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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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OBJECTIVE: The receptor-type tyrosine-protein phosphatase κ (PTPRK) is a candidate tumor suppressor involved in the tumorigenesis of various organs. However, its expression and biological roles in non-small-cell lung cancer (NSCLC) have not yet been investigated. METHODS: PTPRK expression in NSCLC tissues and cell lines was examined using real-time PCR and western blotting. In addition, the effects of PTPRK on cell migration, invasion, and proliferation were evaluated in vitro. Furthermore, we explored whether the downregulation of PTPRK led to STAT3 activation in NSCLC cell lines by western blotting. The expression of phospho-STAT3Tyr705 in primary human NSCLC tissues was evaluated by immunohistochemistry. RESULTS: The results showed that PTPRK expression was frequently reduced in NSCLC tissues with lymph node metastasis and cell lines. The inhibition of PTPRK expression resulted in increased proliferation, invasion, and migration of NSCLC cells in vitro. Additionally, after silencing of PTPRK, phospho-STAT3Tyr705 was significantly increased in NSCLC cells. Moreover, the phospho-STAT3Tyr705 levels of NSCLC tissues were positively correlated with lymph node metastasis and significantly inversely correlated with the expression of PTPRK (p < 0.05). CONCLUSIONS: These results suggested that PTPRK functions as a novel tumor suppressor in NSCLC, and its suppressive ability may be involved in STAT3 activation.
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Carcinoma Pulmonar de Células não Pequenas/metabolismo , Metástase Linfática/patologia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Fator de Transcrição STAT3/metabolismo , Células A549 , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Metástase Linfática/genética , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Fator de Transcrição STAT3/genética , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
Non-small cell lung cancer (NSCLC) is one of the most common malignancies worldwide. In this study, we investigated Ultrahigh Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry and Gas Chromatography Time-of-Flight/Mass Spectrometry-based non-targeted metabolomic profiles of serum samples obtained from early-stage NSCLC patients and healthy controls (HC). Metabolic pathways and the biological relevance of potential biomarkers were extensively studied to gain insights into dysregulated metabolism in NSCLC. The identified biomarker candidates were further externally validated via a targeted metabolomics analysis. The global metabolomics profiles could clearly distinguish NSCLC patients from HC. Phosphatidylcholine (PC) levels were found to be dysregulated in glycerophospholipid (GPL) metabolism, which was the top altered pathway in early-stage NSCLC. Compared with those in HC, significant increases in the levels of saturated and monounsaturated PCs such as PC (15:0/18:1), PC (18:0/16:0) and PC (18:0/20:1) were observed in NSCLC. Additionally, relative to those in HC, the levels of 9 polyunsaturated PCs, namely, PC (17:2/2:0), PC (18:4/3:0), and PC (15:0/18:2), and so on were significantly decreased in NSCLC patients. A panel of 12 altered PCs had good diagnostic performance in differentiating early-stage NSCLC patients from HC, and these PCs may thus be used as serum biomarkers for the early diagnosis of NSCLC.
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Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Fosfatidilcolinas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Fosfatidilcolinas/isolamento & purificaçãoRESUMO
OBJECTIVE: Non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancer cases which cause most of cancer-related deaths globally. As our previous study discovered miR-1260b can be regarded as a specific signature for metastasis in NSCLC patients. However, the molecular mechanisms of miR-1260b underlying NSCLC progression and metastasis remain dismal. METHODS: The expression of miR-1260b in NSCLC tissues and cell lines were examined by real-time PCR, the effects of miR-1260b on cell migration, invasion and proliferation were evaluated in vitro. Furthermore, luciferase reporter assay was performed to identify the targets of miR-1260b, and the association between miR-1260b and its target gene was determined by real-time PCR and western blot assay. RESULTS: The results showed that miR-1260b was significantly upregulated in NSCLC cell lines. The inhibition of miR-1260b expression decreased the migratory and invasive rates in A549 cells while miR-1260b overexpression had the opposite effect. Furthermore, PTPRK was identified as a direct target of miR-1260b, and PTPRK expression was inversely correlated with miR-1260b in NSCLC cell lines and clinical tissues. CONCLUSIONS: These results suggested that miR-1260b may play an important role in NSCLC metastasis progression and could serve as a putative target for diagnosis and treatment of NSCLC.
