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Citrate, which is obtained from oxaloacetate and acetyl-CoA by citrate synthase in mitochondria, plays a key role in both normal and cancer cell metabolism. In this work, we investigated the effect of 10 mM extracellular citrate supplementation on HepG2 cells. Gene expression reprogramming was evaluated by whole transcriptome analysis using gene set enrichment analysis (GSEA). The transcriptomic data were validated through analyzing changes in the mRNA levels of selected genes by qRT-PCR. Citrate-treated cells exhibited the statistically significant dysregulation of 3551 genes; 851 genes were upregulated and 822 genes were downregulated. GSEA identified 40 pathways affected by differentially expressed mRNAs. The most affected biological processes were related to lipid and RNA metabolism. Several genes of the cytochrome P450 family were upregulated in treated cells compared to controls, including the CYP3A5 gene, a tumor suppressor in hepatocellular carcinoma (HCC) that plays an important protective role in HCC metastasis. The citrate-induced dysregulation of cytochromes could both improve the effectiveness of chemotherapeutics used in combination and reduce the aggressiveness of tumors by diminishing cell migration and invasion.
Assuntos
Movimento Celular , Ácido Cítrico , Regulação Neoplásica da Expressão Gênica , Humanos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Hep G2 , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ácido Cítrico/farmacologia , Ácido Cítrico/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/tratamento farmacológico , Invasividade Neoplásica , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Transcriptoma , Perfilação da Expressão GênicaRESUMO
BACKGROUND/AIM: Bladder cancer (BC) is the most prevalent malignant tumor in the urinary tract, classified mainly into muscle-invasive BC (MIBC) and non-MIBC (NMIBC). Recent studies highlight the important role of changes in transcriptome activity in carcinogenesis, aiding in the identification of additional differentially regulated candidate genes, improving our understanding of the molecular basis of gene regulation in BC. This study aimed to evaluate the transcriptome of MIBC patients compared with normal subjects. MATERIALS AND METHODS: mRNA sequencing was conducted using the Illumina NovaSeq 6000 Dx system in a case series comprising 11 subjects with MIBC and 19 healthy controls matched for age and sex. For functional analysis, the pathfindR package was utilized to comprehensively identify pathways enriched in omics data within active subnetworks. RESULTS: Our results demonstrated the presence of differentiated pathways, including spliceosome activity, oxidative phosphorylation, and chemical carcinogenesis due to reactive oxygen species, in MIBC patients compared with controls. CONCLUSION: The identification of novel molecular pathways in MIBC patients could prove useful in defining cancer predisposition factors and exploring potential therapeutic options.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Transcriptoma , Neoplasias da Bexiga Urinária , Humanos , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Invasividade Neoplásica/genética , Estudos de Casos e Controles , Biomarcadores Tumorais/genética , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Biologia Computacional/métodosRESUMO
Lung cancer is the leading cause of cancer-related death worldwide. Non-coding RNAs are emerging as critical players for the onset and progression of cancer. Analyses of three different datasets revealed that the lncRNA JPX was overexpressed in adenocarcinoma tissues in comparison to normal lungs, as expected for an oncogene. Intriguingly, the predicted binding miR-378a-3p showed a significant inverse correlation with JPX expression. The lncRNA/miRNA physical interaction was validated by reporter vectors. Then, the oncogenic activity of JPX, the tumor-suppressive role of miR-378a-3p, and the contribution of their functional interaction to cancer hallmarks were demonstrated using assays for cell proliferation, migration, invasion, and 3D-spheroid formation. Finally, molecular circuits were investigated by boosting the expression of both JPX and miR-378a-3p, singularly and in combination, demonstrating that JPX counteracted miR-378a-3p silencing activity toward its oncogenic targets GLUT1, NRP1, YY1, and Wnt5a. Overall, the data unveil a novel ceRNET (competing endogenous RNA network), wherein JPX acts as a ceRNA by binding to miR-378a-3p, thus reducing the miRNA silencing activity toward its downstream targets, and eliciting oncogenic pathways driving lung cancer. The knowledge of the network may pave the way to develop new diagnostic panels, and innovative RNA-targeted and RNA-based therapeutic strategies.
