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BACKGROUND: The therapeutic value of targeted therapies in patients with lung cancer is reduced when tumours acquire secondary resistance after an initial period of successful treatment. However, the molecular events behind the resistance to targeted therapies in lung cancer remain largely unknown. AIMS: To discover the important role and mechanism of lncRNA BC in promoting tumor metastasis and influencing clinical prognosis of LUAD. MATERIALS & METHODS: Microarrays were used to screen a comprehensive set of lncRNAs with differential expression profiles in lung cancer cells. The functional role and mechanism of lncRNA were further investigated by gain- and loss-of-function assays. RNA pull-down, protein assays, and mass spectrometry were used to identify proteins that interacted with lncRNA. TaqMan PCR was used to measure lncRNA in lung adenocarcinoma and adjacent nontumor tissues from 428 patients. The clinical significance of lncRNA identified was statistically confirmed in this cohort of patients. RESULTS: In this study, we show that the long non-coding RNA BC009639 (BC) is involved in acquired resistance to EGFR-targeted therapies. Among the 235 long non-coding RNAs that were differentially expressed in lung cancer cell lines, with different metastatic potentials, BC promoted growth, invasion, metastasis, and resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKIs), both in vitro and in vivo. BC was highly expressed in 428 patients with lung adenocarcinoma (LUAD) and high BC expression correlated with reduced efficacy of EGFR-TKI therapy. To uncover the molecular mechanism of BC-mediated EGFR-TKI resistance in lung cancer, we screened and identified nucleolin and hnRNPK that interact with BC. BC formed the splicing complex with nucleolin and hnRNPK to facilitate the production of a non-protein-coding inositol monophosphatase domain containing 1 (IMPAD1) splice variant, instead of the protein-coding variant. The BC-mediated alternative splicing (AS) of IMPAD1 resulted in the induction of the epithelial-mesenchymal transition and resistance to EGFR-TKI in lung cancer. High BC expression correlated with clinical progress and poor survival among 402 patients with LUAD. DISSCUSSION: Through alternative splicing, BC boosted the non-coding IMPAD1-203 transcript variant while suppressing the IMPAD1-201 variant. In order to control the processing of pre-mRNA, BC not only attracted RNA binding proteins (NCL, IGF2BP1) or splicing factors (hnRNPK), but also controlled the formation of the splicing-regulator complex by creating RNA-RNA-duplexes. CONCLUSION: Our results reveal an important role for BC in mediating resistance to EGFR-targeted therapy in LUAD through IMPAD1 AS and in implication for the targeted therapy resistance.
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Adenocarcinoma , Processamento Alternativo , Neoplasias Pulmonares , RNA Longo não Codificante , Humanos , Adenocarcinoma/genética , Adenocarcinoma/patologia , Processamento Alternativo/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Pulmão/metabolismo , Neoplasias Pulmonares/patologia , RNA Longo não Codificante/metabolismoRESUMO
Moringa oleifera leaves are a kind of new food raw materials, rich in functional factors, M. oleifera leaves aqueous extract have antioxidant activity and M. oleifera leave protein is an important active ingredient in the aqueous extract. Numerous studies have shown that peptides have strong antioxidant activity. To reveal the antioxidant effects of M. oleifera (MO) leaves peptides, MO leave antioxidant peptides were isolated and prepared to clarify their antioxidant activity. MLPH1 (<1 kDa), MLPH3 (1~3 kDa), MLPH5 (3~5 kDa), and MLPH10 (5~10 kDa) fractions were obtained by the membrane ultrafiltration classification of MO leaves proteolytic hydrolysate (MLPH). MLPH1 was further separated by centrifugal filters, and the fraction separated by <1 kDa (MLPH1-1) was identified and analyzed by LC-MS/MS. The purpose of this study was to investigate the effect of MO leaves antioxidant peptide pretreatment on H2O2-treated HepG2 cells and to refine the antioxidant activity. The results showed that MLPH1 had the strongest antioxidant activity, and three MO leaves antioxidant peptides (LALPVYN, LHIAALVFQ, and FHEEDDAKLF) were obtained. The peptide with the sequence LALPVYN and a molecular weight of 788.44 Da had the strongest antioxidant activity. After 24 h of LALPVYN pretreatment, the cell viability and the CAT, GSH-Px, and SOD enzyme activity were significantly increased, and the MDA, ROS, and apoptosis rates were significantly decreased. These results provide a theoretical basis for further research on the antioxidant mechanism of MO leaves peptides.
