RESUMO
The radial growth of trees in alpine timberline is particularly sensitive to climate change. We sampled and disposed tree-ring cores of three coniferous tree species including Juniperus saltuaria, Abies forrestii, and Larix potaninii at alpine timberline in Yading Nature Reserve. The standard tree-ring chronology was used to explore the response of radial growth of different timberline species to climate change. The results showed that radial growth of L. potaninii increased after 2000, while that of A. forrestii declined after 2002, and J. saltuaria showed a significant decreasing growth trend in the past 10 years. Such results indicated divergent growth responses to climate factors among the three tree species at alpine timberline. The radial growth of J. saltuaria was sensitive to temperature, and was positively correlated with the minimum temperature from previous October to current August, the mean tempera-ture from previous November to current April and from current July to October, but was negatively associated with the relative humidity from current July to October. The radial growth of A. forrestii showed negative correlation with mean temperature and the maximum temperature from May to June in the current year, while it exhibited positive association with the relative humidity and the Palmer drought severity index from May to June in the current year. L. potaninii radial growth was positively associated with mean temperature and the maximum temperature of November-December in the previous year, the maximum temperature of current March and mean temperature of current August. The temporal stability of climate-growth relationship varied among different timberline species. The positive correlation between radial growth of A. forrestii and J. saltuaria and temperature gradually decreased, while the posi-tive relationship of L. potaninii radial growth and temperature gradually increased. Under the background of climate warming, rapid rise in surface air temperatures may promote the radial growth of L. potaninii, while inhibit that of J. saltuaria and A. forrestii, which may change the position of regional timberline.
Assuntos
Mudança Climática , Larix , China , Larix/crescimento & desenvolvimento , Juniperus/crescimento & desenvolvimento , Abies/crescimento & desenvolvimento , Ecossistema , Árvores/crescimento & desenvolvimento , Conservação dos Recursos Naturais , Temperatura , Caules de Planta/crescimento & desenvolvimento , AltitudeRESUMO
Global warming would significantly impact tree growth in the Tibetan Plateau. However, the specific effects of climate change on the radial growth of Pinus wallichiana in Mount Qomolangma are still uncertain. To investigate the responses of radial growth of P. wallichiana to climate change, we analyzed tree-ring samples in Mount Qomolangma. We removed the age-related growth trends and established three chronologies by using the modified negative exponential curve, basal area index, and regional curve standardization, and conducted Pearson correlation and moving correlation analyses to examine the association between radial growth of P. wallichiana and climatic factors. The results showed that this region had experienced a significant upward trend in temperature and that the Palmer drought severity index (PDSI) indicated a decreasing trend since 1980s, while the relative humi-dity changed from a significant upward to a downward trend around 2004, implying the climate shifted toward warmer and drier. Results of Pearson correlation analysis indicated a significant and positive relationship between the radial growth of P. wallichiana and the minimum temperature of April-June and July-September, and precipitation of January-April in the current year. The radial growth of P. wallichiana was significantly and negatively associated with the relative humidity of June, July, and August in the current year. As temperature rose after 1983, the relationship between radial growth of P. wallichiana and the minimum temperature in July and September of the current year increased from a non-significant association to a significant and positive association, while the relationship between radial growth of P. wallichiana and relative humidity in August and precipitation in September of the current year changed from non-significant correlation to a significant and negative correlation. Results of the moving correlation analysis suggested that the radial growth of P. wallichiana showed a significant and stable correlation with the July-September minimum temperature of the current year. Under the background of climate warming, the rapid increases of temperature would accelerate the radial growth of P. wallichiana in Mount Qomolangma.
