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BACKGROUND: Diabetic complications have been studied extensively in recent years. There are very few biomarkers in body fluids that can pinpoint a distinct diabetic complication due to insufficient known specific biomarkers for ischemia. OBJECTIVE: Identifying microRNA in animal models for each complication could enable early diagnosis of a given complication if verified in humans. MicroRNA (miRNA) profiling has been done in rodent models for a number of diabetic complications, like diabetic glomerular injury, atherosclerosis, cognitive impairment, diabetic wound healing, angiopathy and other complications. Due to multiple differences between rodents and humans, the changes in rabbit skin, considered closer to humans than even pigs, may better simulate human diabetic complications of ischemia. METHODS: To study the miRNA profile of rabbits in which diabetes was induced or ischemia was surgically generated, we studied whether diabetes or ischemia-induced specific miRNA could be detected. MicroRNA from the blood of diabetic rabbits and rabbits with local ischemia was collected in PAXgene Blood RNA tubes specifically designed for miRNA isolation and extracted using the PAX gene miRNA extraction kit. The isolated RNA was quality controlled using an RNA analyzer, and further, using RNA seq technology, it was analyzed for distinct miRNAs that were detected in diabetic and non-diabetic rabbits induced with ischemia. RESULTS: A miRNA that was found to be expressed in diabetic rabbits and ischemic rabbits but not in untreated rabbits was miRNA-183. Several miRNAs were differentially expressed across comparison groups, and several upregulated miRNAs were identified being unique to each comparison. In rabbits with a potential diabetic complication of a long-term ischemic model, there was one distinct microRNA, which was highly significantly upregulated in ischemia rabbit (miRNA-133-3p). One miRNA that was highly significantly upregulated in diabetic rabbit but not in ischemic rabbits was miRNA-3074-5p. Only statistically significant results have been considered and analyzed. CONCLUSION: These findings could lead to a precise and timely diagnosis of a potential single diabetic complication without invasive tissue biopsies and could be a novel tool in the management of diabetic patients developing complications due to the progression of diabetes.
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Complicações do Diabetes , Diabetes Mellitus , MicroRNAs , Humanos , Coelhos , Animais , Suínos , MicroRNAs/genética , Isquemia/genética , Isquemia/patologia , Complicações do Diabetes/genética , Biomarcadores , Perfilação da Expressão GênicaRESUMO
Long-read sequencing technologies such as isoform sequencing can generate highly accurate sequences of full-length mRNA transcript isoforms. Such long-read transcriptomics may be especially useful in investigations of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes are readily available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified 4 lymphocyte populations (CD4+ T, CD8+ T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN > 8) for isoform sequencing and parallel RNA-Seq analyses. Many novel polyadenylated transcript isoforms, supported by both isoform sequencing and RNA-Seq data, were identified within each sample. The datasets met several metrics of high quality and have been deposited to the Gene Expression Omnibus database (GSE202327, GSE202328, GSE202329) as both raw and processed files to serve as long-read reference transcriptomes for future studies of human circulating lymphocytes.
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Perfilação da Expressão Gênica , Transcriptoma , Humanos , Masculino , Sequenciamento de Nucleotídeos em Larga Escala , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análise de Sequência de RNA , Subpopulações de Linfócitos/metabolismoRESUMO
PURPOSE: Improving our understanding of the immunologic response to cancer cells within the sentinel lymph nodes (SLN) of primary tumors is expected to identify new approaches to stimulate clinically meaningful cancer immunity. EXPERIMENTAL DESIGN: We used mass cytometry by time-of-flight (CyTOF), flow cytometry, and T-cell receptor immunosequencing to conduct simultaneous single-cell analyses of immune cells in the SLNs of patients with melanoma. RESULTS: We found increased effector-memory αß T cells, TCR clonality, and γδ T cells selectively in the melanoma-bearing SLNs relative to non-melanoma-bearing SLNs, consistent with possible activation of an antitumor immune response. However, we also observed a markedly immunotolerant environment in the melanoma-bearing SLNs indicated by reduced and impaired NK cells and increased levels of CD8+CD57+PD-1+ cells, which are known to display low melanoma killing capabilities. Other changes observed in melanoma-bearing SLNs when compared with non-melanoma-bearing SLNs include (i) reduced CD8+CD69+ T cell/T regulatory cell ratio, (ii) high PD-1 expression on CD4+ and CD8+ T cells, and (iii) high CTLA-4 expression on γδ T cells. CONCLUSIONS: Our data suggest that these immunologic changes compromise antimelanoma immunity and contribute to a high relapse rate. We propose the development of clinical trials to test the neo-adjuvant administration of anti-PD-1 antibodies prior to SLN resection in patients with stage III melanoma. See related commentary by Lund, p. 1996.