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Urotensin II (UII) could increase blood pressure and heart rate via increased central reactive oxygen species (ROS) levels. We reported previously that hydrogen sulfide (H2S) exerts an antihypertensive effect by suppressing ROS production. The aim of the current study is to further examine the effects of endogenous and exogenous H2S on UII-induced cardiovascular effects by using an integrated physiology approach. We also use cell culture and molecular biological techniques to explore the inhibitory role of H2S on UII-induced cardiovascular effects. In this study, we found that cystathionine-ß-synthase (CBS), the main H2S synthesizing enzyme in CNS, was expressed in neuronal cells of the rostral ventrolateral medulla (RVLM) area. Cellular distribution of CBS and urotensin II receptor (UT) in SH-SY5Y cells that are confirmed as glutamatergic were identified by immunofluorescent and Western blots assay. In Sprague-Dawley rats, administration of UII into the RVLM resulted in an increase in mean arterial pressure (MAP), heart rate (HR), ROS production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and phosphorylation of p47phox, extracellular signal-regulated protein kinase (ERK)1/2 and p38MAPK, but not stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK). These effects of UII were attenuated by application into the RVLM of endogenous (L-cysteine, SAM) or exogenous (NaHS) H2S. These results were confirmed in SH-SY5Y cells. UII-induced cardiovascular effects were also significantly abolished by pretreatment with microinjection of Tempol, Apocynin, SB203580, or PD98059 into the RVLM. Preincubated SH-SY5Y cells with Apocynin before administration of UII followed by Western blots assay showed that ROS is in the upstream of p38MAPK/ERK1/2. Gao activation assay in SH-SY5Y cells suggested that H2S may exert an inhibitory role on UII-induced cardiovascular effects by inhibiting the activity of Gαo. These results suggest that both endogenous and exogenous H2S attenuate UII-induced cardiovascular effects via Gαo-ROS-p38MAPK/ERK1/2 pathway.
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AIMS: Attention-deficit/hyperactivity disorder (ADHD) is a prevalent disorder of neurodevelopment in children. The diagnosis of ADHD mainly relies on the symptoms and some may be misdiagnosed due to age-based variation in behaviours. This study aimed to explore biomarkers that are greatly needed for the accurate diagnosis of ADHD. METHODS: Seven hundred and forty-two samples were retrospectively investigated in three independent cohorts, screening, training, and validation, for circulation microRNA measurement using microarray, Taqman polymerase chain reaction, and regression analysis. RESULTS: A panel of five miRNAs (miR-4516, miR-6090, miR-4763-3p, miR-4281, and miR-4466) were identified as ADHD independent risk factors that provided a high diagnostic accuracy and specificity of ADHD (AUC = 0.940 and 0.927 in the training and validation datasets, respectively). This panel of miRNAs differentiated ADHD well from control groups. After clinical improvement by treatment, the panel of miRNAs in patients and AUC changed significantly and were close to those in healthy controls. Importantly, the targets of the miRNAs identified were commonly enriched in receptor signalling pathways, ion channels, and synapse structures. CONCLUSION: Our study identified a useful panel of miRNAs that have considerable clinical value in evaluating ADHD and provide important evidence for aberrant epigenetic regulation in ADHD.