Assuntos
Mudança Climática , Tibet , Pinus/crescimento & desenvolvimento , Ecossistema , Temperatura , Caules de Planta/crescimento & desenvolvimento , Aquecimento GlobalRESUMO
Strong evidence has indicated that upregulation of chemokine (CC motif) ligand-2 (CCL2) expression and the presence of an inflammatory tumor microenvironment significantly contribute to the migratory and invasive properties of oral squamous cell carcinoma, specifically oral tongue squamous cell carcinoma (OTSCC). However, the precise epigenetic mechanism responsible for enhanced CCL2 expression in response to the inflammatory mediator tumor necrosis factor alpha (TNF-α) in OTSCC remains inadequately elucidated. We have demonstrated that the production of CCL2 can be induced by TNF-α, and this induction is mediated by the chromatin remodel protein BRG1. Through the use of a chromatin immunoprecipitation (ChIP) assay, we have found that BRG1 was involved in the recruitment of acetylated histones H3 and H4 at the CCL2 promoter, thereby activating TNF-α-induced CCL2 transcription. Furthermore, we have observed that recruitment of NF-κB p65 to the CCL2 promoter was increased following BRG1 overexpression and decreased after BRG1 knockdown in OTSCC cells. Our Re-ChIP assay has shown that BRG1 knockdown completely inhibits the recruitment of both acetylated histone H3 or H4 and NF-κB to the CCL2 promoter. In summary, the findings of our study demonstrate that BRG1 plays a significant role in mediating the production of CCL2 in OTSCC cells in response to TNF-α stimulation. This process involves the cooperative action of acetylated histone and NF-κB recruitment to the CCL2 promoter site. Our data suggest that BRG1 serves as a critical epigenetic mediator in the regulation of TNF-α-induced CCL2 transcription in OTSCC cells.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias da Língua , Fator de Necrose Tumoral alfa , Humanos , Carcinoma de Células Escamosas/genética , Quimiocina CCL2/metabolismo , Epigênese Genética , Histonas/metabolismo , Neoplasias Bucais , NF-kappa B/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Neoplasias da Língua/genética , Microambiente Tumoral , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Liver fibrosis is caused by a variety of chronic liver injuries and has caused significant morbidity and mortality in the world with increasing tendency. Elucidation of the molecular mechanism of liver fibrosis is the basis for intervention of this pathological process and drug development. Nucleophosmin (NPM) is a widely expressed nucleolar phosphorylated protein, which is particularly important for cell proliferation, differentiation and survival. The biological role of NPM in liver fibrosis remains unknown. Here we show that NPM promotes liver fibrosis through multiple pathways. Our study found that NPM was up-regulated in cirrhosis tissues and activated in hepatic stellate cells (HSCs). NPM inhibition reduced liver fibrosis markers expression in HSCs and inhibited the HSCs proliferation and migration. In mice model, NPM knockdown in HSCs or application of specific NPM inhibitor can remarkably attenuate hepatic fibrosis. Mechanistic analysis showed that NPM promotes hepatic fibrosis by inhibiting HSCs apoptosis through Akt/ROS pathway and by upregulating TGF-ß2 through Akt-induced lncMIAT. LncMIAT up-regulated TGF-ß2 mRNA by competitively sponging miR-16-5p. In response to liver injury, hepatocytes, Kupffer cells and HSCs up-regulated NPM to increase TGF-ß2 secretion to activate HSCs in a paracrine or autocrine manner, leading to increased liver fibrosis. Our study demonstrated that NPM regulated hepatotoxin-induced fibrosis through Akt/ROS-induced apoptosis of HSCs and via the Akt/lncMIAT-up-regulated TGF-ß2. Inhibition of NPM or application of NPM inhibitor CIGB300 remarkably attenuated liver fibrosis. NPM serves a potential new drug target for liver fibrosis.
Assuntos
Células Estreladas do Fígado , Nucleofosmina , Animais , Camundongos , Espécies Reativas de Oxigênio , Fator de Crescimento Transformador beta2 , Proteínas Proto-Oncogênicas c-akt , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Proteínas Nucleares/genética , ApoptoseRESUMO
To investigate the plant community characteristics of alpine cutting blanks under different restoration approaches, we conducted a field survey on cutting blanks experienced either natural restoration (40 years) or artificial restoration (30, 40 and 50 years) in western Sichuan, with natural forests as the reference. Our results showed that after 40 years natural succession, cutting blank was replaced by the secondary shrub of Spiraea alpina, while artificial restoration plantation was dominated by Picea likiangensis var. rubescens. The similarity indices between these communities and natural forests were low (0.19) and medium (0.28-0.49), respectively. Cutting blank through natural and artificial restoration had lower species diversity in the shrub layer but higher diversity in the herb layer than that of natural forests. With the increases of recovery time, total cross-sectional area at breast height, wood volume, index of species diameter class distribution, diversity indices, and similarity indices between plantations and natural forests gradually increased, while stand density gradually decreased. Compared with natural forests, plantations were facing with problems including high stand density, unreasonable structure, pure stands of cohorts and poor regeneration.
