RESUMO
IFNL3, which encodes interferon-λ3 (IFN-λ3), has received considerable attention in the hepatitis C virus (HCV) field, as many independent genome-wide association studies have identified a strong association between polymorphisms near IFNL3 and clearance of HCV. However, the mechanism underlying this association has remained elusive. In this study, we report the identification of a functional polymorphism (rs4803217) in the 3' untranslated region (UTR) of IFNL3 mRNA that dictated transcript stability. We found that this polymorphism influenced AU-rich element (ARE)-mediated decay (AMD) of IFNL3 mRNA, as well as the binding of HCV-induced microRNAs during infection. Together these pathways mediated robust repression of the unfavorable IFNL3 polymorphism. Our data reveal a previously unknown mechanism by which HCV attenuates the antiviral response and indicate new potential therapeutic targets for HCV treatment.
Assuntos
Elementos Ricos em Adenilato e Uridilato/genética , Interleucinas/genética , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Estabilidade de RNA/genética , Regiões 3' não Traduzidas/genética , Sequência de Bases , Linhagem Celular Tumoral , Citometria de Fluxo , Genótipo , Células Hep G2 , Hepacivirus/fisiologia , Hepatite C/genética , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Humanos , Interferons , Interleucinas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido NucleicoRESUMO
The immunologic mechanisms promoting eosinophilic granulomatosis with polyangiitis (EGPA) are unclear. To characterize the mechanisms underlying pulmonary EGPA, we examined and compared EGPA paraffin-embedded lung biopsies with normal lung biopsies, using immunostaining, RNA sequencing, and RT-PCR. The results revealed novel type 2 as well as immuneregulatory features. These features included basophils and increased mast cell contents; increased immunostaining for tumor necrosis factor ligand superfamily member 14; sparse mast cell degranulation; numerous forkhead box protein P3 (FoxP3)+ regulatory T cells and IgG4 plasma cells; and abundant arachidonate 15-lipoxygenase and 25-hydroxyvitamin D-1 α hydroxylase, mitochondrial. Significantly decreased 15-hydroxyprostaglandin dehydrogenase [NAD(+)], which degrades eicosanoids, was observed in EGPA samples. In addition, there was significantly increased mRNA for chemokine (C-C motif) ligands 18 and 13 and major collagen genes, IgG4-rich immune complexes coating alveolar macrophages, and increased immunostaining for phosphorylated mothers against decapentaplegic homolog 2/SMAD2, suggesting transforming growth factor-ß activation. These findings suggest a novel self-promoting mechanism of activation of alveolar macrophages by arachidonate 15-lipoxygenase-derived eicosanoids to express chemokines that recruit a combined type 2/immunoregulatory immune response, which produces these eicosanoids. These results suggest that the pulmonary EGPA immune response resembles the immune response to a tissue-invasive parasite infection.
Assuntos
Síndrome de Churg-Strauss/imunologia , Granulomatose com Poliangiite/imunologia , Imunoglobulina G/imunologia , Plasmócitos/imunologia , Adulto , Síndrome de Churg-Strauss/patologia , Feminino , Granulomatose com Poliangiite/patologia , Humanos , MasculinoRESUMO
Sessile serrated adenoma/polyps (SSA/Ps) of the colon account for 20-30% of all colon cancers. Small non-coding RNAs, including microRNAs (miRNAs), may function as oncogenes or tumor suppressor genes involved in cancer development. Small RNA sequencing (RNA-seq) was used to characterize miRNA profiles in SSA/Ps, hyperplastic polyps (HPs), adenomatous polyps and paired uninvolved colon. Our 108 small RNA-seq samples' results were compared to small RNA-seq data from 212 colon cancers from the Cancer Genome Atlas. Twenty-three and six miRNAs were differentially expressed in SSA/Ps compared to paired uninvolved colon and HPs, respectively. Differential expression of MIR31-5p, MIR135B-5p and MIR378A-5p was confirmed by RT-qPCR. SSA/P-specific miRNAs are similarly expressed in colon cancers containing genomic aberrations described in serrated cancers. Correlation of miRNA expression with consensus molecular subtypes suggests more than one subtype is associated with the serrated neoplasia pathway. Canonical pathway analysis suggests many of these miRNAs target growth factor signaling pathways.