Assuntos
Melanoma , Linfonodo Sentinela , Neoplasias Cutâneas , Humanos , Tolerância Imunológica , Melanoma/patologia , Receptor de Morte Celular Programada 1/uso terapêutico , Linfonodo Sentinela/patologia , Biópsia de Linfonodo Sentinela , Neoplasias Cutâneas/patologia , Microambiente TumoralRESUMO
Throughout the course of the ongoing SARS-CoV-2 pandemic there has been a need for approaches that enable rapid monitoring of public health using an unbiased and minimally invasive means. A major way this has been accomplished is through the regular assessment of wastewater samples by qRT-PCR to detect the prevalence of viral nucleic acid with respect to time and location. Further expansion of SARS-CoV-2 wastewater monitoring efforts to include the detection of variants of interest/concern through next-generation sequencing has enhanced the understanding of the SARS-CoV-2 outbreak. In this report, we detail the results of a collaborative effort between public health and metropolitan wastewater management authorities and the University of Louisville to monitor the SARS-CoV-2 pandemic through the monitoring of aggregate wastewater samples over a period of 28 weeks. Through the use of next-generation sequencing approaches the polymorphism signatures of Variants of Concern/Interest were evaluated to determine the likelihood of their prevalence within the community on the basis of their relative dominance within sequence datasets. Our data indicate that wastewater monitoring of water quality treatment centers and smaller neighborhood-scale catchment areas is a viable means by which the prevalence and genetic variation of SARS-CoV-2 within a metropolitan community of approximately one million individuals may be monitored, as our efforts detected the introduction and emergence of variants of concern in the city of Louisville. Importantly, these efforts confirm that regional emergence and spread of variants of interest/concern may be detected as readily in aggregate wastewater samples as compared to the individual wastewater sheds. Furthermore, the information gained from these efforts enabled targeted public health efforts including increased outreach to at-risk communities and the deployment of mobile or community-focused vaccination campaigns.
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Key elements for viral pathogenesis include viral strains, viral load, co-infection, and host responses. Several studies analyzing these factors in the function of disease severity of have been published; however, no studies have shown how all of these factors interplay within a defined cohort. To address this important question, we sought to understand how these four key components interplay in a cohort of COVID-19 patients. We determined the viral loads and gene expression using high throughput sequencing and various virological methods. We found that viral loads in the upper respiratory tract in COVID-19 patients at an early phase of infection vary widely. While the majority of nasopharyngeal (NP) samples have a viral load lower than the limit of detection of infectious viruses, there are samples with an extraordinary amount of SARS-CoV-2 RNA and a high viral titer. No specific viral factors were identified that are associated with high viral loads. Host gene expression analysis showed that viral loads were strongly correlated with cellular antiviral responses. Interestingly, however, COVID-19 patients who experience mild symptoms have a higher viral load than those with severe complications, indicating that naso-pharyngeal viral load may not be a key factor of the clinical outcomes of COVID-19. The metagenomics analysis revealed that the microflora in the upper respiratory tract of COVID-19 patients with high viral loads were dominated by SARS-CoV-2, with a high degree of dysbiosis. Finally, we found a strong inverse correlation between upregulation of interferon responses and disease severity. Overall our study suggests that a high viral load in the upper respiratory tract may not be a critical factor for severe symptoms; rather, dampened antiviral responses may be a critical factor for a severe outcome from the infection.