Assuntos
Florestas , Picea , Biodiversidade , China , EcossistemaRESUMO
HnRNPA2/B1 is highly expressed in many tumors. However, the role of hnRNPA2/B1 in breast cancer is not clear. In this study, we found the proliferation rate was decreased after knockout of hnRNPA2/B1 by CRISPR-CAS9 in MCF-7 cells, as demonstrated by the reduced expression of CDK4 and p-AKT, and the increased expression of P27. Besides this, the western blot results showed that knockout of hnRNPA2/B1 increased the rate of apoptosis and declined autophagy. By in vivo assay, we found that knockout of hnRNPA2/B1 suppressed tumor growth in a xenograft mouse model. Immunohistochemical staining results confirmed knockout of hnRNPA2/B1 impaired tumor angiogenesis, as illustrated by downregulated expression of VEGF-A. Besides this, interacting proteins with hnRNPA2/B1 were identified by mass spectrometry and the PPI network was constructed. GO analysis suggests that the Interacting proteins are mainly enriched in the Wnt signaling pathway, tumor necrosis factor-mediated signaling pathway, translation, and so on. We then identified hnRNPA2/B1 interacted with signal transducer and activator of transcription 3 (STAT3), as supported by the colocalization of hnRNPA2/B1 and STAT3. Meanwhile, knockout of hnRNPA2/B1 inhibited the phosphorylation of STAT3. Collectively, our results demonstrate that hnRNPA2/B1 promotes tumor cell growth in vitro and in vivo by activating the STAT3 pathway, regulating apoptosis and autophagy.
Assuntos
Neoplasias da Mama/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Células MCF-7 , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The RNA-binding protein hnRNPA2/B1 is a member of the hnRNPs family and is widely expressed in various tissues. hnRNPA2/B1 recognizes and binds specific RNA substrates and DNA motifs and is involved in the transcription, splicing processing, transport, stability, and translation regulation of a variety of RNA molecules and in regulating the expression of a large number of genes. hnRNPA2/B1 is also involved in telomere maintenance and DNA repair, while its expression changes and mutations are involved in the development of various tumors and neurodegenerative and autoimmune diseases. This paper reviews the role and mechanism of hnRNPA2/B1 in RNA metabolism, tumors, and neurodegenerative and autoimmune diseases. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA in Disease and Development > RNA in Disease.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , RNA , Biologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares HeterogêneasRESUMO
BACKGROUND: Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is an important RNA-binding protein that affects the RNA processing, splicing, transport and stability of many genes. hnRNPA2/B1 is expressed during proliferation and metastasis of various cancer types and promotes such processes. However, the precise role and mechanism of hnRNPA2/B1 in breast cancer remain unclear. METHODS: The association of hnRNPA2/B1 with breast cancer metastasis was assessed using tissue chips, mouse models and publicly available data. The role and mechanism of hnRNPA2/B1 in breast cancer metastasis were studied in cell lines and mouse models. FINDINGS: In contrast to other cancer research findings, hnRNPA2/B1 expression was negatively correlated with breast cancer metastasis. hnRNPA2/B1 inhibited MDA-MB-231 triple-negative breast cancer (TNBC) cell metastasis in vitro and in vivo. hnRNPA2/B1 knockout activated ERK-MAPK/Twist and GR-beta/TCF4 pathways but inhibited STAT3 and WNT/TCF4 signalling pathways. Profilin 2 (PFN2) promoted breast cancer cell migration and invasion, whereas hnRNPA2/B1 bound directly to the UAGGG locus in the 3'-untranslated region of PFN2 mRNA and reduced the stability of PFN2 mRNA. INTERPRETATION: Our data supported the role of hnRNPA2/B1 in tumour metastasis risk and survival prediction in patients with breast cancer. The inhibitory role of hnRNPA2/B1 in metastasis was a balance of downstream multiple genes and signalling pathways. PFN2 downregulation by hnRNPA2/B1 might partly explain the inhibitory mechanism of hnRNPA2/B1 in breast cancer metastasis. Therefore, hnRNPA2/B1 might be used as a new prognostic biomarker and valuable molecular target for breast cancer treatments.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Genes Neoplásicos , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Transdução de Sinais , Regiões 3' não Traduzidas/genética , Actinas/metabolismo , Animais , Neoplasias da Mama/diagnóstico , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Profilinas/genética , Profilinas/metabolismo , Prognóstico , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de SobrevidaRESUMO
Reactive oxygen species (ROS) play both deleterious and beneficial roles in cancer cells. Nucleophosmin (NPM) is heavily implicated in cancers of diverse origins, being its gene over-expression in solid tumors or frequent mutations in hematological malignancies. However, the role and regulatory mechanism of NPM in oxidative stress are unclear. Here, we found that NPM regulated the expression of peroxiredoxin 6 (PRDX6), a member of thiol-specific antioxidant protein family, consequently affected the level and distribution of ROS. Our data indicated that NPM knockdown caused the increase of ROS and its relocation from cytoplasm to nucleoplasm. In contrast, overexpression or cytoplasmic localization of NPM upregulated PRDX6, and decreased ROS. In addition, NPM knockdown decreased peroxiredoxin family proteins, including PRDX1, PRDX4, and PRDX6. Co-immunoprecipitation further confirmed the interaction between PRDX6 and NPM. Moreover, NSC348884, an inhibitor specifically targeting NPM oligomerization, decreased PRDX6 and significantly upregulated ROS. These observations demonstrated that the expression and localization of NPM affected the homeostatic balance of oxidative stress in tumor cells via PRDX6 protein. The regulation axis of NPM/PRDX/ROS may provide a novel therapeutic target for cancer treatment. J. Cell. Biochem. 