Assuntos
Adenoma/genética , Neoplasias do Colo/genética , Pólipos do Colo/genética , MicroRNAs/genética , Adenoma/patologia , Idoso , Animais , Biomarcadores Tumorais/genética , Neoplasias do Colo/patologia , Pólipos do Colo/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNA , Thoracica/genéticaRESUMO
Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by HCV are not defined. We provide a systems biology analysis with multiple lines of evidence to indicate that interleukin-1ß (IL-1ß) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1ß compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells, resident hepatic macrophages, are the primary cellular source of hepatic IL-1ß during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages, and Kupffer cells recovered from normal donor liver, produce IL-1ß after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1ß secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggers MyD88-mediated TLR7 signaling to induce IL-1ß mRNA expression. HCV uptake concomitantly induces a potassium efflux that activates the NLRP3 inflammasome for IL-1ß processing and secretion. RNA sequencing analysis comparing THP1 cells and chronic hepatitis C patient liver demonstrates that viral engagement of the NLRP3 inflammasome stimulates IL-1ß production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identify intrahepatic IL-1ß production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1ß activity could offer therapeutic actions to reduce hepatic inflammation and mitigate disease.
Assuntos
Proteínas de Transporte/metabolismo , Hepatite C Crônica/imunologia , Inflamassomos/imunologia , Interleucina-1beta/biossíntese , Células de Kupffer/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Quimiocinas/biossíntese , Citocinas/biossíntese , Ativação Enzimática , Hepacivirus/imunologia , Humanos , Interleucina-1beta/sangue , Interleucina-1beta/genética , Células de Kupffer/imunologia , Fígado/imunologia , Fígado/metabolismo , Fígado/virologia , Hepatopatias/imunologia , Hepatopatias/virologia , Fator 88 de Diferenciação Mieloide/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fagocitose , RNA Mensageiro/biossíntese , Transdução de Sinais , Tetraspanina 28 , Receptor 7 Toll-Like/metabolismoRESUMO
Serrated polyposis syndrome (SPS) presents with multiple sessile serrated lesions (SSL) in the large intestine and confers increased colorectal cancer (CRC) risk. However, the etiology of SPS is not known. SSL-derived organoids have not been previously studied but may help provide insights into SPS pathogenesis and identify novel biomarkers and chemopreventive strategies. This study examined effects of EGFR and COX pathway inhibition in organoid cultures derived from uninvolved colon and polyps of SPS patients. We also compared with organoids representing the hereditary gastrointestinal syndromes, Familial Adenomatous Polyposis (FAP) and Lynch syndrome (LS). Eighteen total organoid colon cultures were generated from uninvolved colon and polyps in SPS, FAP, LS, and non-syndromic screening colonoscopy patients. BRAF and KRAS mutation status was determined for each culture. Erlotinib (EGFR inhibitor) and sulindac (COX inhibitor) were applied individually and in combination. A 44-target gene custom mRNA panel (including WNT and COX pathway genes) and a 798-gene microRNA gene panel were used to quantitate organoid RNA expression by NanoString analysis. Erlotinib treatment significantly decreased levels of mRNAs associated with WNT and MAPK kinase signaling in organoids from uninvolved colon from all four patient categories and from all SSL and adenomatous polyps. Sulindac did not change the mRNA profile in any culture. Our findings suggest that EGFR inhibitors may contribute to the chemopreventive treatment of SSLs. These findings may also facilitate clinical trial design using these agents in SPS patients. Differentially expressed genes identified in our study (MYC, FOSL1, EGR1, IL33, LGR5 and FOXQ1) may be used to identify other new molecular targets for chemoprevention of SSLs.