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COVID-19/patologia , Interferons/metabolismo , SARS-CoV-2/genética , Adulto , Idoso , COVID-19/virologia , Disbiose/etiologia , Feminino , Humanos , Masculino , Metagenômica , Microbiota/genética , Pessoa de Meia-Idade , Nasofaringe/virologia , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real , Sistema Respiratório/microbiologia , Sistema Respiratório/virologia , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , Transcriptoma , Regulação para Cima , Carga ViralRESUMO
Age is an important factor for determining the outcome of melanoma patients. Sentinel lymph node (SLN) status is also a strong predictor of survival for melanoma. Paradoxically, older melanoma patients have a lower incidence of SLN metastasis but a higher mortality rate when compared with their younger counterparts. The mechanisms that underlie this phenomenon remain unknown. This study uses three independent datasets of RNA samples from patients with melanoma metastatic to the SLN to identify age-related transcriptome changes in SLNs and their association with outcome. Microarray was applied to the first dataset of 97 melanoma patients. NanoString was performed in the second dataset to identify the specific immune genes and pathways that are associated with recurrence in younger versus older patients. qRT-PCR analysis was used in the third dataset of 36 samples to validate the differentially expressed genes (DEGs) from microarray and NanoString. These analyses show that FOS, NR4A, and ITGB1 genes were significantly higher in older melanoma patients with positive SLNs. IRAK3- and Wnt10b-related genes are the major pathways associated with recurrent melanoma in younger and older patients with tumor-positive SLNs, respectively. This study aims to elucidate age-related differences in SLNs in the presence of nodal metastasis.
Assuntos
Melanoma/genética , Linfonodo Sentinela/patologia , Neoplasias Cutâneas/genética , Adulto , Fatores Etários , Idoso , Proteínas Relacionadas à Autofagia/genética , Moléculas de Adesão Celular/genética , Feminino , Humanos , Integrina beta1/genética , Quinases Associadas a Receptores de Interleucina-1/genética , Lectinas Tipo C/genética , Metástase Linfática , Masculino , Melanoma/patologia , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-fos/genética , Receptores Imunológicos/genética , Transdução de Sinais , Neoplasias Cutâneas/patologia , Transcriptoma , Proteínas Wnt/genéticaRESUMO
Periodontitis is an irreversible, bacteria-induced, chronic inflammatory disease that compromises the integrity of tooth-supporting tissues and adversely affects systemic health. As the immune system's first line of defense against bacteria, neutrophils use their microbicidal functions in the oral cavity to protect the host against periodontal disease. However, periodontal pathogens have adapted to resist neutrophil microbicidal mechanisms while still propagating inflammation, which provides essential nutrients for the bacteria to proliferate and cause disease. Advances in sequencing technologies have recognized several newly appreciated bacteria associated with periodontal lesions such as the Gram-positive anaerobic rod, Filifactor alocis. With the discovery of these oral bacterial species, there is also a growing need to assess their pathogenic potential and determine their contribution to disease progression. Currently, few studies have addressed the pathogenic mechanisms used by oral bacteria to manipulate the neutrophil functional responses at the level of the transcriptome. Thus, this study aims to characterize the global changes at the gene expression level in human neutrophils during infection with F. alocis. Our results indicate that the challenge of human neutrophils with F. alocis results in the differential expression of genes involved in multiple neutrophil effector functions such as chemotaxis, cytokine and chemokine signaling pathways, and apoptosis. Moreover, F. alocis challenges affected the expression of components from the TNF and MAPK kinase signaling pathways. This resulted in transient, dampened p38 MAPK activation by secondary stimuli TNFα but not by fMLF. Functionally, the F. alocis-mediated inhibition of p38 activation by TNFα resulted in decreased cytokine production but had no effect on the priming of the respiratory burst response or the delay of apoptosis by TNFα. Since the modulatory effect was characteristic of viable F. alocis only, we propose this as one of F. alocis' mechanisms to control neutrophils and their functional responses.