118: 4697-4707, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Antioxidantes/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Peroxirredoxina VI/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Humanos , Indóis/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/genética , Nucleofosmina , Peroxirredoxina VI/antagonistas & inibidores , Peroxirredoxina VI/genéticaRESUMO
Tumor growth and metastasis are angiogenesis dependent. Angiogenic growth involves endothelial cell proliferation, migration, and invasion. Ephrin-B2 is a ligand for Eph receptor tyrosine kinases and is an important mediator in vascular endothelial growth factor-mediated angiogenesis. However, research offer controversial information regarding effects of ephrin-B2 on vascular endothelial cells. In this paper, proteome analyses showed that ephrin-B2/Fc significantly activates multiple signaling pathways related to cell proliferation, survival, and migration and suppresses apoptosis and cell death. Cytological experiments further confirm that ephrin-B2/Fc stimulates endothelial cell proliferation, triggers dose-dependent migration, and suppresses cell apoptosis. Results demonstrate that soluble dose-dependent ephrinB2 can promote proliferation and migration and inhibit apoptosis of human umbilical vein endothelial cells. These results also suggest that ephrinB2 prevents ischemic disease and can potentially be a new therapeutic target for treating angiogenesis-related diseases and tumors.
Assuntos
Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Células Cultivadas , Efrina-B2/genética , Efrina-B2/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Nucleophosmin(NPM), heavily implicated in diverse solid tumors, is an important multifunctional protein mainly located in the nucleolus. Our previous study confirmed that NPM can also localize and accumulate in the cytoplasm of liver cancer cells. However, the role of cytoplasmic NPM (NPMc +) is unclear. Here, we showed that both nucleolar NPM and NPMc+ could promote cell proliferation, although the effect of NPMc+ was weaker than that of NPM. Cell adhesion ability of hepatoma cells was significantly reduced to a greater extent by NPMc+ expression. Nucleolar NPM enhanced cell migration and invasion, whereas NPMc+ impeded cell migration and invasion. The investigation of NPM interactional proteins by proteomic method demonstrated that the NPM was involved in multiple biological processes. By contrast, the interactional proteins of NPMc+ were mainly implicated in tRNA amino acylation regulation. The interactional network of NPMc+ was significantly small and simple. These results suggested that relocation of NPM altered its interactional network and consequently disturbed the primary functions, including cell proliferation, adhesion, migration, and invasion. NPM plays a promotional role in cancer and the reducing relocation may be a potential therapeutic target for hepatocellular carcinoma. J. Cell. Biochem. 118: 3225-3236, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Carcinoma Hepatocelular/metabolismo , Movimento Celular , Proliferação de Células , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Carcinoma Hepatocelular/patologia , Adesão Celular , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/patologia , Nucleofosmina , Transporte ProteicoRESUMO
The observation that prolonged inflammation plays a causative role in cancer development has been well documented. However, an incremental process that leads from healthy to malignant phenotypes has not yet been described. Experimentally induced hepatocellular carcinoma is considered one of the representative laboratory models for studying this process. Hepatic exposure to viral infection or toxic reagents leads to chronic inflammation and gradual transformation into hepatocellular carcinoma. Here we present metabolomic profiles of hepatic cells at different stages during inflammation-induced cellular transformation by N-nitrosodiethylamine. Using gas chromatography-mass spectrometry, we quantitatively assessed the changes in cellular metabolites during the transformation process in hepatitis and liver cirrhosis. Further pathway analysis of the differentially expressed metabolites showed that carbohydrate metabolism and lipid metabolism were greatly altered in hepatitis and liver cirrhosis, respectively. Additionally, the enhanced inflammation in cirrhosis was associated with a shift from carbohydrate metabolism to lipid and amino acid metabolism. Among the differentially expressed metabolites found in diseased mouse livers, d-glucose and d-mannitol showed the most significant changes, highlighting them as potential early-diagnostic biomarkers of hepatocellular carcinoma development. Taken together, these investigations into the dynamic metabolic changes that occur during the precancerous stages of hepatocellular carcinoma add to and refine understanding of how chronic inflammation ultimately leads to cancer. Furthermore, the findings set the stage for identifying metabolites that may serve as early-diagnostic indicators of these unfolding events.