RESUMO
The molecular mechanisms behind human liver disease progression to cirrhosis remain elusive. Nuclear receptor small heterodimer partner (SHP/Nr0b2) is a hepatic tumor suppressor and a critical regulator of liver function. SHP expression is diminished in human cirrhotic livers, suggesting a regulatory role in human liver diseases. The goal of this study was to identify novel SHP-regulated genes that are involved in the development and progression of chronic liver disease. To achieve this, we conducted the first comprehensive RNA sequencing (RNA-seq) analysis of Shp(-/-) mice, compared the results with human hepatitis C cirrhosis RNA-seq and nonalcoholic steatohepatitis (NASH) microarray datasets, and verified novel results in human liver biospecimens. This approach revealed new gene signatures associated with chronic liver disease and regulated by SHP. Several genes were selected for validation of physiological relevance based on their marked upregulation, novelty with regard to liver function, and involvement in gene pathways related to liver disease. These genes include peptidoglycan recognition protein 2, dual specific phosphatase-4, tetraspanin 4, thrombospondin 1, and SPARC-related modular calcium binding protein-2, which were validated by qPCR analysis of 126 human liver specimens, including steatosis, fibrosis, and NASH, alcohol and hepatitis C cirrhosis, and in mouse models of liver inflammation and injury. This RNA-seq analysis identifies new genes that are regulated by the nuclear receptor SHP and implicated in the molecular pathogenesis of human chronic liver diseases. The results provide valuable transcriptome information for characterizing mechanisms of these diseases.
Assuntos
Perfilação da Expressão Gênica , Genoma Humano , Hepatopatias/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Biópsia , Análise por Conglomerados , Biologia Computacional , Bases de Dados Genéticas , Progressão da Doença , Fígado Gorduroso/genética , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Hepatite C Crônica/genética , Humanos , Imuno-Histoquímica , Cirrose Hepática/genética , Cirrose Hepática Experimental/genética , Hepatopatias/patologia , Hepatopatias Alcoólicas/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNARESUMO
The water disinfection byproduct bromate (BrO3(-)) produces cytotoxic and carcinogenic effects in rat kidneys. Our previous studies demonstrated that BrO3(-) caused sex-dependent differences in renal gene and protein expression in rats and the elimination of brominated organic carbon in their urine. The present study examined changes in renal cell apoptosis and protein expression in male and female F344 rats treated with BrO3(-) and associated these changes with accumulation of 3-bromotyrosine (3-BT)-modified proteins. Rats were treated with 0, 11.5, 46 and 308 mg/L BrO3(-) in drinking water for 28 days and renal sections were prepared and examined for apoptosis (TUNEL-staining), 8-oxo-deoxyguanosine (8-oxoG), 3-BT, osteopontin, Kim-1, clusterin, and p-21 expression. TUNEL-staining in renal proximal tubules increased in a dose-related manner beginning at 11.5mg BrO3(-)/L in female rats and 46 mg/L in males. Increased 8-oxoG staining was observed at doses as low as 46 mg/L. Osteopontin expression also increased in a dose-related manner after treatment with 46 mg/L, in males only. In contrast, Kim-1 expression increased in a dose-related manner in both sexes, although to a greater extent in females at the highest dose. Clusterin and p21 expression also increased in a dose-related manner in both sexes. The expression of 3-BT-modified proteins only increased in male rats, following a pattern previously reported for accumulation of α-2u-globulin. Increases in apoptosis in renal proximal tubules of male and female rats at the lowest doses suggest a common mode of action for renal carcinogenesis for the two sexes that is independent of α-2u-globulin nephropathy.