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Clostridiales/imunologia , Neutrófilos/fisiologia , Periodontite/imunologia , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , Explosão Respiratória , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition in estrogen receptor-positive (ER+) breast cancer remains a significant clinical challenge. Efforts to uncover the mechanisms underlying resistance are needed to establish clinically actionable targets effective against resistant tumors. In this study, we sought to identify differentially expressed genes (DEGs) associated with acquired resistance to palbociclib in ER+ breast cancer. We performed next-generation transcriptomic RNA sequencing (RNA-seq) and pathway analysis in ER+ MCF7 palbociclib-sensitive (MCF7/pS) and MCF7 palbociclib-resistant (MCF7/pR) cells. We identified 2183 up-regulated and 1548 down-regulated transcripts in MCF7/pR compared to MCF7/pS cells. Functional analysis of the DEGs using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified several pathways associated with breast cancer, including 'cell cycle', 'DNA replication', 'DNA repair' and 'autophagy'. Additionally, Ingenuity Pathway Analysis (IPA) revealed that resistance to palbociclib is closely associated with deregulation of several key canonical and metabolic pathways. Further studies are needed to determine the utility of these DEGs and pathways as therapeutics targets against ER+ palbociclib-resistant breast cancer.
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Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Piperazinas/farmacologia , Piridinas/farmacologia , Receptores de Estrogênio/metabolismo , Transcriptoma/efeitos dos fármacos , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Biologia Computacional , Feminino , Humanos , Células Tumorais CultivadasRESUMO
Abundant with organelle-like membranous structures, the tumor microenvironment is composed of cancer cells that secrete exosomes. Studies have shown that these secreted exosomes transport RNA and active molecules to other cells to reshape the tumor microenvironment and promote tumor growth. In fact, we found that exosomes derived from melanoma cells drive pre-malignant transition in primary melanocytes. However, there is little available in the scientific literature on how exosomes modulate melanocytes in the microenvironment to optimize conditions for tumor progression and metastasis. We therefore focused this current study on identifying these conditions genetically. Through RNA sequencing, we analyzed gene expression levels of melanocytes driven by exosomes derived from melanoma and lung cancer cells compared with those without exosome controls. Significant differences were found in gene expression patterns of melanocytes driven by exosomes derived from melanoma and lung cancer cells. In the melanocytes responding to exosomes derived from melanoma cells, genes of lipopolysaccharide and regulation of leukocyte chemotaxis were predominant. In the melanocytes responding to exosomes derived from lung cancer cells, genes of DNA replication and mitotic nuclear division played an important role. These results provide further mechanistic understanding of tumor progression promoted by tumor-derived exosomes. This will also help identify potential therapeutic targets for melanoma progression.
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Exossomos/metabolismo , Melanócitos/metabolismo , Melanoma/genética , Transcriptoma , Células A549 , Células Cultivadas , Exossomos/genética , Exossomos/patologia , Perfilação da Expressão Gênica , Humanos , Microambiente TumoralRESUMO
Exposure to ionizing radiation associated with highly energetic and charged heavy particles is an inherent risk astronauts face in long duration space missions. We have previously considered the transcriptional effects that three levels of radiation (0.3 Gy, 1.5 Gy, and 3.0 Gy) have at an immediate time point (1 hr) post-exposure [1]. Our analysis of these results suggest effects on transcript levels that could be modulated at lower radiation doses [2]. In addition, a time dependent effect is likely to be present. Therefore, in order to develop a lab-on-a-chip approach for detection of radiation exposure in terms of both radiation level and time since exposure, we developed a time- and dose-course study to determine appropriate sensitive and specific transcript biomarkers that are detectable in blood samples. The data described herein was developed from a study measuring exposure to 0.15 Gy, 0.30 Gy, and 1.5 Gy of radiation at 1 hr, 2 hr, and 6 hr post-exposure using Affymetrix® GeneChip® PrimeView™ microarrays. This report includes raw gene expression data files from the resulting microarray experiments representing typical radiation exposure levels an astronaut may experience as part of a long duration space mission. The data described here is available in NCBI's Gene Expression Omnibus (GEO), accession GSE63952.