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The aim of this study was to analyze signaling pathways associated with differentially expressed messenger RNAs in people with restless legs syndrome (RLS). Seventeen RLS patients and 18 controls were enrolled. Coding RNA expression profiling of 12,857 gene transcripts by next-generation sequencing was performed. Enrichment analysis by pathfindR tool was carried-out, with p-adjusted ≤0.001 and fold-change ≥2.5. Nine main different network groups were significantly dysregulated in RLS: infections, inflammation, immunology, neurodegeneration, cancer, neurotransmission and biological, blood and metabolic mechanisms. Genetic predisposition plays a key role in RLS and evidence indicates its inflammatory nature; the high involvement of mainly neurotropic viruses and the TORCH complex might trigger inflammatory/immune reactions in genetically predisposed subjects and activate a series of biological pathways-especially IL-17, receptor potential channels, nuclear factor kappa-light-chain-enhancer of activated B cells, NOD-like receptor, mitogen-activated protein kinase, p53, mitophagy, and ferroptosis-involved in neurotransmitter mechanisms, synaptic plasticity, axon guidance, neurodegeneration, carcinogenesis, and metabolism.
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Parkinson's disease (PD) stands as the most prevalent degenerative movement disorder, marked by the degeneration of dopaminergic neurons in the substantia nigra of the midbrain. In this study, we conducted a transcriptome analysis utilizing post mortem mRNA extracted from the substantia nigra of both PD patients and healthy control (CTRL) individuals. Specifically, we acquired eight samples from individuals with PD and six samples from CTRL individuals, with no discernible pathology detected in the latter group. RNA sequencing was conducted using the TapeStation 4200 system from Agilent Technologies. A total of 16,148 transcripts were identified, with 92 mRNAs displaying differential expression between the PD and control groups. Specifically, 33 mRNAs were significantly up-regulated, while 59 mRNAs were down-regulated in PD compared to the controls. The identification of statistically significant signaling pathways, with an adjusted p-value threshold of 0.05, unveiled noteworthy insights. Specifically, the enriched categories included cardiac muscle contraction (involving genes such as ATPase Na+/K+ transporting subunit beta 2 (ATP1B2), solute carrier family 8 member A1 (SLC8A1), and cytochrome c oxidase subunit II (COX2)), GABAergic synapse (involving GABA type A receptor-associated protein-like 1 (GABARAPL1), G protein subunit beta 5 (GNB5), and solute carrier family 38 member 2 (SLC38A2), autophagy (involving GABARAPL1 and tumor protein p53-inducible nuclear protein 2 (TP53INP2)), and Fc gamma receptor (FcγR) mediated phagocytosis (involving amphiphysin (AMPH)). These findings uncover new pathophysiological dimensions underlying PD, implicating genes associated with heart muscle contraction. This knowledge enhances diagnostic accuracy and contributes to the advancement of targeted therapies.