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Carcinoma Pulmonar de Células não Pequenas/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , MicroRNAs/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/genética , Humanos , Neoplasias Pulmonares/genética , Regulação para CimaRESUMO
With accumulating public omics data, great efforts have been made to characterize the genetic heterogeneity of breast cancer. However, identifying novel targets and selecting the best from the sizeable lists of candidate targets is still a key challenge for targeted therapy, largely owing to the lack of economical, efficient and systematic discovery and assessment to prioritize potential therapeutic targets. Here, we describe an approach that combines the computational evaluation and objective, multifaceted assessment to systematically identify and prioritize targets for biological validation and therapeutic exploration. We first establish the reference gene expression profiles from breast cancer cell line MCF7 upon genome-wide RNA interference (RNAi) of a total of 3689 genes, and the breast cancer query signatures using RNA-seq data generated from tissue samples of clinical breast cancer patients in the Cancer Genome Atlas (TCGA). Based on gene set enrichment analysis, we identified a set of 510 genes that when knocked down could significantly reverse the transcriptome of breast cancer state. We then perform multifaceted assessment to analyze the gene set to prioritize potential targets for gene therapy. We also propose drug repurposing opportunities and identify potentially druggable proteins that have been poorly explored with regard to the discovery of small-molecule modulators. Finally, we obtained a small list of candidate therapeutic targets for four major breast cancer subtypes, i.e., luminal A, luminal B, HER2+ and triple negative breast cancer. This RNAi transcriptome-based approach can be a helpful paradigm for relevant researches to identify and prioritize candidate targets for experimental validation.
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BACKGROUND: Mounting evidence indicates that nuclear targeting by growth factors plays an indispensable role on their biological activities. Midkine (MK) is a multifunctional growth factor and has been discovered to play important roles in carcinogenesis. MK has been reported to localize to the nucleus and nucleolus of HepG2 cells and is involved in cell proliferation and apoptosis. METHODS: The interaction was reconfirmed by in vitro pull down and in vivo coimmunoprecipitation (Co-IP), also by the colocalization in the HepG2 cells. The proliferation and migration was determined by MTT and trans-well assay. RESULTS: PLSCR1 was identified as a novel MK-interacting protein. Notably, PLSCR1 interacted with MK in the cell nucleus and regulated hepatic carcinoma cell proliferation and migration. CONCLUSIONS: This study suggests that PLSCR1 positively regulates hepatic carcinoma cell proliferation and migration through interacting with MK, thus deepening our understanding on the regulation of midkine during hepatic carcinoma growth and metastasis.
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Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Movimento Celular , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Microscopia de Fluorescência , Midkina , Metástase Neoplásica , Sais de Tetrazólio/química , Tiazóis/química , Técnicas do Sistema de Duplo-Híbrido , CicatrizaçãoRESUMO
Midkine (MK) is a heparin-binding growth factor involved in growth, survival, migration, and differentiation of various target cells and dysregulation of MK signaling is implicated in a variety of inflammatory diseases and cancers. Although MK has been reported to act on endothelial cells and to have proangiogenic effects, the exact role of MK in angiogenesis is poorly defined. Progranulin (PGRN) is a secreted glycoprotein that functions as an important regulator of development, cell cycle progression, cell motility, tumorigenesis, angiogenesis. We screened the PGRN from the hepatic cancer cell cDNA library which was interacted with MK, and confirmed the binding by co-immunoprecipitation and co-localization. During our study, the interaction between MK and PGRN had the important role on the HUVECs proliferation, migration, and tubulogenesis, which indicated the interaction may regulate the angiogenesis, also the in vivo angiogenesis model CAM showed the promotion effect stimulated by MK and PGRN. These findings provide the first evidence linking the association of MK and PGRN and may identify the mechanism of MK during the hepatocellular carcinoma angiogenesis.
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Carcinoma Hepatocelular/irrigação sanguínea , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Hepáticas/irrigação sanguínea , Neovascularização Patológica/metabolismo , Fatores de Crescimento Neural/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Humanos , Imunoprecipitação , Midkina , Progranulinas , Transdução de Sinais/fisiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Previous studies have revealed several targets of miR-10b, such as syndecan-1, HOXD10, TBX5, and E-cadherin. In this study, we aimed to assess whether Krüppel-like factor 4 (KLF4) is a target gene of miR-10b in gastric cancer (GC). Targeting of KLF4 by miR-10b was confirmed by dual-luciferase reporter assays. The expression levels of miR-10b and KLF4 mRNA in 5 different gastric cancer cell lines and 65 pairs of gastric cancer tissues were detected by Real-time PCR. In addition, KLF4 protein in gastric cancer cell lines and 30 GC tissues was measured by western blotting and immunochemistry, respectively. KLF4 is a direct target gene of miR-10b in GC, and its expression is reduced by miR-10b at both mRNA and protein levels. In addition, the expression level of miR-10b was tendentiously upregulated in GC tissues while the expression levels of KLF4 mRNA and protein were decreased in gastric cancer tissues compared with normal adjacent tissue. There was a dramatically inverse correlation between the expression levels of miR-10b and KLF4 mRNA in GC (r=-0.339, P=0.006). These findings indicate that miR-10b was upregulated in GC and may have a key role in GC pathogenesis and development through the downregulation of its target gene KLF4.