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Glyphosate is a broad-spectrum herbicide that is used worldwide. It represents a potential harm to surface water, and when commercially mixed with surfactants, its uptake is greatly magnified. The most well-known glyphosate-based product is Roundup. This herbicide is potentially an endocrine disruptor and many studies have shown the cytotoxicity potential of glyphosate-based herbicides. In breast cancer (BC) cell lines it has been demonstrated that glyphosate can induce cellular proliferation via estrogen receptors. Therefore, we aimed to identify gene expression changes in ER+ and ER- BC cell lines treated with Roundup and AMPA, to address changes in canonical pathways that would be related or not with the ER pathway, which we believe could interfere with cell proliferation. Using the Human Transcriptome Arrays 2.0, we identified gene expression changes in MCF-7 and MDA-MB-468 exposed to low concentrations and short exposure time to Roundup Original and AMPA. The results showed that at low concentration (0.05% Roundup) and short exposure (48h), both cell lines suffered deregulation of 11 canonical pathways, the most important being cell cycle and DNA damage repair pathways. Enrichment analysis showed similar results, except that MDA-MB-468 altered mainly metabolic processes. In contrast, 48h 10mM AMPA showed fewer differentially expressed genes, but also mainly related with metabolic processes. Our findings suggest that Roundup affects survival due to cell cycle deregulation and metabolism changes that may alter mitochondrial oxygen consumption, increase ROS levels, induce hypoxia, damage DNA repair, cause mutation accumulation and ultimately cell death. To our knowledge, this is the first study to analyze the effects of Roundup and AMPA on gene expression in triple negative BC cells. Therefore, we conclude that both compounds can cause cellular damage at low doses in a relatively short period of time in these two models, mainly affecting cell cycle and DNA repair.
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Neoplasias da Mama/genética , Glicina/análogos & derivados , Transdução de Sinais/genética , Transcriptoma/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Estrogênios/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicina/farmacologia , Herbicidas/efeitos adversos , Herbicidas/farmacologia , Humanos , Células MCF-7 , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , GlifosatoRESUMO
The importance of gut microbiota in human health and pathophysiology is undisputable. Despite the abundance of metagenomics data, the functional dynamics of gut microbiota in human health and disease remain elusive. Urolithin A (UroA), a major microbial metabolite derived from polyphenolics of berries and pomegranate fruits displays anti-inflammatory, anti-oxidative, and anti-ageing activities. Here, we show that UroA and its potent synthetic analogue (UAS03) significantly enhance gut barrier function and inhibit unwarranted inflammation. We demonstrate that UroA and UAS03 exert their barrier functions through activation of aryl hydrocarbon receptor (AhR)- nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways to upregulate epithelial tight junction proteins. Importantly, treatment with these compounds attenuated colitis in pre-clinical models by remedying barrier dysfunction in addition to anti-inflammatory activities. Cumulatively, the results highlight how microbial metabolites provide two-pronged beneficial activities at gut epithelium by enhancing barrier functions and reducing inflammation to protect from colonic diseases.
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Cumarínicos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas de Junções Íntimas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células CACO-2 , Cumarínicos/química , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Organismos Livres de Patógenos Específicos , Proteínas de Junções Íntimas/genéticaRESUMO
It is clear that obesity increases the risk of many types of cancer, including breast cancer. However, the underlying molecular mechanisms by which obesity is linked to cancer risk remain to be defined. Herein, we report that circulating adipose fatty acid binding protein (A-FABP) promotes obesity-associated breast cancer development. Using clinical samples, we demonstrated that circulating A-FABP levels were significantly increased in obese patients with breast cancer in comparison with those without breast cancer. Circulating A-FABP released by adipose tissue directly targeted mammary tumor cells, enhancing tumor stemness and aggressiveness through activation of the IL-6/STAT3/ALDH1 pathway. Importantly, genetic deletion of A-FABP successfully reduced tumor ALHD1 activation and obesity-associated mammary tumor growth and development in different mouse models. Collectively, these data suggest circulating A-FABP as a new link between obesity and breast cancer risk, thereby revealing A-FABP as a potential new therapeutic target for treatment of obesity-associated cancers.