Assuntos
Doença de Parkinson , Humanos , Doença de Parkinson/genética , Análise em Microsséries , Perfilação da Expressão Gênica , Mesencéfalo , Substância Negra , Proteínas NuclearesRESUMO
There is growing evidence that various ATP-binding cassette (ABC) transporters contribute to the growth and development of tumors, but relatively little is known about how the ABC transporter family behaves in hepatocellular carcinoma (HCC), one of the most common cancers worldwide. Cellular model studies have shown that ABCC6, which belongs to the ABC subfamily C (ABCC), plays a role in the cytoskeleton rearrangement and migration of HepG2 hepatocarcinoma cells, thus highlighting its role in cancer biology. Deep knowledge on the molecular mechanisms underlying the observed results could provide therapeutic insights into the tumors in which ABCC6 is modulated. In this study, differential expression levels of mRNA transcripts between ABCC6-silenced HepG2 and control groups were measured, and subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Real-Time PCR and Western blot analyses confirmed bioinformatics; functional studies support the molecular mechanisms underlying the observed effects. The results provide valuable information on the dysregulation of fundamental cellular processes, such as the focal adhesion pathway, which allowed us to obtain detailed information on the active role that the down-regulation of ABCC6 could play in the biology of liver tumors, as it is involved not only in cell migration but also in cell adhesion and invasion.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Células Hep G2 , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de AdenosinaRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disorder. The number of cases of PD is expected to double by 2030, representing a heavy burden on the healthcare system. Clinical symptoms include the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain, which leads to striatal dopamine deficiency and, subsequently, causes motor dysfunction. Certainly, the study of the transcriptome of the various RNAs plays a crucial role in the study of this neurodegenerative disease. In fact, the aim of this study was to evaluate the transcriptome in a cohort of subjects with PD compared with a control cohort. In particular we focused on mRNAs and long non-coding RNAs (lncRNA), using the Illumina NextSeq 550 DX System. Differential expression analysis revealed 716 transcripts with padj ≤ 0.05; among these, 630 were mRNA (coding protein), lncRNA, and MT_tRNA. Ingenuity pathway analysis (IPA, Qiagen) was used to perform the functional and pathway analysis. The highest statistically significant pathways were: IL-15 signaling, B cell receptor signaling, systemic lupus erythematosus in B cell signaling pathway, communication between innate and adaptive immune cells, and melatonin degradation II. Our findings further reinforce the important roles of mitochondria and lncRNA in PD and, in parallel, further support the concept of inverse comorbidity between PD and some cancers.
Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Doença de Parkinson/genética , RNA Longo não Codificante/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNARESUMO
Parkinson's disease (PD) is a neurodegenerative synucleinopathy that has a not yet fully understood molecular pathomechanism behind it. The role of risk genes regulated by small non-coding RNAs, or microRNAs (miRNAs), has also been highlighted in PD, where they may influence disease progression and comorbidities. In this case-control study, we analyzed miRNAs on peripheral blood mononuclear cells by means of RNA-seq in 30 participants, with the aim of identifying miRNAs differentially expressed in PD compared to age-matched healthy controls. Additionally, we investigated the pathways influenced by differentially expressed miRNAs and assessed whether a specific pathway could potentially be associated with PD susceptibility (enrichment analyses performed using the Ingenuity Pathway Analysis tools). Overall, considering that the upregulation of miRNAs might be related with the downregulation of their messenger RNA targets, and vice versa, we found several putative targets of dysregulated miRNAs (i.e., upregulated: hsa-miR-1275, hsa-miR-23a-5p, hsa-miR-432-5p, hsa-miR-4433b-3p, and hsa-miR-4443; downregulated: hsa-miR-142-5p, hsa-miR-143-3p, hsa-miR-374a-3p, hsa-miR-542-3p, and hsa-miR-99a-5p). An inverse connection between cancer and neurodegeneration, called "inverse comorbidity", has also been noted, showing that some genes or miRNAs may be expressed oppositely in neurodegenerative disorders and in some cancers. Therefore, it may be reasonable to consider these miRNAs as potential diagnostic markers and outcome measures.
Assuntos
MicroRNAs , Doença de Parkinson , Humanos , Doença de Parkinson/genética , Estudos de Casos e Controles , Leucócitos Mononucleares/metabolismo , MicroRNAs/metabolismo , Regulação para Baixo/genéticaRESUMO
BACKGROUND: Prostate cancer (PCa) is one of the leading causes of death in Western countries. Environmental and genetic factors play a pivotal role in PCa etiology. Timely identification of the genetic causes is useful for an early diagnosis. Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder; it is associated with the presence of Lewy bodies and genetic factors are involved in its pathogenesis. Several studies have indicated that the expression of target genes in patients with PD is inversely related to cancer development; this phenomenon has been named "inverse comorbidity". The present study was undertaken to evaluate whether a genetic dysregulation occurs in opposite directions in patients with PD or PCa. METHODS AND RESULTS: In the present study, next-generation sequencing transcriptome analysis was used to assess whether a genetic dysregulation in opposite directions occurs in patients with PD or PCa. The genes SLC30A1, ADO, SRGAP2C, and TBC1D12 resulted up-regulated in patients with PD compared to healthy donors as controls and down-regulated in patients with PCa compared with the same control group. CONCLUSIONS: These results support the hypothesis of the presence of inverse comorbidity between PD and PCa.