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Transtorno do Deficit de Atenção com Hiperatividade , MicroRNAs , Transtorno do Deficit de Atenção com Hiperatividade/diagnóstico , Transtorno do Deficit de Atenção com Hiperatividade/genética , Biomarcadores , Biomarcadores Tumorais , Criança , Epigênese Genética , Perfilação da Expressão Gênica , Humanos , MicroRNAs/genética , Estudos RetrospectivosRESUMO
LncRNA (long noncoding RNA) is often dysregulated in tumors especially hepatocellular carcinoma (HCC). However, the dysregulation mechanism of lncRNAs is largely unknown. Here, we showed that lncRNA lncAY expression was stimulated in HCC by either endogenous or exogenous sulfatide. Elevated lncAY promoted HCC cell migration or angiogenesis, whereas lncAY silence suppressed HCC cell migration and proliferation. Interestingly, the activity of lncAY gene promoter was enhanced by sulfatide. Then Myb and MEF2C were identified as the transcription factors responsible for the stimulation of lncAY promoter activity and transcription by sulfatide. Both Myb and MEF2C enrichment on lncAY promoter was further confirmed, and their occupancy on lncAY promoter was strengthened by sulfatide for Myb or MEF2C was acetylated. Mutant Myb-K456A exhibited reduced acetylation and weak stimulation for lncAY transcription. However, Myb mutation K456/503A prevented Myb from acetylation induced by sulfatide. The mutant Myb K456/503A further was unable to occupy lncAY promoter and enhance lncAY transcription. In conclusion, this study demonstrated lncAY transcription was abnormally upregulated by sulfatide in HCC through Myb/MEF2C to promote HCC progression.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Fatores de Transcrição MEF2 , Proteínas Proto-Oncogênicas c-myb , RNA Longo não Codificante , Acetilação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Fatores de Transcrição MEF2/genética , Proteínas Proto-Oncogênicas c-myb/genética , RNA Longo não Codificante/genética , Sulfoglicoesfingolipídeos/metabolismoRESUMO
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver. Long non-coding RNAs as master gene regulators play important roles in tumorigenesis and progression. However, the significance of lncRNAs and their regulatory mechanisms in HCC are largely unknown. Our study was to define the role of lncAY (long noncoding RNA AY927503) in HCC. METHODS: Methylated RNA immunoprecipitation qPCR combined with bioinformatics were used to identify the m6A modification of lncAY. qRT-PCR, western blotting and immunofluorescence were used to identify the expression of the lncAY/YTHDF2/BMI1/Wnt axis in HCC tissues and cell lines. Gain- and loss-of functions of lncAY and BMI1 were implemented to confirm their roles in the behaviors of HCC cells. RESULTS: Our findings suggested that m6A-modified lncAY expression relied on m6A "reader" protein YTHDF2. LncAY upregulated BMI1 expression in HCC cells and a notably positive relevance is evident between lncAY and BMI1 expression in TCGA HCC datasets. BMI1 was upregulated in HCC tissues and patients with higher BMI1 expression had a poor clinical prognosis. Besides, GSEA analysis showed remarkable enrichment of high BMI1 expression in gene sets associated with Wnt/ß-catenin signaling. Rescue results revealed that BMI1 reversed the suppressive effects of lncAY depletion in HCC cells. CONCLUSIONS: Our work suggested that lncAY might elevate BMI1 expression and further activate the Wnt/ß-catenin signaling. BMI1 reverses the suppressive effects of lncAY depletion in HCC cells. Collectively, our work uncovers a novel undefined regulatory signaling pathway, namely lncAY/BMI1/Wnt/ß-catenin axis, involved in liver cancer progression.
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Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Complexo Repressor Polycomb 1/genética , RNA Longo não Codificante/genética , Via de Sinalização Wnt , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Complexo Repressor Polycomb 1/metabolismo , beta Catenina/metabolismoRESUMO
INTRODUCTION: Antimicrobial resistance (AMR) of Neisseria gonorrhoeae (N. gonorrhoeae) becomes a grave public health problem in the world. A strengthened Antimicrobial Resistance Surveillance Program is needed to track the trend of AMR development. However, the lack of a proper antimicrobial susceptibility test (AST) method is a barrier to expand the AMR surveillance in China. Traditional agar dilution (AD) method is laborious and E-test strips have no approval license for clinical use. Herein, a Chinese group modified the microdilution (MD) method for clinical ASTs. The objective of this study is to compare the MD method with the AD method for N. gonorrhoeae AST. MATERIALS AND METHODS: A total of 166 clinical isolates were tested for antimicrobial susceptibility of ceftriaxone, spectinomycin, azithromycin, ciprofloxacin, tetracycline, and penicillin using MD and AD method simultaneously. Results of MD method were read manually or automatically. Rates of essential agreement (EA), category agreement (CA), minor error, and very major error were compared. RESULTS: The total EAs (compared with results read manually) of penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone, and azithromycin were 90.4%, 97.0%, 85.5%, 100.0%, 94%, and 72.3%; and CAs were 82.5%, 94.0%, 100%, 100%, 95.2%, and 94%, respectively. CONCLUSION: We conclude that the MD method might be an alternative for clinical AST of N. gonorrhoeae in China. In particular, MD method has the potency of accurate differentiation of isolates resistant to ceftriaxone or azithromycin, which were empirically recommended for gonococcal treatment, but its quality remained suboptimal, and further improvement is needed for clinical use.