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Carcinoma/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , MicroRNAs/metabolismo , Neoplasias Gástricas/metabolismo , Regiões 3' não Traduzidas , Sítios de Ligação , Carcinoma/genética , Carcinoma/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Transfecção , Regulação para CimaRESUMO
BACKGROUND: To investigate the inhibitory effect of midkine-binding peptides on human umbilical vein endothelial cells (HUVECs) proliferation and angiogenesis of xenograft tumor. METHODS: The midkine-binding peptides were panned by Ph.D.-7(™) Phage Display Peptide Library Kit, and the specific binding activities of positive clones to target protein were examined by phage ELISA. The effect of midkine-binding peptides on proliferation of HUVECs was confirmed by MTT test. The xenograft tumor model was formed in BALB/c mice with the murine hepatocarcinoma cells H22 (H22). Microvessel density (MVD) was analyzed by immunohistochemistry of factor VIII staining. RESULTS: Midkine-binding peptides have the inhibitory effects on tumor angiogenesis, a proliferation assay using human umbilical vein endothelial cells (HUVECs) indicated that particular midkine-binding peptides significantly inhibited the proliferation of the HUVECs. Midkine-binding peptides were also observed to efficiently suppress angiogenesis induced by murine hepatocarcinoma H22 cells in BALB/c nude mice. CONCLUSION: The midkine-binding peptides can inhibit solid tumor growth by retarding the formation of new blood vessels. The results indicate that midkine-binding peptides may represent potent anti-angiogenesis agents in vivo.
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Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Midkina , Neovascularização Patológica/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lung cancer is one of the most common causes of cancer death, for which no validated tumor biomarker is sufficiently accurate to be useful for diagnosis. Additionally, the metabolic alterations associated with the disease are unclear. In this study, we investigated the construction, interaction, and pathways of potential lung cancer biomarkers using metabolomics pathway analysis based on the Kyoto Encyclopedia of Genes and Genomes database and the Human Metabolome Database to identify the top altered pathways for analysis and visualization. We constructed a diagnostic model using potential serum biomarkers from patients with lung cancer. We assessed their specificity and sensitivity according to the area under the curve of the receiver operator characteristic (ROC) curves, which could be used to distinguish patients with lung cancer from normal subjects. The pathway analysis indicated that sphingolipid metabolism was the top altered pathway in lung cancer. ROC curve analysis indicated that glycerophospho-N-arachidonoyl ethanolamine (GpAEA) and sphingosine were potential sensitive and specific biomarkers for lung cancer diagnosis and prognosis. Compared with the traditional lung cancer diagnostic biomarkers carcinoembryonic antigen and cytokeratin 19 fragment, GpAEA and sphingosine were as good or more appropriate for detecting lung cancer. We report our identification of potential metabolic diagnostic and prognostic biomarkers of lung cancer and clarify the metabolic alterations in lung cancer.
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Biomarcadores Tumorais/sangue , Endocanabinoides/sangue , Neoplasias Pulmonares/sangue , Prognóstico , Esfingosina/sangue , Idoso , Idoso de 80 Anos ou mais , Bases de Dados de Proteínas , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Redes e Vias Metabólicas/genética , Metaboloma/genética , Pessoa de Meia-IdadeRESUMO
PURPOSE: Lung cancer is one of the most common causes of death from cancer. Serum markers that enable diagnosis of the disease in the early stage have not been found. METHODS: Serum samples were collected from 30 healthy volunteers and from 30 lung cancer patients preoperatively and postoperatively. Samples were subjected to metabolomic analysis using liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry and gas chromatography/mass spectrometry. Differences in metabolomic profiles among the three groups were characterized by multivariate statistical techniques such as principal components analysis and partial least squares discriminant analysis (PLS-DA). An independent t test was used to determine whether levels of biomarker candidates identified using PLS-DA modeling were significantly different among groups at the univariate analysis level (p < 0.05). RESULTS: Based on pattern recognition results and univariate analysis, we showed that levels of ten potential biomarkers in serum were significantly different in the preoperative lung cancer patients compared with healthy volunteers and/or the postoperative lung cancer patients. The levels of sphingosine, phosphorylcholine, glycerophospho-N-arachidonoyl ethanolamine, γ-linolenic acid, 9,12-octadecadienoic acid, oleic acid, and serine were significantly different in preoperative lung cancer patients compared to healthy volunteers and to postoperative lung cancer patients. For prasterone sulfate, α-hydroxyisobutyric acid, 2,3,4-trihydroxybutyric acid, the levels were statistically different in preoperative and postoperative lung cancer patients compared with the healthy volunteers. CONCLUSIONS: Our study identified potential metabolic biomarkers for diagnosis of lung cancer.
