-cdc2 (Y15), and triggers G2 phase arrest in both MGC-803 cells and SMMC-7721 cells. It can also activate the mitochondria-related cell apoptosis pathway through the caspase-8/Bid pathway, inhibiting the ERK/Bad pathway and promoting apoptosis in both of these two cell types.
Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Harmina/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Citometria de Fluxo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Regulação para Cima , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Modification of cell cycle in entomopathogenic fungi is likely crucial for host infection and environmental adaptation. Here we show that Wee1 and Cdc25 can balance cell cycle-required cyclin-dependent kinase 1 (Cdk1) activity in Beauveria bassiana. The Cdk1 phosporylation signal was strong in Δcdc25 but very weak in Δwee1 and absent in Δwee1Δcdc25. Consequently, cell cycles, septation patterns and many septation-dependent gene transcripts of these mutants were reversely changed. Hyphal cells were short in Δwee1, slender in Δcdc25 and short and swollen in Δwee1Δcdc25. Conidiation was most defective in Δwee1, followed by Δcdc25. Their conidia and yeast-like blastospores also altered antagonistically in both size and complexity, accompanied with abnormally branched germlings in Δwee1 and Δwee1Δcdc25. Conidial thermotolerance and UV-B resistance decreased much more in Δwee1Δcdc25 than in Δwee1 but significantly increased in Δcdc25. The double deletion and the point mutation Cdk1(T14A/P15F) for inhibitory phosphorylation caused most defective virulence, followed by wee1 deletion. All the changes were restored by ectopic gene complementation. Virulence changes in all the mutants and control strains were highly correlated to those in blastospore size or complexity. Taken together, Wee1 and Cdc25 control cell cycle, morphogenesis, asexual development, stress tolerance and virulence of B. bassiana by balancing the Cdk1 activity.
Assuntos
Beauveria , Proteína Quinase CDC2/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fosfatases cdc25/metabolismo , Beauveria/enzimologia , Beauveria/crescimento & desenvolvimento , Beauveria/patogenicidade , Proteína Quinase CDC2/genética , Ciclo Celular/genética , Proteínas Fúngicas/genética , Hifas/metabolismo , Fosforilação , Proteínas Tirosina Quinases/genética , Esporos Fúngicos/metabolismo , Estresse Fisiológico/genética , Virulência , Fosfatases cdc25/genéticaRESUMO
Two conserved 14-3-3 proteins orthologous to Saccharomyces cerevisiaeâ Bmh1/2 are poorly understood in filamentous fungi. Here we show that Bmh1 and Bmh2 contribute equally to the fundamental biology and physiology of Beauveria bassiana by targeting many sets of proteins/enzymes. Single Bmh deletion caused similar upregulation of another. Excellent knockdown (â¼91%) expressions of Bmh1 in ΔBmh2 and Bmh2 in ΔBmh1 resulted in equally more severe multiphenotypic defects than the single deletions, including G2 /M transition, blastospore size, carbon/nitrogen utilization, conidiation, germination and conidial tolerances to high osmolarity, oxidation, cell wall stress, high temperature and UV-B irradiation. All the deletion and deletion/knockdown mutants showed similar defects in blastospore yield and density, hyphal septation and cell size, hyphal responses to most chemical stresses and virulence. All the defects were evident with altered transcripts of phenotype-related genes and well restored by each Bmh complementation. Our Bmh1- and Bmh2-specific transcriptomes generated under osmotic and oxidative stresses revealed up to 6% genes differentially expressed by at least twofold in the fungal genome. Many of those were greatly depressed or co-depressed in ΔBmh1 and ΔBmh2. Our findings provide a thorough insight into the functions and complementary effects of the two 14-3-3 proteins in the filamentous entomopathogen.
Assuntos
Proteínas 14-3-3/metabolismo , Beauveria/metabolismo , Beauveria/patogenicidade , Proteínas Fúngicas/metabolismo , Mariposas/microbiologia , Esporos Fúngicos/crescimento & desenvolvimento , Proteínas 14-3-3/genética , Animais , Beauveria/genética , Beauveria/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Hifas/patogenicidade , Morfogênese , Estresse Oxidativo , Controle Biológico de Vetores , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Esporos Fúngicos/patogenicidade , VirulênciaRESUMO
New 2p-4f and 2p-3d-4f compounds [Tb(hfac)3(NIT-PhNO2)2]· 0.5C7H16 (1) and [Ln(hfac)3(NIT-PhNO2)2]2[Cu(hfac)2(NIT-PhNO2)2] (Ln(III) = Gd 2, Tb 3; hfac = hexafluoroacetylacetonate; NIT-PhNO2 = 2-(p-nitrophenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) have been obtained. Complex 1 consists of mononuclear trispin [Tb(hfac)3(NIT-PhNO2)2] units in which two radical ligands are ligated to the Tb(III) ion as monodentate ligands through the NO groups, while complexes 2 and 3 contain two kinds of trispin moieties, namely, [Ln(hfac)3(NIT-PhNO2)2] and [Cu(hfac)2(NIT-PhNO2)2]. In the [Cu(hfac)2(NIT-PhNO2)2] moiety, the radicals are bonded to the copper(II) ion in the axial positions via the nitroxides. For three compounds, 1D supramolecular chains are formed via the π-π stacking interactions involving the radical ligands. Magnetic investigations show that both Tb complexes exhibit slow relaxation of magnetization at low temperature; strikingly, complex 3 displays a higher energy barrier than that of 1. It represents the first example to use the paramagnetic complex to tune magnetic relaxation of 4f-based compounds.