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Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias , Doença de Parkinson , Neoplasias da Próstata , RNA-Seq , Idoso , Idoso de 80 Anos ou mais , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismoRESUMO
Down syndrome (DS) is defined by the presence of a third copy of chromosome 21. Several comorbidities can be found in these patients, such as intellectual disability (ID), muscle weakness, hypotonia, congenital heart disease, and autoimmune diseases. The molecular mechanisms playing a role in the development of such comorbidities are still unclear. The regulation and expression of genes that map to chromosome 21 are dynamic and complex, so it is important to perform global gene expression studies with high statistical power to fully characterize the transcriptome in DS patients. This study was undertaken to evaluate mRNAs and lncRNA expression in patients with DS versus a matched cohort of healthy subjects. RNA sequencing was used to perform this transcriptome study. Differential expression analysis revealed 967 transcripts with padj ≤ 0.05. Among them, 447 transcripts were differentially expressed in patients with DS compared to controls. Particularly, 203 transcripts were down expressed (151 protein-coding mRNAs, 45 lncRNAs, 1 microRNA, 1 mitochondrial tRNA, 1 ribozyme, and 1 small nuclear RNA) and 244 were over expressed (210 protein-coding mRNAs and 34 lncRNAs). Interestingly, deregulated lncRNAs are involved in pathways that play a role in developmental disorders, neurological diseases, DNA replication and repair mechanisms, and cancer development in DS patients. In conclusion, these results suggest a role of lncRNAs in the phenotype of DS patients.
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Síndrome de Down/genética , Perfilação da Expressão Gênica/métodos , RNA Longo não Codificante/genética , Transcriptoma/genética , Adulto , Cromossomos Humanos Par 21/genética , Estudos de Coortes , Comorbidade , Reparo do DNA/genética , Replicação do DNA/genética , Deficiências do Desenvolvimento/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Doenças do Sistema Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sicília , Adulto JovemRESUMO
Trisomy 21, also known as Down Syndrome (DS), is the most common chromosome abnormality and causes intellectual disability. Long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5), whose differential expression has recently been reported in patients with Klinefelter syndrome, has been addressed to play a role in the development of inflammatory and autoimmune diseases, vascular endothelial cells apoptosis and atherosclerosis, all being common features in patients with DS. Therefore, the aim of this study was to assess the lncRNA GAS5 expression profile in DS patients and in controls. lncRNA GAS5 levels were evaluated by qRT-PCR assay in 23 patients with DS and 23 age-matched controls. A significant lncRNA GAS5 down-regulation was observed in patients with DS by RT-PCR analysis, The RNA sequencing experiments confirmed the qRT-PCR data. LncRNA GAS5 down-expression may play a role in the development of some typical features of the patients with DS and, particularly, in inflammatory and autoimmune diseases.
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Síndrome de Down/genética , Síndrome de Down/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNARESUMO
The widelyvariable phenotypic spectrum and the different severity of symptoms in men with Klinefelter syndrome (KS) suggest a role for epigenetic mediators. Therefore, the aim of this study is to evaluate the possible involvement of miRNAs in the clinical manifestations of KS. To accomplish this, we performed a transcriptome analysis in peripheral blood mononuclear cells (PBMCs) of 10 non-mosaic KS patients, 10 aged-matched healthy men and 10 aged-matched healthy female controls with normal karyotype. After RNA extraction from PBMC and the preparation of RNA libraries, the samples were sequenced using next generation high-throughput sequencing technology. Expression profiling analysis revealed a significant differential expression of 2 miRNAs in KS compared to male controls. In particular, MIR3648 resulted significantly (q-value < 0.0001) down-regulated by -19.084- fold, while MIR3687was strongly down-regulated (q-value < 0.0001) considering KS patients. These results were confirmed by qRT-PCR. The functional analysis of the two transcripts showed that they seem to play a role in breast cancer, hemopoietic abnormalities, immune defects and adipocyte differentiation and fat cell maturation. Therefore, we speculate that both miRNAs may play a role in the immune and metabolic disorders and in the risk of breast cancer development in men with KS.