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Neoplasias da Mama/sangue , Neoplasias da Mama/etiologia , Proteínas de Ligação a Ácido Graxo/sangue , Obesidade/complicações , Família Aldeído Desidrogenase 1 , Animais , Biomarcadores Tumorais/sangue , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Progressão da Doença , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Humanos , Interleucina-6/metabolismo , Isoenzimas/metabolismo , Camundongos Endogâmicos C57BL , Invasividade Neoplásica/patologia , Obesidade/sangue , Obesidade/metabolismo , Obesidade/patologia , Retinal Desidrogenase/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Current risk assessment tools to estimate the risk of nonsentinel lymph node metastases after completion lymphadenectomy for a positive sentinel lymph node (SLN) biopsy in cutaneous melanoma are based on clinical and pathologic factors. We identified a novel genetic signature that can predict non-SLN metastases in patients with cutaneous melanoma staged with a SLN biopsy. METHODS: RNA was collected for tumor-positive SLNs in patients staged by SLN biopsy for cutaneous melanoma. All patients with a tumor-positive SLN biopsy underwent completion lymphadenectomy. A 1:10 case:control series of positive and negative non-SLN patients was analyzed by microarray and quantitative RT-PCR. Candidate differentially expressed genes were validated in a 1:3 case:control separate cohort of positive and negative non-SLN patients. RESULTS: The 1:10 case:control discovery set consisted of 7 positive non-SLN cases matched to 70 negative non-SLN controls. The cases and controls were similar with regards to important clinicopathologic factors, such as gender, primary tumor site, age, ulceration, and thickness. Microarray and RT-PCR identified six potential differentially expressed genes for validation. In the 40-patient separate validation set, 10 positive non-SLN patients were matched to 30 negative non-SLN controls based on gender, ulceration, age, and thickness. Five of the six genes were differentially expressed. The five gene panel identified patients at low (7.1%) and high risk (66.7%) for non-SLN metastases. CONCLUSIONS: A novel, non-SLN gene score based on differential expressed genes in a tumor-positive SLN can identify patients at high and low risk for non-SLN metastases.
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Melanoma/genética , Melanoma/secundário , Linfonodo Sentinela , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Transcriptoma , Adulto , Área Sob a Curva , Estudos de Casos e Controles , Feminino , Humanos , Excisão de Linfonodo , Linfonodos/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Curva ROC , Linfonodo Sentinela/patologia , Linfonodo Sentinela/cirurgia , Biópsia de Linfonodo SentinelaRESUMO
Arsenic is a widely distributed toxic natural element. Chronic arsenic ingestion causes several cancers, especially skin cancer. Arsenic-induced cancer mechanisms are not well defined, but several studies indicate that mutation is not the driving force and that microRNA expression changes play a role. Chronic low arsenite exposure malignantly transforms immortalized human keratinocytes (HaCaT), serving as a model for arsenic-induced skin carcinogenesis. Early changes in miRNA expression in HaCaT cells chronically exposed to arsenite will reveal early steps in transformation. HaCaT cells were maintained with 0/100 nM NaAsO2 for 3 and 7 weeks. Total RNA was purified. miRNA and mRNA expression was assayed using Affymetrix microarrays. Targets of differentially expressed miRNAs were collected from TargetScan 6.2, intersected with differentially expressed mRNAs using Partek Genomic Suite software, and mapped to their pathways using MetaCore software. MDM2, HMGB1 and TP53 mRNA, and protein levels were assayed by RT-qPCR and Western blot. Numerous miRNAs and mRNAs involved in carcinogenesis pathways in other systems were differentially expressed at 3 and 7 weeks. A TP53 regulatory network including MDM2 and HMGB1 was predicted by the miRNA and mRNA networks. Total TP53 and TP53-S15-phosphorylation were induced. However, TP53-K382-hypoacetylation suggested that the induced TP53 is inactive in arsenic exposed cells. Our data provide strong evidence that early changes in miRNAs and target mRNAs may contribute to arsenic-induced carcinogenesis.