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Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/cirurgia , Espectrometria de Massas/métodos , Metabolômica/métodos , Idoso , Biomarcadores Tumorais/sangue , Humanos , Neoplasias Pulmonares/diagnóstico , Pessoa de Meia-Idade , Período Pós-Operatório , Período Pré-OperatórioRESUMO
The increasing evidence supported the role of Enhancer of Zeste Homolog 2 (EZH2) in the cancer development and progression. However, its precise role in the tumorigenesis of Nasopharyngeal Carcinoma (NPC) remains to be elucidated. EZH2 was depleted by retroviral infection in the NPC cells (HK-1, CNE-2, CNE-1 and C666-1). The degree of EZH2 knockdown was then assessed by real-time quantitative PCR and Western Blot analysis. Cell proliferation was assessed using the soluble tetrazolium salt (MTS) cell proliferation assay, and cell cycle was measured by FACS test. The methylation status of p16(INK4a) was determined by bisulphate treatment of the DNA, followed by MSP. EZH2 was over-expressed in NPC cells, and the expression in undifferentiated-derived NPC cells (CNE-1, C666-1) was more significant than differentiated-derived NPC cells (HK-1, CNE-2). EZH2 was successfully depleted after retroviral infection in C666-1 cells, and the EZH2 depletion could inhibit the proliferation and arrested G1/S phase of NPC cells. In addition, both mRNA and protein levels of p16(INK4a) increased significantly in presence of EZH2 depletion. The further Methylation-Specific Polymerase Chain Reaction (MSP) assay suggested that over-expressed EZH2 may contribute to the reduction of p16(INK4a) expression by hyper methylating its promoter. EZH2 is overexpressed in NPC and reduces expression of p16(INK4a) by influencing methylation, opening therapeutic options.
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Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação Neoplásica da Expressão Gênica , Complexo Repressor Polycomb 2/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste , Pontos de Checagem da Fase G1 do Ciclo Celular , Expressão Gênica , Inativação Gênica , Humanos , Neoplasias NasofaríngeasRESUMO
BACKGROUND AND OBJECTIVES: Midkine (MK) mRNA was highly expressed in various human cancer tissues and cells. The present study aimed to investigate whether MK mRNA level in peripheral blood mononuclear cells (PBMC) could serve as a diagnostic biomarker for patients having primary non-small cell lung cancer (NSCLC). METHODS: MK mRNA level in PBMC from 87 patients with primary NSCLC, 35 patients with lung benign lesion (LEL), and 30 healthy volunteers was analyzed by real-time quantitative RT-PCR. The levels of serum carcinoembryonic antigen, carbohydrate antigen 125 (CA125), and neuron-specific enolase were detected by chemiluminescent microparticle enzyme immunoassay. RESULTS: PBMC MK mRNA level was significantly higher in patients with primary NSCLC than that in other groups (P < 0.001), while there was no significant difference between LEL patients and healthy volunteers (P > 0.05). Higher MK mRNA level was correlated with clinical stages (P = 0.026), differentiation (P = 0.025), and lymph node metastasis (P = 0.022) of NSCLC. Using a cutoff of 0.0063, the sensitivity and specificity of MK mRNA levels to differentiate between patients with NSCLC and patients with LEL were 57.47 and 93.33 %,and it were 56.32 and 93.33 % for patients with NSCLC and healthy volunteers, respectively. Furthermore, multivariate analysis indicated that PBMC MK mRNA level above the cutoff value presented a chance of 11-fold higher for NSCLC occurrence. CONCLUSIONS: MK mRNA level in PBMC may be a potential non-invasive molecular marker for the diagnosis of primary NSCLC.