Assuntos
Apoptose/efeitos dos fármacos , Bromatos/toxicidade , Carcinógenos Ambientais/toxicidade , Túbulos Renais Proximais/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Tirosina/análogos & derivados , Poluentes Químicos da Água/toxicidade , Administração Oral , Animais , Relação Dose-Resposta a Droga , Feminino , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Ratos , Ratos Endogâmicos F344 , Caracteres Sexuais , Tirosina/biossínteseRESUMO
Bromate (BrO3(-)), a by-product of ozonation of drinking water, induces nephrotoxicity in male rats at much lower doses than in female rats. This difference appears to be related to the development of α-2u-globulin nephropathy in males. To determine sex-dependent changes in mRNA and protein expression in the renal cortex attributable to α-2u-globulin nephropathy, we performed microarray and immunohistochemical analyses in proximal renal tubules of male and female F344 rats treated with KBrO3 for 28 days. Particular attention was paid to molecular biomarkers of renal tubular injury. Microarray analysis of male and female rats treated with BrO3(-) at low doses (125 mg/L KBrO3) displayed marked sex-dependent changes in renal gene expression. The greatest differences were seen in genes encoding for cellular differentiation, apoptosis, ion transport, and cell proliferation. Differences by sex were especially prominent for the cell cycle checkpoint gene p21, the renal injury protein Kim-1, and the kidney injury and cancer biomarker protein osteopontin. Dose-related nephrotoxicity, assessed by hematoxylin and eosin staining, was greater in males compared to female rats, as was cellular proliferation, assessed by bromodeoxyuridine staining. The fraction of proximal renal cells with elevated 8-oxodeoxyguanosine (8-OH-dG) was only increased at the high dose and did not differ by sex. Dose-dependent increases in the expression of osteopontin were detected immunohistochemically only in male rats and were localized in proximal tubule cells. Similarly, BrO3(-) treatment increased clusterin and Kim-1 staining in the proximal tubules; however, staining for these proteins did not differ appreciably between males and females. These data demonstrate both qualitative and quantitative differences in the response of male versus female kidneys to BrO3(-)-treatment. These sex-dependent effects likely contribute to renal carcinogenesis of BrO3(-) in the male rat.
Assuntos
Bromatos/toxicidade , Córtex Renal/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/biossíntese , 8-Hidroxi-2'-Desoxiguanosina , Animais , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Proliferação de Células/efeitos dos fármacos , Clusterina/biossíntese , Clusterina/genética , Desoxiguanosina/análogos & derivados , Feminino , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Córtex Renal/efeitos dos fármacos , Córtex Renal/patologia , Nefropatias/induzido quimicamente , Nefropatias/patologia , Túbulos Renais/patologia , Masculino , Análise em Microsséries , Proteína Oncogênica p21(ras)/biossíntese , Proteína Oncogênica p21(ras)/genética , Osteopontina/biossíntese , Osteopontina/genética , Reação em Cadeia da Polimerase , Ratos , Ratos Endogâmicos F344RESUMO
Alcohol-associated liver disease (ALD) represents a spectrum of histopathological changes, including alcoholic steatosis, steatohepatitis, and cirrhosis. One of the early responses to excessive alcohol consumption is lipid accumulation in the hepatocytes. Lipid ω-hydroxylation of medium- and long-chain fatty acid metabolized by the cytochrome P450 4A (CYP4A) family is an alternative pathway for fatty acid metabolism. The molecular mechanisms of CYP4A in ALD pathogenesis have not been elucidated. In this study, WT and Shp-/- mice were fed with a modified ethanol-binge, National Institute on Alcohol Abuse and Alcoholism model (10 days of ethanol feeding plus single binge). Liver tissues were collected every 6 hours for 24 hours and analyzed using RNA-Seq. The effects of REV-ERBα agonist (SR9009, 100 mg/kg/d) or CYP4A antagonist (HET0016, 5 mg/kg/d) in ethanol-fed mice were also evaluated. We found that hepatic Cyp4a10 and Cyp4a14 expression were significantly upregulated in WT mice, but not in Shp-/- mice, fed with ethanol. ChIP quantitative PCR and promoter assay revealed that REV-ERBα is the transcriptional repressor of Cyp4a10 and Cyp4a14. Rev-Erbα-/- hepatocytes had a marked induction of both Cyp4a genes and lipid accumulation. REV-ERBα agonist SR9009 or CYP4A antagonist HET0016 attenuated Cyp4a induction by ethanol and prevented alcohol-induced steatosis. Here, we have identified a role for the SHP/REV-ERBα/CYP4A axis in the pathogenesis of ALD. Our data also suggest REV-ERBα or CYP4A as the potential therapeutic targets for ALD.