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Rationale: Tumor metastasis is the main cause for cancer-related death. However, the driving molecules of metastasis remain largely unknown. Here, we aim to identify long non-coding RNAs (lncRNAs) critical for human hepatocellular carcinoma (HCC) metastasis. Methods: Microarrays were used to screen a comprehensive set of lncRNAs with differential expression profiles in sulfatide-treated cells. Mass spectrometry, protein arrays, and RNA pull-down experiments were used to identify proteins that interacted with lncRNA. Epigenetic analysis was used to study lncRNA-mediated regulation mechanisms. Results: We identified lncRNA AY927503 (AY) as a metastasis-associated molecule that was highly expressed in human hepatocellular carcinoma (HCC) and correlated with metastatic events and poor prognosis in patients with HCC. AY promoted HCC cell migration, stemness, 5-fluorouracil resistance, and metastasis in mice. However, knockdown of integrin αV (ITGAV) abolished AY-stimulated migration, cell viability in HCC cells or tube formation. AY strongly promoted ITGAV transcription and αVß3 expression by interacting with the ITGAV promoter specifically and stimulating its activity. AY was identified to interact with histone 1FX (H1FX), but deletion of the central domain of AY (AY∆371-522) abolished H1FX binding and ITGAV promoter stimulation. AY significantly enriched H3K4Me3 and acH3K9/14 but reduced H3K27Me3 and H1FX occupancy on the ITGAV promoter, which remodeled chromatin structures for RNA polymerase II recruitment. Knockdown of H1FX abrogated ITGAV transcription stimulated by AY. Conclusions: Our findings suggested that lncRNA AY promoted HCC metastasis via induction of chromatin modification for ITGAV transcription as a pioneer factor and was a potential molecular signature for metastasis or poor prognosis in patients with HCC.
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Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Integrina alfaV/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , RNA Longo não Codificante/metabolismo , Transcrição Gênica , Animais , Carcinoma Hepatocelular/irrigação sanguínea , Linhagem Celular Tumoral , Proliferação de Células , Montagem e Desmontagem da Cromatina/genética , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/irrigação sanguínea , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Neovascularização Patológica/genética , RNA Longo não Codificante/genética , Regulação para Cima/genéticaRESUMO
OBJECTIVES: The aim of the study is to identify ceftriaxone resistance-related genes in Neisseria gonorrhoeae. METHODS: Differences in gene expression were compared between ceftriaxone-susceptible N. gonorrhoeae isolates [minimum inhibitory concentration (MIC)=0.002-0.004mg/L] and isolates with decreased ceftriaxone susceptibility (MIC=0.125-0.5mg/L) using RNA-Seq (RNA sequencing). RESULTS: Total RNA of 10 clinical isolates was used to make libraries and generated an average of 24.07Mb reads per sample; these were assembled into 1871 mRNA genes. Moreover, 21 differentially expressed genes (DEGs) were found between the N. gonorrhoeae isolates with susceptibility and decreased susceptibility to ceftriaxone with a fold change of ≥2 (P<0.05), among which 11 were upregulated and 10 were downregulated. Furthermore, all DEGs were verified by quantitative reverse transcription PCR (qRT-PCR), which detected 25 clinical isolates with decreased ceftriaxone susceptibility and 21 ceftriaxone-susceptible isolates. In addition, seven DEGs revealed relative expression levels by 2-ΔΔCt and showed a statistical significance (P≤0.05). Analysis of Gene Ontology (GO) terms and KEGG pathway for functional enrichment showed that six DEGs were related to the cellular component and one DEG was related to the biosynthesis of antibiotics, and these results might be related to ceftriaxone resistance. CONCLUSIONS: Examining ceftriaxone resistance-related genes in N. gonorrhoeae is necessary owing to the high morbidity and antimicrobial resistance of N. gonorrhoeae, especially its eventual resistance to third-generation extended-spectrum cephalosporins (cefixime and ceftriaxone). Moreover, this report provides a new direction for the study and control of ceftriaxone-resistant N. gonorrhoeae.