Assuntos
Transformação Celular Neoplásica/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Inflamação/metabolismo , Fígado/metabolismo , Metabolômica/métodos , Animais , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/genética , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Análise por Conglomerados , Dietilnitrosamina , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hepatite Animal/induzido quimicamente , Hepatite Animal/metabolismo , Humanos , Inflamação/genética , Lipopolissacarídeos , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Metaboloma/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Pirrolidinas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TiocarbamatosRESUMO
Nucleophosmin (NPM, also known as B23), mainly localized in the nucleolus, has been reported to be overexpressed in many types of human cancer, including colon, ovarian, prostate and gastric cancer. NPM was identified while screening the differential nuclear matrix proteins during HMBA-induced differentiation of human liver cancer cells. We investigated the aberrant expression and subcellular localization of NPM in clinical liver cancer tissues and a cell line with the aim of providing more evidence for revealing the roles of NPM on regulating liver cancer cell proliferation and differentiation. In addition, we studied the potential interaction between NPM and several important proteins. Our results revealed that NPM protein was overexpressed in cancer cells, which was in accordance with the overexpressed mRNA in cancer tissues compared to the corresponding non-cancer tissues. We also found a decrease of NPM in protein and mRNA levels upon treatment with the differentiation reagent HMBA. We focused on the aberrant localization of NPM. Immunochemistry and immunofluorescence revealed aberrant cytoplasmic and nucleoplasm localization of NPM in liver cancer tissues and its colocalization with c-Myc, c-Fos, P53 and Rb in the SMMC-7721 cell line. The interactions between NPM and the above proteins were confirmed by GST pull-down assay and co-immunoprecipitation assay. These findings indicate that NPM plays a regulatory role in liver cancer, which deserves in-depth investigation.