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Adenocarcinoma/diagnóstico , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Fatores de Crescimento Neural/genética , Derrame Pleural Maligno/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Midkina , Fatores de Crescimento Neural/metabolismo , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/metabolismo , Prognóstico , Estudos Prospectivos , RNA Mensageiro/genética , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Midkine is a heparin-binding growth factor that promotes the proliferation, survival, migration and differentiation of various target cells. Midkine plays an important role in tumorigenesis and tumor progression, and is overexpressed in many human malignant tumors. Patients with high tumor midkine expression frequently have a worse prognosis than those with low expression. The present study was designed to investigate the interaction network of midkine in hepatic cancer cells, and to elucidate its role in hepatocellular carcinoma. METHODS: DNA encoding full-length midkine was cloned into pDBLeu vector to serve as bait in yeast two-hybrid screening to identify interacting proteins. Candidate proteins were examined on SC-Leu-Trp-His+3-AT (20 mmol/L) plates and assayed for X-gal activity, then sequenced and classified according to the GenBank. Finally, identified proteins were expressed by the in vitro expression system pCMVTnT, and protein interactions were confirmed by co-immunoprecipitation. RESULTS: Using the yeast two-hybrid system, we found 6 proteins that interacted with midkine: NK-kappa-B inhibitor alpha (I-κ-B-alpha), Dvl-binding protein naked cuticle 2, granulin, latent active TGF-beta binding protein 3, latent active TGF-beta binding protein 4, and phospholipid scramblase 1. In vitro co-immunoprecipitation demonstrated that all identified proteins directly interacted with midkine. CONCLUSION: The identification of midkine-interacting proteins in hepatic cancer cells indicates that midkine is a multifunctional factor that may participate in cell migration, differentiation, and proliferation, and is also associated with the multicellular response feedback during the development of hepatocellular carcinoma.
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Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Fatores de Crescimento Neural/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ligação ao Cálcio , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/metabolismo , Clonagem Molecular , Humanos , Proteínas I-kappa B/metabolismo , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Ligação a TGF-beta Latente/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Midkina , Inibidor de NF-kappaB alfa , Fatores de Crescimento Neural/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Progranulinas , Ligação ProteicaRESUMO
We prepared rabbit monoclonal antibodies that target human midkine (MK). The MK gene was amplified by PCR from the plasmid pEGFP-MK and subcloned into the prokaryotic expression vector pGEX-1λT to generate an N-terminally glutathione S-transferase (GST)-tagged fusion protein construct. Expression of the GST-MK fusion protein was achieved by IPTG induction in Escherichia coli cells. The expressed protein was purified using the GST system. After verifying purification, the fusion protein was used to immunize rabbits to prepare monoclonal antibodies against human MK by the rabbit hybridoma technique. The hybridomas generated were screened by an enzyme-link immunoassay (ELISA) for specificity, which was further characterized by Western blotting and ELISA. SDS-PAGE analysis showed that the purified protein corresponds to the calculated molecular weight. The GST-MK fusion protein was prepared. At least one hybridoma cell line secreting anti-MK MAb was obtained. Western blotting analysis confirmed the identity of the MAb. The titer of the MAbs measured by an indirect ELISA was 1:64,000. The affinity constant, which was measured by a non-competitive ELISA, was found to be 3.0 × 10(9) M(-1). Western blotting and immunohistochemistry analysis showed that the produced MAbs bind to the MK protein in cancerous tissues. The GST-MK fusion protein was successfully expressed and purified. The MAbs against MK were subsequently prepared, which should further aid research and the application of MK MAbs in clinical settings.
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Anticorpos Monoclonais/imunologia , Citocinas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Western Blotting , Linhagem Celular , Clonagem Molecular , Citocinas/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Glutationa Transferase/metabolismo , Humanos , Hibridomas/imunologia , Imuno-Histoquímica , Midkina , Plasmídeos/genética , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
AIM: To investigate the ultrastructural location of midkine (MK) in nucleolus and function corresponding to its location. METHODS: To investigate the ultrastructural location of MK in nucleolus with immunoelectronic microscopy. To study the role that MK plays in ribosomal biogenesis by real-time PCR. The effect of MK on anti-apoptotic activity of HepG2 cells was studied with FITC-conjugated annexin V and propidium iodide PI double staining through FACS assay. RESULTS: MK mainly localized in the granular component (GC), dense fibrillar component (DFC) and the border between the DFC and fibrillar center (FC). The production of 45S precursor rRNA level was decreased significantly in the presence of MK antisense oligonucleotide in the HepG2 cells. Furthermore, it was found that exogenous MK could protect HepG2 from apoptosis significantly. CONCLUSION: MK was constitutively translocated to the nucleolus of HepG2 cells, where it accumulated and mostly distributed at DFC, GC components and at the region between FC and DFC, MK played an important role in rRNA transcription, ribosome biogenesis, and cell proliferation in HepG2 cells. MK might serve as a molecular target for therapeutic intervention of human carcinomas.