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Antibacterianos/farmacologia , Ceftriaxona/farmacologia , Farmacorresistência Bacteriana/genética , Neisseria gonorrhoeae/genética , Expressão Gênica , Gonorreia/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/efeitos dos fármacos , Reação em Cadeia da Polimerase , RNA-SeqRESUMO
Paired amphipathic helix protein (SIN3B) is a transcription corepressor for many genes. Here we show a different regulation mechanism of integrin αV gene expression by SIN3B in human hepatocellular carcinoma (HCC). We first observed a close relationship between Integrin αV and SIN3B expressions in HCC patients and tumor cell lines with different metastatic potentials. Overexpression of SIN3B significantly accelerated the cell migration rate of SMMC-7721, but failed when integrin αV expression was silenced. Interestingly, SIN3B stimulated integrin αV subunit promoter activity only in the presence of sulfatide. Importantly, SIN3B was identified in the complex with sulfatide by mass spectrometry. Fat blot assay indicated that SIN3B specifically interacted with sulfatide. Molecular modeling suggested that sulfatide induced the conformational change of SIN3B from compacted α-helices to a relaxed ß-sheet in PAH2 domain. The data of immunoprecipitation and ChIP assay indicated that altered SIN3B lost the binding affinity with MAD1 and HDAC2, which reduced the recruitment of HDAC2 on integrin αV gene promoter and prevented the deacetylation of the histone 3. In conclusion, this study demonstrated that SIN3B promoted the transcriptional activation of the integrin αV subunit gene promoter by reducing interaction with HDAC2.
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Carcinoma Hepatocelular/patologia , Integrina alfaV/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Repressoras/metabolismo , Acetilação , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Histona Desacetilase 2/química , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Humanos , Integrina alfaV/genética , Neoplasias Hepáticas/metabolismo , Simulação de Dinâmica Molecular , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Sulfoglicoesfingolipídeos/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Integrin αV gene expression is often dysregulated in cancers especially in hepatocellular carcinoma (HCC); however, the mechanism of regulation is poorly understood. Here, it is demonstrated that sulfatide activated integrin αV gene transcription, through histone H3K9/14 acetylation at the promoter, and high integrin αV expression are closely associated with poor prognosis. To elucidate the mechanism of regulation of acetylation, sulfatide-bound proteins were screened by mass spectrometry (MS), and bromodomain containing protein 1 (BRD1) was identified as an interacting protein that also colocalized with sulfatide in HCC cells. BRD1 was also formed a complex with Sp1, which was recruited to the integrin αV gene promoter. Sulfatide was also found to induce BRD1, monocytic leukemia zinc finger (MOZ) and histone acetyltransferase binding to ORC1 (HBO1) acetyltransferase multiprotein complex recruitment to the integrin αV promoter, which is responsible for histone H3K9/14 acetylation. Finally, knockdown of BRD1 limited sulfatide-induced H3K9/14 acetylation and occupancy of MOZ or HBO1 on integrin αV gene promoter.Implications: This study demonstrates that sulfatide interaction with BRD1 mediates acetylation and is important for regulation of integrin αV gene expression. Mol Cancer Res; 16(4); 610-22. ©2018 AACR.
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Carcinoma Hepatocelular/metabolismo , Integrina alfaV/genética , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Regulação para Cima , Acetilação , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Histona Acetiltransferases , Chaperonas de Histonas , Humanos , Integrina alfaV/metabolismo , Neoplasias Hepáticas/genética , Masculino , Modelos Moleculares , Metástase Neoplásica , Proteínas Nucleares/química , Prognóstico , Análise de Sobrevida , Análise Serial de TecidosRESUMO
BACKGROUND AND OBJECTIVE: Gonorrhea is a sexually transmitted disease caused by the bacterium Neisseria gonorrhoeae. Rapid detection is crucial for effective prevention and treatment. This study developed and tested a low-cost effective method for detecting N. gonorrhoeae, especially in developing countries. METHODS: DNA from a N. gonorrhoeae standard strain, as well as from 26 genital secretion samples of gonorrhea patients, were isolated and used for loop-mediated isothermal amplification (LAMP) assay, which was conducted using either an automatic real-time PCR analyzer or a water bath. The amplified porA pseudogene sequence was compared with the NCBI database and the LAMP results were compared with that of the traditional culture method for its sensitivity and specificity. RESULTS: LAMP was able to detect Neisseria DNA at a concentration as low as 1 pg/µL (1 × 103 CFU/mL cells). The LAMP assay results obtained using an automatic real-time PCR analyzer was similar to that of the water bath. Relative to traditional culture, the sensitivity and specificity of the LAMP assay were 94.7 and 85.7%, respectively. CONCLUSION: LAMP was sensitive and reliable for detecting the porA gene of N. gonorrhoeae. It could be used as a rapid, low cost, and effective method for detecting N. gonorrhoeae.