Assuntos
Regulação da Expressão Gênica , Síndrome de Klinefelter/genética , MicroRNAs/genética , Cariótipo Anormal , Adulto , Biomarcadores , Estudos de Casos e Controles , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Síndrome de Klinefelter/sangue , Síndrome de Klinefelter/diagnóstico , Leucócitos/metabolismo , Leucócitos Mononucleares , Masculino , Pessoa de Meia-Idade , TranscriptomaRESUMO
BACKGROUND: The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERß) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. RESULTS: Applying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERß in gene regulation, we identify AGO2 as a novel partner of ERß in human BC cells. ERß-AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERß binding sites, and total and nascent RNA-Seq in ERß + vs ERß - cells, and before and after AGO2 knock-down in ERß + cells, reveals a widespread involvement of this factor in ERß-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERß-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability. CONCLUSIONS: These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERß, indicating that both factors are endowed with multiple roles in the control of key cellular functions.
Assuntos
Proteínas Argonautas/metabolismo , Neoplasias da Mama/genética , Receptor beta de Estrogênio/metabolismo , Regulação da Expressão Gênica , Splicing de RNA , Complexo de Inativação Induzido por RNA/metabolismo , Transcrição Gênica , Neoplasias da Mama/metabolismo , Genoma Humano , Humanos , Células MCF-7RESUMO
Hepatocellular carcinoma (HCC) is the result of a stepwise process, often beginning with development within a cirrhotic liver of premalignant lesions, morphologically characterized by low- (LGDN) and high-grade (HGDN) dysplastic nodules. PIWI-interacting RNAs (piRNAs) are small noncoding RNAs (sncRNAs), 23-35 nucleotide-long, exerting epigenetic and post-transcriptional regulation of gene expression. Recently the PIWI-piRNA pathway, best characterized in germline cells, has been identified also in somatic tissues, including stem and cancer cells, where it influences key cellular processes.Small RNA sequencing was applied to search for liver piRNAs and to profile their expression patterns in cirrhotic nodules (CNs), LGDN, HGDN, early HCC and progressed HCC (pHCC), analyzing 55 samples (14 CN, 9 LGDN, 6 HGDN, 6 eHCC and 20 pHCC) from 17 patients, aiming at identifying possible relationships between these sncRNAs and liver carcinogenesis. We identified a 125 piRNA expression signature that characterize HCC from matched CNs, correlating also to microvascular invasion in HCC. Functional analysis of the predicted RNA targets of deregulated piRNAs indicates that these can target key signaling pathways involved in hepatocarcinogenesis and HCC progression, thereby affecting their activity. Interestingly, 24 piRNAs showed specific expression patterns in dysplastic nodules, respect to cirrhotic liver and/or pHCC.The results demonstrate that the PIWI-piRNA pathway is active in human liver, where it represents a new player in the molecular events that characterize hepatocarcinogenesis, from early stages to pHCC. Furthermore, they suggest that piRNAs might be new disease biomarkers, useful for differential diagnosis of dysplastic and neoplastic liver lesions.
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Carcinoma Hepatocelular/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Lesões Pré-Cancerosas/genética , RNA Interferente Pequeno/genética , Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Análise por Conglomerados , Diagnóstico Diferencial , Humanos , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Lesões Pré-Cancerosas/patologiaRESUMO
The KCNT1 gene encodes for subunits contributing to the Na(+)-activated K(+) current (KNa), expressed in many cell types. Mutations in KCNT1 have been found in patients affected with a wide spectrum of early-onset epilepsies, including Malignant Migrating Partial Seizures in Infancy (MMPSI), a severe early-onset epileptic encephalopathy characterized by pharmacoresistant focal seizures migrating from one brain region or hemisphere to another and neurodevelopment arrest or regression, resulting in profound disability. In the present study we report identification by whole exome sequencing (WES) of two de novo, heterozygous KCNT1 mutations (G288S and, not previously reported, M516V) in two unrelated MMPSI probands. Functional studies in a heterologous expression system revealed that channels formed by mutant KCNT1 subunits carried larger currents when compared to wild-type KCNT1 channels, both as homo- and heteromers with these last. Both mutations induced a marked leftward shift in homomeric channel activation gating. Interestingly, the KCNT1 blockers quinidine (3-1000µM) and bepridil (0.03-10µM) inhibited both wild-type and mutant KCNT1 currents in a concentration-dependent manner, with mutant channels showing higher sensitivity to blockade. This latter result suggests two genotype-tailored pharmacological strategies to specifically counteract the dysfunction of KCNT1 activating mutations in MMPSI patients.