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Carcinoma Hepatocelular/metabolismo , Nucléolo Celular/metabolismo , Neoplasias Hepáticas/metabolismo , Fatores de Crescimento Neural/metabolismo , RNA Ribossômico/metabolismo , Transcrição Gênica , Transporte Ativo do Núcleo Celular , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/ultraestrutura , Linhagem Celular Tumoral , Nucléolo Celular/ultraestrutura , Proliferação de Células , Separação Celular , Citometria de Fluxo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/ultraestrutura , Microscopia Imunoeletrônica , Midkina , Fatores de Crescimento Neural/genética , Oligonucleotídeos Antissenso/metabolismo , Reação em Cadeia da PolimeraseRESUMO
AIM: To evaluate the in vivo antitumor effects of antisense oligonucleotides targeting midkine (MK-AS). METHODS: An in situ human hepatocellular carcinoma (HCC) model was established in mice livers orthotopically. The MK-AS and 5- fluorouracil (5-Fu) were administered intravenously. The tumor sizes and plasma alpha-fetoprotein (AFP) were measured by calipers and radiation immunoassay respectively. The morphology of tumors was evaluated by hematoxylin-eosin staining of histological sections. Human MK, p53, Bax, Bcl-2, and caspase-3 protein content were detected by Western blotting. RESULTS: MK-AS significantly inhibited in situ human HCC growth in mice compared with the saline group in a dose-dependent manner. After the treatment with MK-AS or with 5-Fu, the plasma AFP concentration decreased in a dose-dependent manner. Interestingly, MK-AS also clearly downregulated the protein level of Bcl-2, and upregulated p53, Bax, and caspase-3 in the hepatocellular carcinoma tissue. CONCLUSION: These results demonstrated that MK-AS was an effective antitumor antisense oligonucleotide in vivo in mice; its antitumor effect is associated with the increase of pro-apoptotic proteins, such as p53, Bax, and caspase-3, and the decrease of the anti-apoptotic protein, Bcl-2.
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Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Citocinas/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Carcinoma Hepatocelular/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Midkina , Ensaios Antitumorais Modelo de Xenoenxerto , alfa-Fetoproteínas/biossíntese , alfa-Fetoproteínas/genéticaRESUMO
AIM: To construct an expression plasmid encoding human wild-type midkine (MK) and enhanced green fluorescence protein (EGFP) fusion protein (MK-EGFP), and to analyze the subcellular localization of MK in different carcinoma cell lines. METHODS: Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells, respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected. RESULTS: Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines. CONCLUSION: This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.
Assuntos
Carcinoma Hepatocelular , Nucléolo Celular/metabolismo , Citocinas/genética , Citocinas/metabolismo , Neoplasias Hepáticas , Proteínas Recombinantes de Fusão/genética , Neoplasias da Mama , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Midkina , Plasmídeos/genética , Neoplasias da PróstataRESUMO
AIM: To investigate the effect of antisense compounds (AS) targeting human p53 mRNA on radiosensitivity of MCF-7 cells. METHODS: Western blotting and RTPCR were used to analyze the protein content and mRNA level. Additionally, cell proliferation, cell cycle and cell apoptosis were all analyzed in irradiated or sham-irradiated cells. RESULTS: Among the five antisense compounds (AS), AS3 was identified to efficiently inhibit p53 mRNA level and protein content. Interestingly, AS3 transfer has little effect on cell proliferation in DU-145 cells (mutant p53) after ionizing radiation (IR). In contrast, a marked increase of cell apoptosis and growth inhibition were observed in MCF-7 cells (wild-type p53), suggesting that AS3 can increase radiosensitivity of MCF-7 cells. Additionally, it was also observed that the transfection of AS3 decreased the fraction of G1 phase cells, and increased the proportion of S phase cells compared to untreated cells 24 h after IR in MCF-7 cell lines. CONCLUSION: AS3 transfection increases MCF-7 cell apoptosis induced by 5 Gy-radiation, and this mechanism may be closely associated with abrogation of G1 phase arrest.