Assuntos
Citocromo P-450 CYP4A/metabolismo , Ácidos Graxos/metabolismo , Hepatopatias Alcoólicas/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Amidinas , Animais , Citocromo P-450 CYP4A/antagonistas & inibidores , Modelos Animais de Doenças , Etanol/administração & dosagem , Etanol/efeitos adversos , Hepatócitos , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipidômica , Fígado/efeitos dos fármacos , Fígado/patologia , Hepatopatias Alcoólicas/tratamento farmacológico , Hepatopatias Alcoólicas/patologia , Masculino , Camundongos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Cultura Primária de Células , Pirrolidinas/administração & dosagem , RNA-Seq , Receptor EphB2 , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais/efeitos dos fármacos , Tiofenos/administração & dosagem , Regulação para CimaRESUMO
We have previously observed that a chronic drinking water exposure to monomethylarsonous acid [MMA(III)], a cellular metabolite of inorganic arsenic, increases tumor frequency in the skin of keratin VI/ornithine decarboxylase (K6/ODC) transgenic mice. To characterize gene expression profiles predictive of MMA(III) exposure and mode of action of carcinogenesis, skin and papilloma RNA was isolated from K6/ODC mice administered 0, 10, 50, and 100 ppm MMA(III) in their drinking water for 26 weeks. Following RNA processing, the resulting cRNA samples were hybridized to Affymetrix Mouse Genome 430A 2.0 GeneChips(R). Micoarray data were normalized using MAS 5.0 software, and statistically significant genes were determined using a regularized t-test. Significant changes in bZIP transcription factors, MAP kinase signaling, chromatin remodeling, and lipid metabolism gene transcripts were observed following MMA(III) exposure as determined using the Database for Annotation, Visualization and Integrated Discovery 2.1 (DAVID) (Dennis et al., Genome Biol 2003;4(5):P3). MMA(III) also caused dose-dependent changes in multiple Rho guanine nucleotide triphosphatase (GTPase) and cell cycle related genes as determined by linear regression analyses. Observed increases in transcript abundance of Fosl1, Myc, and Rac1 oncogenes in mouse skin support previous reports on the inducibility of these oncogenes in response to arsenic and support the relevance of these genomic changes in skin tumor induction in the K6/ODC mouse model.
Assuntos
Perfilação da Expressão Gênica , Queratina-6/fisiologia , Oncogenes , Compostos Organometálicos/toxicidade , Ornitina Descarboxilase/fisiologia , Papiloma/induzido quimicamente , Neoplasias Cutâneas/induzido quimicamente , Pele/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Teorema de Bayes , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Feminino , Proteínas de Choque Térmico HSP90/genética , Modelos Lineares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Papiloma/genética , Análise de Componente Principal , Neoplasias Cutâneas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
The receptor for advanced glycation end products (RAGE), a cell membrane receptor, recognizes ligands produced by cigarette smoke (CS) and has been implicated in the pathogenesis of COPD. We demonstrate that deletion or pharmacologic inhibition of RAGE prevents development of CS-induced emphysema. To identify molecular pathways by which RAGE mediates smoking related lung injury we performed unbiased gene expression profiling of alveolar macrophages (AM) obtained from RAGE null and C57BL/6 WT mice exposed to CS for one week or four months. Pathway analysis of RNA expression identified a number of genes integral to the pathogenesis of COPD impacted by the absence of RAGE. Altered expression of antioxidant response genes and lung protein 4-HNE immunostaining suggest attenuated oxidative stress in the RAGE null mice despite comparable CS exposure and lung leukocyte burden as the WT mice. Reduced endoplasmic reticulum stress in response to CS exposure also was observed in the AM from RAGE null mice. These findings provide novel insight into the sources of oxidative stress, macrophage activation, and the pathogenesis of lung disease due to CS exposure.