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Arsenitos/toxicidade , Carcinógenos Ambientais/toxicidade , Transformação Celular Neoplásica/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , MicroRNAs/genética , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Regulação da Expressão Gênica , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , FosforilaçãoRESUMO
MicroRNAs are biomarkers and potential therapeutic targets for breast cancer. Anacardic acid (AnAc) is a dietary phenolic lipid that inhibits both MCF-7 estrogen receptor α (ERα) positive and MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation with IC50s of 13.5 and 35 µM, respectively. To identify potential mediators of AnAc action in breast cancer, we profiled the genome-wide microRNA transcriptome (microRNAome) in these two cell lines altered by the AnAc 24:1n5 congener. Whole genome expression profiling (RNA-seq) and subsequent network analysis in MetaCore Gene Ontology (GO) algorithm was used to characterize the biological pathways altered by AnAc. In MCF-7 cells, 69 AnAc-responsive miRNAs were identified, e.g., increased let-7a and reduced miR-584. Fewer, i.e., 37 AnAc-responsive miRNAs were identified in MDA-MB-231 cells, e.g., decreased miR-23b and increased miR-1257. Only two miRNAs were increased by AnAc in both cell lines: miR-612 and miR-20b; however, opposite miRNA arm preference was noted: miR-20b-3p and miR-20b-5p were upregulated in MCF-7 and MDA-MB-231, respectively. miR-20b-5p target EFNB2 transcript levels were reduced by AnAc in MDA-MB-231 cells. AnAc reduced miR-378g that targets VIM (vimentin) and VIM mRNA transcript expression was increased in AnAc-treated MCF-7 cells, suggesting a reciprocal relationship. The top three enriched GO terms for AnAc-treated MCF-7 cells were B cell receptor signaling pathway and ribosomal large subunit biogenesis and S-adenosylmethionine metabolic process for AnAc-treated MDA-MB-231 cells. The pathways modulated by these AnAc-regulated miRNAs suggest that key nodal molecules, e.g., Cyclin D1, MYC, c-FOS, PPARγ, and SIN3, are targets of AnAc activity.
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Ácidos Anacárdicos/farmacologia , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Estudo de Associação Genômica Ampla , MicroRNAs/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Interferência de RNA , RNA Mensageiro/genética , TranscriptomaRESUMO
BACKGROUND: Arsenic is naturally prevalent in the earth's crust and widely distributed in air and water. Chronic low arsenic exposure is associated with several cancers in vivo, including skin cancer, and with transformation in vitro of cell lines including immortalized human keratinocytes (HaCaT). Arsenic also is associated with cell cycle dysregulation at different exposure levels in multiple cell lines. In this work, we analyzed gene expression in HaCaT cells to gain an understanding of gene expression changes contributing to transformation at an early time point. METHODS: HaCaT cells were exposed to 0 or 100nM NaAsO2 for 7weeks. Total RNA was purified and analyzed by microarray hybridization. Differential expression with fold change≥|1.5| and p-value≤0.05 was determined using Partek Genomic Suite™ and pathway and network analyses using MetaCore™ software (FDR≤0.05). Cell cycle analysis was performed using flow cytometry. RESULTS: 644 mRNAs were differentially expressed. Cell cycle/cell cycle regulation pathways predominated in the list of dysregulated pathways. Genes involved in replication origin licensing were enriched in the network. Cell cycle assay analysis showed an increase in G2/M compartment in arsenite-exposed cells. CONCLUSIONS: Arsenite exposure induced differential gene expression indicating dysregulation of cell cycle control, which was confirmed by cell cycle analysis. The results suggest that cell cycle dysregulation is an early event in transformation manifested in cells unable to transit G2/M efficiently. Further study at later time points will reveal additional changes in gene expression related to transformation processes.