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Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Espasmos Infantis/genética , Animais , Células CHO , Cricetinae , Cricetulus , Exoma , Humanos , Lactente , Ativação do Canal Iônico , Masculino , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Canais de Potássio Ativados por Sódio , Espasmos Infantis/diagnósticoRESUMO
Small non-coding RNAs (sncRNAs) represent a heterogeneous group of <200nt-long transcripts comprising microRNAs, PIWI-interacting RNAs (piRNAs) and small-nucleolar-RNAs (snoRNAs) involved in physiological and pathological processes such as carcinogenesis and tumor progression. Aberrant sncRNA expression in cancer has been associated with specific clinical phenotypes, grading, staging, metastases development and resistance to therapy.Aim of the present work is to study the role of sncRNAs in endometrial carcinogenesis. Changes in sncRNA expression were identified by high-throughput genomic analysis of paired normal, hyperplastic and cancerous endometrial tissues obtained by endometrial biopsies (n = 10). Using smallRNA sequencing and microarrays we identified significant differences in sncRNA expression pattern between normal, hyperplastic and neoplastic endometrium. This led to the definition of a sncRNA signature (129 microRNAs, 2 of which not previously described, 10 piRNAs and 3 snoRNAs) of neoplastic transformation. Functional bioinformatics analysis identified as downstream targets multiple signaling pathways potentially involved in the hyperplastic and neoplastic tissue responses, including Wnt/ß-catenin, and ERK/MAPK and TGF-ß-Signaling.Considering the regulatory role of sncRNAs, this newly identified sncRNA signature is likely to reflect the events leading to endometrial cancer, which can be exploited to dissect the carcinogenic process including novel biomarkers for early and non-invasive diagnosis of these tumors.
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Biomarcadores Tumorais/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Endométrio/metabolismo , MicroRNAs/genética , Pequeno RNA não Traduzido/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Neoplasias do Endométrio/mortalidade , Endométrio/patologia , Feminino , Seguimentos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Taxa de SobrevidaRESUMO
Estrogen receptor subtypes (ERα and ERß) are transcription factors sharing a similar structure but exerting opposite roles in breast cancer cells. Besides the well-characterized genomic actions of nuclear ERs upon ligand binding, specific actions of ligand-free ERs in the cytoplasm also affect cellular functions. The identification of cytoplasmic interaction partners of unliganded ERα and ERß may help characterize the molecular basis of the extra-nuclear mechanism of action of these receptors, revealing novel mechanisms to explain their role in breast cancer response or resistance to endocrine therapy. To this aim, cytoplasmic extracts from human breast cancer MCF-7 cells stably expressing tandem affinity purification-tagged ERα and ERß and maintained in estrogen-free medium were subject to affinity-purification and MS analysis, leading to the identification of 84 and 142 proteins associated with unliganded ERα and ERß, respectively. Functional analyses of ER subtype-specific interactomes revealed significant differences in the molecular pathways targeted by each receptor in the cytoplasm. This work, reporting the first identification of the unliganded ERα and ERß cytoplasmic interactomes in breast cancer cells, provides novel experimental evidence on the nongenomic effects of ERs in the absence of hormonal stimulus. All MS data have been deposited in the ProteomeXchange with identifier PXD001202 (http://proteomecentral.proteomexchange.org/dataset/PXD001202).