Assuntos
Fumar Cigarros/efeitos adversos , Enfisema/fisiopatologia , Pulmão/patologia , Ativação de Macrófagos , Macrófagos Alveolares/imunologia , Estresse Oxidativo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor para Produtos Finais de Glicação Avançada/deficiência , Fumaça/efeitos adversosRESUMO
OBJECTIVES: Sessile serrated adenoma/polyps (SSA/Ps) contribute up to 30% of all colon cancers. There is considerable histological overlap between SSA/Ps and hyperplastic polyps. Inadequate consensus exists among pathologists, and no molecular biomarkers exist to differentiate these lesions with high accuracy. Lack of reliable diagnosis adversely affects clinical care. We previously defined a novel 7-gene panel by RNA sequencing that differentiates SSA/Ps from hyperplastic polyps. Here, we use the 7-gene panel as a molecular approach to differentiate SSA/Ps and HPs with higher sensitivity and specificity in a large sample set from a tertiary health care center. METHODS: Reverse transcription quantitative polymerase chain reaction of the 7-gene panel was performed on 223 formalin-fixed, paraffin-embedded serrated polyp and normal colon samples. We compare the sensitivity and specificity of the 7-gene panel with the BRAF and KRAS mutation incidence in differentiating SSA/Ps and HPs. We also evaluate the clinical data of patients with SSA/Ps showing high and low expression of the gene panel. RESULTS: The 7-gene RNA expression panel differentiates SSA/Ps and HPs with 89.2% sensitivity and 88.4% specificity. The gene panel outperforms BRAF mutation in identification of SSA/Ps. Clinical data suggest that expression of the 7-gene panel correlates with the development of SSA/Ps in the future. DISCUSSION: This study describes a novel 7-gene panel that identifies SSA/Ps with improved accuracy. Our data show that RNA markers of SSA/Ps advance the distinction of serrated lesions and contribute to the study of the serrated pathway to colon cancer.
Assuntos
Adenoma/diagnóstico , Neoplasias do Colo/prevenção & controle , Pólipos do Colo/diagnóstico , Perfilação da Expressão Gênica , Adenoma/genética , Biomarcadores/análise , Neoplasias do Colo/genética , Pólipos do Colo/genética , Colonoscopia , Análise Mutacional de DNA , Diagnóstico Diferencial , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
To identify gene expression biomarkers and pathways targeted by sulindac and erlotinib given in a chemoprevention trial with a significant decrease in duodenal polyp burden at 6 months (P < 0.001) in familial adenomatous polyposis (FAP) patients, we biopsied normal and polyp duodenal tissues from patients on drug versus placebo and analyzed the RNA expression. RNA sequencing was performed on biopsies from the duodenum of FAP patients obtained at baseline and 6-month endpoint endoscopy. Ten FAP patients on placebo and 10 on sulindac and erlotinib were selected for analysis. Purity of biopsied polyp tissue was calculated from RNA expression data. RNAs differentially expressed between endpoint polyp and paired baseline normal were determined for each group and mapped to biological pathways. Key genes in candidate pathways were further validated by quantitative RT-PCR. RNA expression analyses of endpoint polyp compared with paired baseline normal for patients on placebo and drug show that pathways activated in polyp growth and proliferation are blocked by this drug combination. Directly comparing polyp gene expression between patients on drug and placebo also identified innate immune response genes (IL12 and IFNγ) preferentially expressed in patients on drug. Gene expression analyses from tissue obtained at endpoint of the trial demonstrated inhibition of the cancer pathways COX2/PGE2, EGFR, and WNT. These findings provide molecular evidence that the drug combination of sulindac and erlotinib reached the intended tissue and was on target for the predicted pathways. Furthermore, activation of innate immune pathways from patients on drug may have contributed to polyp regression. Cancer Prev Res; 11(1); 4-15. ©2017 AACRSee related editorial by Shureiqi, p. 1.