Assuntos
Neoplasias Hepáticas/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Acetamidas/farmacologia , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Citoplasma/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/metabolismo , NucleofosminaRESUMO
Prohibitin (PHB), also known as inhibin, is important in cell proliferation, differentiation and apoptosis. This protein localizes to the inner membrane of mitochondria, where it acts as a chaperone protein, and is also found in the nucleus, where it negatively regulates transcription. The tumor-suppressive role of PHB in cell proliferation appears to be contradictory. In this study, we investigated the existence, localization and alterations in the expression of PHB in the whole cell and nuclear matrix and analyzed its co-localization with the expression products of related genes. The western blot analysis results revealed that PHB exists in the composition of nuclear matrix proteins and that the expression level of PHB is significantly increased in the whole cell and markedly decreased in the nuclear matrix after curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) treatment. The laser confocal scanning microscope results demonstrated the co-localization of PHB with p53, c-Myc, Bax, and Fas in HaCaT cells, and this co-localization region was transferred as a result of curcumin treatment. In addition, the results of the GST pull-down assay demonstrated the direct interaction of PHB with p53, c-Myc and Bax but not Fas in vitro. Results of the present study confirmed that the expression and distribution of PHB, which is a nuclear matrix protein, affect the apoptosis of HaCaT cells and its co-localization with specific gene products connected with cell apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Matriz Nuclear/metabolismo , Proteínas Repressoras/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Curcumina/farmacologia , Células Epidérmicas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Matriz Nuclear/genética , Proibitinas , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/químicaRESUMO
Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is involved in the synthesis of RNA. Its expression is up-regulated in many tumor cell lines. In this study, we investigated the distribution of hnRNP A2/B1 in the nuclear matrix, including its co-localization with expression products of related genes. Results from 2-DE PAGE and MS showed that hnRNP A2/B1 is involved with components of nuclear matrix proteins of SK-N-SH cells, and that its expression level is down-regulated after retinoic acid (RA) treatment. Protein immunoblotting results further confirm the existence of hnRNP A2/B1 in the nuclear matrix, as well as its down-regulation after RA treatment. Immunofluorescence microscopy observation showed that hnRNP A2/B1 localized in nuclear matrix of SK-N-SH cells and its distribution regions were altered after RA treatment. Laser scanning confocal microscopy observation showed that hnRNP A2/B1 co-localized with c-Myc, c-Fos, P53, and Rb in SK-N-SH cells. The co-localized region was altered as a result of RA treatment. Our data proved that hnRNP A2/B1 is a nuclear matrix protein and can be up-regulated in human neuroblastoma. The expression and distribution of hnRNP A2/B1 can affect the differentiation of SK-N-SH cells, as well as its co-localization with related oncogenes and tumor suppressor genes.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Matriz Nuclear/metabolismo , Transporte Proteico/efeitos dos fármacos , Tretinoína/farmacologia , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Filamentos Intermediários/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína do Retinoblastoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The nuclear matrix-intermediate filament system of human neuroblastoma SK-N-SH cells before and after retinoic acid (RA) treatment was selectively extracted and the distribution of prohibitin (PHB) in the nuclear matrix, as well as its colocalization with related genes, was observed. Results of two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS) identification, and protein immunoblotting all confirm that PHB was present in the components of SK-N-SH nuclear matrix proteins and was down-regulated after RA treatment. Immunofluorescence microscopy observations show that PHB was localized in the nuclear matrix and its distribution was altered due to RA treatment. Laser confocal microscopy results reveal that PHB colocalized with the expression products of c-myc, c-fos, p53, and Rb, but the colocalization region was altered after RA treatment. Our results prove that PHB is a nuclear matrix protein and is localized in nuclear matrix fibers. The distribution of PHB in SK-N-SH cells and its colocalization with related proto-oncogenes and tumor suppressor genes suggest that PHB plays pivotal roles in the differentiation of SK-N-SH cells and deserves further study.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Matriz Nuclear/metabolismo , Proteínas Repressoras/metabolismo , Tretinoína/farmacologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Humanos , Immunoblotting , Filamentos Intermediários/efeitos dos fármacos , Filamentos Intermediários/metabolismo , Microscopia de Fluorescência , Proteínas Associadas à Matriz Nuclear/metabolismo , Proibitinas , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/química , Reprodutibilidade dos Testes , Proteína do Retinoblastoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND AND AIM: Nuclear-matrix proteins can be proteomic markers for cancer lesions. The present study aimed to determine the roles of heterogeneous nuclear ribonucleoproteins--A2 and B1 (hnRNP-A2/B1) in human gastric carcinogenesis. METHODS: Human gastric cancer and non-cancerous tissues were collected for immunohistochemical analysis. Proteomics technique, Western blot, laser confocal microscope, and real-time quantitative reverse transcription-polymerase chain reaction were performed to determine the aberrant expression of nuclear-matrix proteins. RESULTS: hnRNP-A2/B1 existed in the nuclear matrix of gastric cancer cells, and its expression was enhanced in human gastric cancer and decreased by hexamethylene bisacetamide. The colocalization of hnRNP-A2/B1 with c-myc, c-fos, p53, and Rb was translocated from the nucleolus to the cytoplasm during the differentiation of tumor cells. CONCLUSIONS: hnRNP-A2/B1 affected tumor cell differentiation through interaction with oncogenes and tumor-suppressor genes, and it was overexpressed in human gastric cancer. We postulate that hnRNP-A2/B1 could serve as a biomarker for the diagnosis of human gastric cancer.