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Neoplasias da Mama/metabolismo , Citoplasma/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Mapeamento de Interação de Proteínas/métodos , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7/metabolismo , Espectrometria de MassasRESUMO
PIWI-interacting small non-coding RNAs (piRNAs) are genetic and epigenetic regulatory factors in germline cells, where they maintain genome stability, are involved in RNA silencing and regulate gene expression. We found that the piRNA biogenesis and effector pathway are present in human breast cancer (BC) cells and, analyzing smallRNA-Seq data generated from BC cell lines and tumor biopsies, we identified >100 BC piRNAs, including some very abundant and/or differentially expressed in mammary epithelial compared to BC cells, where this was influenced by estrogen or estrogen receptor ß, and in cancer respect to normal breast tissues. A search for mRNAs targeted by the BC piRNome revealed that eight piRNAs showing a specific expression pattern in breast tumors target key cancer cell pathways. Evidence of an active piRNA pathway in BC suggests that these small non-coding RNAs do exert transcriptional and post-transcriptional gene regulatory actions also in cancer cells.
Assuntos
Proteínas Argonautas/metabolismo , Neoplasias da Mama/genética , RNA Interferente Pequeno/metabolismo , Pequeno RNA não Traduzido/biossíntese , Proteínas Argonautas/genética , Sequência de Bases , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Receptor beta de Estrogênio/metabolismo , Estrogênios/deficiência , Feminino , Humanos , Células MCF-7 , Dados de Sequência Molecular , RNA Interferente Pequeno/genética , Pequeno RNA não Traduzido/genética , Análise de Sequência de RNA , TransfecçãoRESUMO
Sequencing of the KCNT1, PLCB1, SCN1A and TBC1D24 loci was performed in six children with typical features of malignant migrating partial seizures of infancy (MMPSI), to verify the presence of potential disease-causing mutations, including those already reported to be associated with the disease. Sanger sequencing failed to identify in these genes the previously reported pathogenic mutations in these patients, while a comprehensive mutational scanning analysis of these four loci by targeted re-sequencing led to detection of both intronic and exonic new variants. Based on the current knowledge, the sequence variants identified here do not allow to predict functional phenotypes that might explain, at least in part, MMPSI symptoms.
Assuntos
Epilepsias Parciais/diagnóstico , Epilepsias Parciais/genética , Mutação/genética , Proteínas de Transporte/genética , Feminino , Proteínas Ativadoras de GTPase , Variação Genética , Humanos , Lactente , Masculino , Proteínas de Membrana , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Proteínas do Tecido Nervoso/genética , Fenótipo , Fosfolipase C beta/genética , Canais de Potássio/genética , Canais de Potássio Ativados por Sódio , Deleção de SequênciaRESUMO
Anhedonia is a core symptom of depression that also characterizes substance abuse-related mood disorders, in particular those secondary to stimulant abuse. This study investigated the long-lasting condition of cocaine sensitization as an inducing condition for anhedonia in rats. Cortical-mesolimbic dopamine plays a central role in assessing the incentive value of a stimulus and an increased dopamine output in these areas after a novel palatable meal seems to correlate with the ability to acquire an instrumental behaviour aimed at earning it again. This dopaminergic response is associated with consistent modifications in the phosphorylation pattern of some cAMP-dependent protein kinase (PKA) substrates and it is mediated by dopamine D1 receptor stimulation. Thus, since behavioural cocaine sensitization is characterized by tonically increased levels of phospho-Thr75 DARPP-32 that is a potent PKA inhibitor, we hypothesized that cocaine-sensitized rats might reveal deficits in palatable food responding. Indeed, non-food-deprived cocaine-sensitized rats showed no interest in palatable food, no dopaminergic response after a palatable meal in terms of increased dopamine output and DARPP-32 phosphorylation changes, and no ability to acquire a palatable food-sustained instrumental behaviour. Repeated administration of an established antidepressant compound, imipramine, corrected these deficits and reinstated the dopaminergic response in the cortico-mesolimbic areas to control values. Thus, the behavioural modifications observed in cocaine-sensitized rats satisfy some requirements for an experimental model of anhedonia since they are induced by repeated cocaine administration (aetiological validity), they mimic an anhedonia-like symptom (construct validity), and are reversed by the administration of imipramine (predictive validity).