Assuntos
Polipose Adenomatosa do Colo/prevenção & controle , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Ciclo-Oxigenase 1/química , Neoplasias Duodenais/prevenção & controle , RNA Mensageiro/genética , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Adulto , Neoplasias Duodenais/genética , Neoplasias Duodenais/patologia , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib/administração & dosagem , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Sulindaco/administração & dosagem , Adulto JovemRESUMO
Chronic arsenic exposure in humans is associated with cancers of the skin, lung, bladder and other tissues. There is evidence that folate deficiency may increase susceptibility to arsenic effects, including skin lesions. K6/ODC mice develop skin tumors when exposed to 10ppm sodium arsenite for 5 months. In the current study, K6/ODC mice maintained on either a folate deficient or folate sufficient diet were exposed to 0, 1, or 10ppm sodium arsenite in the drinking water for 30 days. Total RNA was isolated from skin samples and gene expression analyzed using Affymetrix Mouse 430 2.0 GeneChips. Data from 24 samples, with 4 mice in each of the 6 treatment groups, were RMA normalized and analyzed by two-way ANOVA using GeneSpring. Top gene ontology (GO) categories for genes responding significantly to both arsenic treatment and folate deficiency include nucleotide metabolism and cell organization and biogenesis. For many of these genes, folate deficiency magnifies the response to arsenic treatment. In particular, expression of markers of epidermal differentiation, e.g., loricrin, small proline rich proteins and involucrin, was significantly reduced by arsenic in the folate sufficient animals, and reduced further or at a lower arsenic dose in the folate deficient animals. In addition, expression of a number of epidermal cell growth/proliferation genes and cellular movement genes was altered. These results indicate that arsenic disrupts the normal balance of cell proliferation and differentiation, and that folate deficiency exacerbates these effects, consistent with the view that folate deficiency is a nutritional susceptibility factor for arsenic-induced skin tumorigenesis.
Assuntos
Arsenitos/toxicidade , Carcinógenos Ambientais/toxicidade , Diferenciação Celular/efeitos dos fármacos , Epiderme/efeitos dos fármacos , Deficiência de Ácido Fólico/genética , Expressão Gênica/efeitos dos fármacos , Compostos de Sódio/toxicidade , Animais , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Epiderme/metabolismo , Feminino , Ácido Fólico/administração & dosagem , Ácido Fólico/sangue , Deficiência de Ácido Fólico/metabolismo , Perfilação da Expressão Gênica , Homocisteína/sangue , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Pele/efeitos dos fármacos , Pele/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Sessile serrated colon adenoma/polyps (SSA/P) are found during routine screening colonoscopy and may account for 20% to 30% of colon cancers. However, differentiating SSA/Ps from hyperplastic polyps (HP) with little risk of cancer is challenging and complementary molecular markers are needed. In addition, the molecular mechanisms of colon cancer development from SSA/Ps are poorly understood. RNA sequencing (RNA-Seq) was performed on 21 SSA/Ps, 10 HPs, 10 adenomas, 21 uninvolved colon, and 20 control colon specimens. Differential expression and leave-one-out cross-validation methods were used to define a unique gene signature of SSA/Ps. Our SSA/P gene signature was evaluated in colon cancer RNA-Seq data from The Cancer Genome Atlas (TCGA) to identify a subtype of colon cancers that may develop from SSA/Ps. A total of 1,422 differentially expressed genes were found in SSA/Ps relative to controls. Serrated polyposis syndrome (n = 12) and sporadic SSA/Ps (n = 9) exhibited almost complete (96%) gene overlap. A 51-gene panel in SSA/P showed similar expression in a subset of TCGA colon cancers with high microsatellite instability. A smaller 7-gene panel showed high sensitivity and specificity in identifying BRAF-mutant, CpG island methylator phenotype high, and MLH1-silenced colon cancers. We describe a unique gene signature in SSA/Ps that identifies a subset of colon cancers likely to develop through the serrated pathway. These gene panels may be utilized for improved differentiation of SSA/Ps from HPs and provide insights into novel molecular pathways altered in colon cancer arising from the serrated pathway. Cancer Prev Res; 9(6); 456-65. ©2016 AACR.