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Tumor metastasis is the major cause of cancer-related death especially in human hepatocellular carcinoma (HCC). Although microRNAs have been implicated in tumor development, the roles of miR-124 in HCC metastasis are still not well understood. We conducted functional analysis in this study to investigate miR-124. We observed that miR-124 significantly retarded the wound healing and migration of HCC SMMC-7721 and BEL-7404 cells. Further analysis indicated miR-124 directly targeting the transcriptional factor Sp1 which is an important transcription factor for the integrin αV subunit gene transcription. Co-transfection of miR-124 with the luciferase reporter containing Sp1 3' untranslated region (UTR) significantly suppressed the luciferase activities. While mutation of the binding site of miR-124 in Sp1 mRNA 3'UTR completely abrogated the suppression of miR-124. Overexpression of miR-124 resulted in robust downregulation of Sp1 and integrin αV expression at either mRNA or protein level. Ectopic expression of miR-124 in HCC dramatically repressed the wound healing and migration in vitro and tumor metastasis in mouse experiments. Our findings demonstrated that miR-124 played as an important role in regulation of integrin αV expression in HCC, and reintroduction of miR-124 might be an alternative therapeutic strategy for controlling integrin αV expression in HCC.
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Carcinoma Hepatocelular/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Integrina alfaV/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , Interferência de RNA , Animais , Apoptose/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Fator de Transcrição Sp1/genéticaRESUMO
Sialic acid modification is a kind of post-translational modification. To investigate the regulation effect of sialic acid on neural differentiation, we used CycloManN propanyl perac (CycloManN pro), a metabolic precursor of sialic acid, to treat PC12 cells. We noted that CycloManN pro indeed robustly promoted global sialylation detected by MAL II lectin blot in PC12 cells. Simultaneously, we interestingly found that the neurite outgrowth of PC12 cells was significantly promoted by the CycloManN pro treatment. The profile analysis of sialylated proteins showed that a protein band at 55KD was greatly enhanced especially in PC12L cells after CycloManN pro treatment. After enrichment with lectin MAL II, the proteins in this band were analyzed by mass spectrometry. The results showed that 23 proteins were in the band, but the score of vimentin was the highest among them. To investigate further the role of vimentin in the process of neurite differentiation, vimentin construct was transfected into PC12 cells. We interestingly observed that ectopic expression of vimentin significantly enhanced the neurite outgrowth induced by CycloManN pro. However, after three potential glycosylation sites (Ser-7, Thr-33, Ser-34:) of vimentin were mutated to alanine, overexpression of the mutated vimentin completely lost the enhancement activity for the neural differentiation even in the presence of CycloManN pro. Taken together, our study demonstrated that vimentin was important in the induction of neural differentiation by CycloManN pro.
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Neuritos/metabolismo , Crescimento Neuronal , Processamento de Proteína Pós-Traducional , Ácidos Siálicos/metabolismo , Vimentina/metabolismo , Animais , Lectinas/metabolismo , Mutação , Células PC12 , Ratos , Vimentina/genéticaRESUMO
Attention deficit hyperactivity disorder (ADHD) is a child developmental and behavioral disorder which seriously hinders their education and development. To investigate the key regulators in the prefrontal cortex (PFC), the major affected areas of ADHD, microRNA (miR)-138,138*, 34c*, 296, and 494, were noted for their significant downregulation in ADHD model rats spontaneously hypertensive rats (SHRs) compared to Wistar Kyoto (WKY) rat control. Based on promoter sequence analysis and activity assay, glucocorticoid receptor (Nr3c1) was identified for the inhibition of the promoter activity of miR-138-1, 34c*, 296, and 494 genes and their transcription. In the PFC of ADHD model rats SHR, Nr3c1 expression was abnormally elevated and reversely correlated with the levels of miR-138-1, 34c, 296, and 494 expression. Luciferase report assays indicated that all miR-138, 138*, 34c*, 296, and 494 targeted the 3' untranslated region of transcription factor Bhlhb2 (Bhlhe40) messenger RNA (mRNA) in common and ectopic expression of miR-138,138*, 34c*, 296, and 494 further suppressed the expression of Bhlhb2 gene. Consistently, Bhlhb2 expression was significantly higher in PFC of ADHD model SHR than control. Overexpressed Bhlhb2 in vitro significantly suppressed PC12 cell differentiation, and silence of Bhlhb2 enhanced the growth of neurite axon and dendrite. To observe the roles of Bhlhb2 further in vivo, Bhlhb2 was silenced in the PFC of nine SHR rats. Interestingly, knockdown of Bhlhb2 significantly improved the hyperactivity behaviors in SHRs compared to control. These findings show that Nr3c1-Bhlhb2 axis dysregulation was involved in the development of attention deficit and hyperactivity.