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Neoplasias Gástricas/metabolismo , Acetamidas/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Biomarcadores Tumorais/genética , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , China , Citoplasma/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Imuno-Histoquímica , Microscopia Confocal , Matriz Nuclear/metabolismo , Prognóstico , Proteômica/métodos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Non-viral vectors have been widely used in gene transfection. However, its drawbacks limit its applications. In this study, a novel cationic polymer was developed as a DNA condensing agent for systemic gene delivery. Its transfection efficiency, cytotoxicity, and biocompatibility were also evaluated. Sofast, novel cationic polymer of branched polyethlenimine, was constructed by chemical methods. Its diameter, zeta potential, nucleic acid binding ability, and anti-nuclease ability were detected by electron microscopy and gel electrophoresis. In vitro, the efficiency of transfection was measured by comparing it with other gene vectors in different cell lines. MTT assay was performed to determine cytotoxicity. The compatibility of Sofast gene vector in the serum and its stability were investigated. Mouse, guinea pig and rabbit were used to process the toxic, allergenic, and pyrogenic properties of the vector in vivo. The in vivo expression was performed in the guinea pig. The results from an in vitro assay proved that the Sofast gene vector had a higher transfection efficiency than other gene vectors in a variety of primary cell cultures and transformed cell lines. The cytotoxicity assay showed a lower cytotoxicity and the cellular survival rate was >90%. The Sofast gene vector possessed compatibility with the serum and was fit to be transported at normal temperature. The results from in vivo tests indicated that the Sofast gene vector had greatly lower cytotoxicity, better biocompatibility, and higher transfection efficiency compared with other gene vectors. Because the Sofast gene vector had higher transfection efficiency, lower cytotoxicity and better compatibility than other gene vectors, it could be used for gene transfection both in vitro and in vivo.
Assuntos
Cátions/química , DNA/administração & dosagem , Polímeros/química , Transfecção , Animais , Cátions/efeitos adversos , Cátions/síntese química , Cátions/metabolismo , Linhagem Celular , Sobrevivência Celular , DNA/metabolismo , Desoxirribonucleases/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Cobaias , Células HEK293 , Humanos , Luciferases/genética , Masculino , Polímeros/efeitos adversos , Polímeros/síntese química , Polímeros/metabolismo , beta-Galactosidase/análiseRESUMO
Nucleophosmin (NPM1) is frequently upregulated and mutated in various tumor cells. To investigate the mechanism of induced differentiation of tumor cells, the nuclear matrix of human hepatocarcinoma SMMC-7721 cells induced by hexamethylene bisacetamide (HMBA) was selectively extracted and subjected to proteomic methodologies. We confirmed that NPM1 existed in nuclear matrix proteins and downregulated after HMBA treatment. By using immunogold electromicroscopy, we found that NPM1 was localized on nuclear matrix-intermediate filaments. Our study also revealed the colocalization between NPM1 and products of oncogenes or tumor suppressor genes including c-Fos, c-Myc, p53, and Rb by using laser scanning confocal microscopy in SMMC-7721 cells.
Assuntos
Acetamidas/farmacologia , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Matriz Nuclear/metabolismo , Nucleoplasminas/biossíntese , Western Blotting , Carcinoma Hepatocelular/ultraestrutura , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Matriz Nuclear/ultraestrutura , Nucleofosmina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
To synthesize a lipid-cationic polymer (LCP) containing brassidic acid side chain and to investigate its transfection efficiency and characteristics as a siRNA gene vector. The LCP was chemically synthesized and its nucleic acid binding capacity was determined by gel electrophoresis. HeLa-EGFP and TH1080-EGFP cell lines were transfected with siRNA against enhanced green fluorescent protein (EGFP) gene using a LCP to investigate the transfection efficiency. An MTT assay was performed to evaluate the cellular toxicity of the LCP vector. Its degradability and stability under acidic conditions were also investigated. The LCP vector possessed high DNA binding capacity. More than 73% of the cellular fluorescence was inhibited by the LCP-mediated transfection of siRNA against EGFP gene, indicating that vector had high transfection efficiency. Cellular viability was about 95% at the optimum transfection efficiency of LCP, suggesting that the cellular toxicity of LCP was very low. The LCP was also observed to be degradable; moreover, it could be easily stored at normal temperature. A gene vector used for the transfection of siRNA was successfully fabricated from synthesized LCP. Its numerous excellent properties entitle values for further scientific research.