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Arsenitos/toxicidade , Ciclo Celular/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Arsenitos/administração & dosagem , Ciclo Celular/fisiologia , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Humanos , Queratinócitos/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Embryonic stem cell (ESC) abnormalities in genome methylation hamper the utility of their therapeutic derivatives; however, the underlying mechanisms are unknown. Here, we show that the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, Sirt1, selectively prevents abnormal DNA methylation of some developmental genes in murine ESCs by antagonizing Dnmt3l. Transcriptome and DNA methylome analyses demonstrated that Sirt1-null (Sirt1-/-) ESCs repress expression of a subset of imprinted and germline genes concomitant with increased DNA methylation of regulatory elements. Dnmt3l was highly expressed in Sirt1-/- ESCs, and knockdown partially rescued abnormal DNA methylation of the Sirt1 target genes. The Sirt1 protein suppressed transcription of Dnmt3l and physically interacted with the Dnmt3l protein, deacetylating and destabilizing Dnmt3l protein. Sirt1 deficiency delayed neurogenesis and spermatogenesis. These differentiation delays were significantly or partially abolished by reintroduction of Sirt1 cDNA or Dnmt3l knockdown. This study sheds light on mechanisms that restrain DNA methylation of developmentally vital genes operating in ESCs.
Assuntos
Diferenciação Celular/fisiologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Metilação de DNA/fisiologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Sirtuína 1/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NAD/metabolismo , Neurogênese/fisiologia , Espermatogênese/fisiologia , Transcrição Gênica/fisiologiaRESUMO
PURPOSE: Melanoma patients with a single microscopically-positive sentinel lymph node (SLN) are classified as stage III and are often advised to undergo expensive and substantially toxic adjuvant therapy. However, the 5-year survival rate for these patients, with or without adjuvant therapy, varies from 14 to 85 %, representing a heterogeneous biological population with a variable prognosis. We aimed to identify an SLN gene signature to aid in risk stratification of patients with tumor-positive SLNs. METHODS: Microarray experiments were performed to screen SLN genes in recurrence (N = 39) versus non-recurrence (N = 58) groups in the training dataset. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) assay was applied to confirm the expression of selected SLN genes, which were further verified using an independent validation cohort (N = 30). Area under the receiver operating characteristic curve (AUC) was calculated to evaluate prognostic accuracy of the selected SLN gene panel, and the prognostic value of our SLN gene signature was also compared with the current American Joint Committee on Cancer (AJCC) staging system. RESULTS: We identified two SLN genes (PIGR and TFAP2A) that provided high prognostic accuracy in SLN-positive melanoma patients (AUC = 0.864). These two SLN genes, along with clinicopathological features, can differentiate the high- and low-risk groups in node-positive melanoma patients in this cohort. CONCLUSION: The two SLN genes, when combined with clinicopathological features, may offer a new tool for personalized patient risk assessment.
Assuntos
Metástase Linfática/patologia , Melanoma/genética , Melanoma/patologia , Receptores de Imunoglobulina Polimérica/genética , Linfonodo Sentinela/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fator de Transcrição AP-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medição de Risco , Biópsia de Linfonodo Sentinela , Taxa de SobrevidaRESUMO
Astronauts participating in long duration space missions are likely to be exposed to ionizing radiation associated with highly energetic and charged heavy particles. Previously proposed gene biomarkers for radiation exposure include phosphorylated H2A Histone Family, Member X (γH2AX), Tumor Protein 53 (TP53), and Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A). However, transcripts of these genes may not be the most suitable biomarkers for radiation exposure due to a lack of sensitivity or specificity. As part of a larger effort to develop lab-on-a-chip methods for detecting radiation exposure events using blood samples, we designed a dose-course microarray study in order to determine coding and non-coding RNA transcripts undergoing differential expression immediately following radiation exposure. The main goal was to elicit a small set of sensitive and specific radiation exposure biomarkers at low, medium, and high levels of ionizing radiation exposure. Four separate levels of radiation were considered: 0 Gray (Gy) control; 0.3 Gy; 1.5 Gy; and 3.0 Gy with four replicates at each radiation level. This report includes raw gene expression data files from the resulting microarray experiments from all three radiation levels ranging from a lower, typical exposure than an astronaut might see (0.3 Gy) to high, potentially lethal, levels of radiation (3.0 Gy). The data described here is available in NCBI's Gene Expression Omnibus (GEO), accession GSE64375.