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Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Homeodomínio/genética , Receptores de Glucocorticoides/genética , Animais , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Redes Reguladoras de Genes/fisiologia , Células HEK293 , Proteínas de Homeodomínio/biossíntese , Humanos , Masculino , Células PC12 , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiopatologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores de Glucocorticoides/biossínteseRESUMO
Objective To observe and analyze the clinical outcomes of perfluoropropane (C3 Fs) injection and laser photocoagulation on myopic foveoschisis.Methods A total of 14 patients (18 eyes) diagnosed as myopic foveoschisis were enrolled in this retrospective study.All patients received intraocular tamponade of 0.5-0.7 mL C3 F8,and after 1 week,underwent macular photocoagulation.These patients were given the best-corrected visual acuity (BCVA) and optical coherence tomography (OCT) examination for central foveal thickness (CFT) and maximal macular thickness (MMT) before and after treatment.Results OCT examination showed that the mean CFT decreased significantly from (494.00 ±454.80) iμm before treatment to (193.61 ± 97.42) μm at the last follow-up,with statistical significance (P =0.01),and the mean MMT decreased from (687.33 ± 385.15)pμn to (331.06 ± 109.31)μm at the same duration,approaching significant difference (P =0.001).The foveoschisis healed completely and partially in 14 eyes at the last follow-up,the mean CFT decreased significantly from (567.36 ±493.01) μm before treatment to (171.43 ± 90.84) μm after treatment,with statistical significance (P =0.006),and the mean MMT decreased from (744.14 ± 417.38)μm to (303.86 ± 8.62)prn at the same duration,approaching significant difference (P =0.002).Patients' BCVA before treatment was (0.94 ± 0.39) logMAR,of which 13 eyes had BCVA < 0.6 logMAR,and increased to (0.92 ± 0.36) logMAR at the last follow-up,with no significant difference (P =0.78).The foveoschisis healed completely and partially in 14 eyes,and the BCVA was (1.04 ± 0.37) logMAR before treatment,up to (0.90 ± 0.34) logMAR after treatment,and the difference was not statistically significant (P =0.16).At the last follow-up,the vision of 4 eyes was increased by 2 lines and above,and unchanged in 10 eyes.All patients had no visual symptoms such as dark spots and no increase in intraocular pressure after treatment.Conclusion Intraocular C3 F8 tamponade and macular photocoagulation can be an satisfying alternative treatment for patients with myopic foveoschisis.
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Integrin αVß3 is a malignant driver of anchorage-independence and tumor angiogenesis, but its dysregulation in hepatocellular carcinoma (HCC) remains unclear. In this study, we observed that sulfatide significantly promoted integrin αV(ITGAV) expression and wound closure in HCC. We also noted that elevated sulfatide profoundly stimulated integrin αVß3 clustering and signaling. In the cells with integrin αVß3 clustering induced by sulfatide, integrin ß3 subunit was phosphorylated. Simultaneously, focal adhesion kinase (FAK), Src and paxillin were also phosphorylated. Treatment with FAK inhibitor resulted in robust suppression of FAK-Y397 and Src-Y416 phosphorylation stimulated by sulfatide, but not suppression of integrin ß3 phosphorylation. Src inhibitors repressed Src-Y416 and FAK Y861 and Y925 phosphorylation, but not FAK-Y397 and integrin ß3 phosphorylation. After mutation of integrin ß3 (Y773F and Y785F), FAK or Src phosphorylation failed to be stimulated by sulfatide. Moreover, ß3 Y773 and Y785 phosphorylation was suppressed by insulin-like growth factor receptor knockdown even in cells stimulated by sulfatide. In assays of immunoprecipitation and immunostaining with integrin αV or ß3 antibody, labeled sulfatide was found in the complex and co-localized with integrin αVß3. Taken together, this study demonstrated that elevated sulfatide bound to integrin αVß3 and induced clustering and phosphorylation of αVß3 instead of matrix ligand binding, triggering outside-in signaling.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Integrina alfaVbeta3/genética , Transdução de Sinais/efeitos dos fármacos , Sulfoglicoesfingolipídeos/farmacologia , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina alfaVbeta3/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Paxilina/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Sulfoglicoesfingolipídeos/metabolismo , Tirosina/genética , Tirosina/metabolismo , Quinases da Família src/metabolismoRESUMO
E-cadherin is often dysregulated in aggressive lung cancer, the mechanism of which cannot always be explained at the level of transcription. In 66 patients with lung cancer, immunohistochemical staining demonstrated that co-localization of E-cadherin and core fucose by Lens culinaris agglutinin was significantly less extensive in tumor than in nontumor tissue. Through gain and loss of fucosylation experiments in the giant lung carcinoma cell lines 95C and 95D, our results revealed that E-cadherin core fucosylation in 95C cells overexpressing α-1, 6-fucosyltransferase (Fut8) inhibited Fut8-95C cell migration, whereas knockdown of Fut8 in 95D cells enhanced migration of short-interfering RNA-targeting Fut8 (siFut8)-95D cells. The level of active Src (phosphorylated Src [Y416]) was significantly reduced in Fut8-95C cells, but elevated in siFut8-95D cells. In protein complexes immunoprecipitated from Fut8-95C cell lysates with anti-E-cadherin, less phosphorylated Src (Y416) and more ß-catenin were observed, but immunoprecipitates from siFut8-95D cells, containing less core fucosylated E-cadherin, contained an elevated level of phospho-Src Y416. In Fut8-95C cells, phosphorylation of Akt (Y315, Y326) and GSK-3ß (S9) was significantly reduced, but ß-catenin (S37) phosphorylation was enhanced. Expression of N-cadherin and Snail1 was also reduced in Fut8-95C cells, but significantly increased in siFut8-95D cells. Intriguingly, when Src kinase activity was inhibited by treatment of cells with PP2 and SU6656, regulation of N-cadherin, Snail1 and cell migration by E-cadherin core fucosylation was abrogated in both Fut8-95C and siFut8-95D cells. Therefore, posttranslational modification of E-cadherin by less core fucosylation recruited and activated Src, and induced an epithelial-mesenchymal transition-like process in lung cancer cells.
Assuntos
Caderinas/metabolismo , Transição Epitelial-Mesenquimal , Fucose/metabolismo , Neoplasias Pulmonares/metabolismo , Processamento de Proteína Pós-Traducional , Quinases da Família src/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Glicosilação , Humanos , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismoRESUMO
Up to date, there has been no molecular signature available in the clinical practice for attention-deficit/hyperactivity disorder (ADHD). To investigate circulating miRNA let-7d significance in ADHD, we investigated serum miRNA let-7d in 35 newly diagnosed ADHD subjects who were randomly selected from 406 patients out of 7450 children, paired with gender- and age-matched control through case-control study. We observed that circulating miRNA let-7d was significantly higher in ADHD subjects than in control (p < 0.05). Higher circulation level of miRNA let-7d was significantly associated with ADHD (odds ratio 16.7; 95% confidence, p < 0.05). Meanwhile, serum galectin-3 level was down-regulated in ADHD subjects and the subjects with low galectin-3 expression accounted for 66% in ADHD. The difference of the serum galectin-3 levels between ADHD and non-ADHD groups reached significance (p < 0.05). In 1-year follow-up, a significantly higher rate of clinical improvement was noted in subjects with low level of circulating miRNA let-7d (p < 0.05) than those with high level of circulating miRNA let-7d. Our data demonstrated that miRNA let-7d was elevated in the serum of ADHD subjects, which might be a novel, useful molecule signature for ADHD.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/sangue , Galectina 3/sangue , MicroRNAs/sangue , Adolescente , Animais , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Modelos Animais de Doenças , Feminino , Galectina 3/genética , Galectina 3/metabolismo , Humanos , Masculino , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estudos de Amostragem , Resultado do TratamentoRESUMO
Galactose-3'-O- sulfation is specific and exists in many important molecules from various human tissues, and the sulfation modification results in alteration of host molecule recognition and interaction with partner molecules which lead to signaling. The modification is thus associated with the regulation of cellular adhesion and interaction, and involved in cell recognition and even in tumor metastasis process since the binding affinity to their extracellular ligands has changed. Sulfated glycoproteins or glycolipids may also trigger signaling in the cells, which is important in regulating cell-cell and cell-extracellular matrix interaction, and in adhesion molecule transcription activation.