Assuntos
Neoplasias do Colo/genética , Pólipos do Colo/genética , Transcriptoma , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Adulto JovemRESUMO
Organic iodides have been shown to induce thyroid hypertrophy and increase alterations in colloid in rats, although the mechanism involved in this toxicity is unclear. To evaluate the effect that free iodide has on thyroid toxicity, we exposed rats for 2 weeks by daily gavage to sodium iodide (NaI). To compare the effects of compounds with alternative mechanisms (increased thyroid hormone metabolism and decreased thyroid hormone synthesis, respectively), we also examined phenobarbital (PB) and propylthiouracil (PTU) as model thyroid toxicants. Follicular cell hypertrophy and pale-staining colloid were present in thyroid glands from PB-treated rats, and more severe hypertrophy/colloid changes along with diffuse hyperplasia were present in thyroid glands from PTU-treated rats. In PB- and PTU-treated rats, thyroid-stimulating hormone (TSH) levels were significantly elevated, and both thyroxine and triiodothyronine hormone levels were significantly decreased. PB induced hepatic uridine diphosphate-glucuronyltransferase (UDPGT) activity almost 2-fold, whereas PTU reduced hepatic 5 -deiodinase I (5 -DI) activity to < 10% of control in support of previous reports regarding the mechanism of action of each chemical. NaI also significantly altered liver weights and UDPGT activity but did not affect thyroid hormone levels or thyroid pathology. Thyroid gene expression analyses using Affymetrix U34A GeneChips, a regularized t-test, and Gene Map Annotator and Pathway Profiler demonstrated significant changes in rhodopsin-like G-protein-coupled receptor transcripts from all chemicals tested. NaI demonstrated dose-dependent changes in multiple oxidative stress-related genes, as also determined by principal component and linear regression analyses. Differential transcript profiles, possibly relevant to rodent follicular cell tumor outcomes, were observed in rats exposed to PB and PTU, including genes involved in Wnt signaling and ribosomal protein expression.
Assuntos
Perfilação da Expressão Gênica , Iodetos/toxicidade , Glândula Tireoide/efeitos dos fármacos , Animais , Antitireóideos/farmacologia , Glucuronosiltransferase/metabolismo , Iodeto Peroxidase/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Masculino , Tamanho do Órgão , Fenobarbital/farmacologia , Propiltiouracila/farmacologia , Ratos , Glândula Tireoide/enzimologia , Glândula Tireoide/metabolismo , Hormônios Tireóideos/metabolismoRESUMO
BACKGROUND: Sessile serrated adenomas/polyps (SSA/Ps) may account for 20-30% of colon cancers. Although large SSA/Ps are generally recognized phenotypically, small (<1 cm) or dysplastic SSA/Ps are difficult to differentiate from hyperplastic or small adenomatous polyps by endoscopy and histopathology. Our aim was to define the comprehensive gene expression phenotype of SSA/Ps to better define this cancer precursor. RESULTS: RNA sequencing was performed on 5' capped RNA from seven SSA/Ps collected from patients with the serrated polyposis syndrome (SPS) versus eight controls. Highly expressed genes were analyzed by qPCR in additional SSA/Ps, adenomas and controls. The cellular localization and level of gene products were examined by immunohistochemistry in syndromic and sporadic SSA/Ps, adenomatous and hyperplastic polyps and controls. We identified 1,294 differentially expressed annotated genes, with 106 increased ≥10-fold, in SSA/Ps compared to controls. Comparing these genes with an array dataset for adenomatous polyps identified 30 protein coding genes uniquely expressed ≥10-fold in SSA/Ps. Biological pathways altered in SSA/Ps included mucosal integrity, cell adhesion, and cell development. Marked increased expression of MUC17, the cell junction protein genes VSIG1 and GJB5, and the antiapoptotic gene REG4 were found in SSA/Ps, relative to controls and adenomas, were verified by qPCR analysis of additional SSA/Ps (nâ=â21) and adenomas (nâ=â10). Immunohistochemical staining of syndromic (n≥11) and sporadic SSA/Ps (n≥17), adenomatous (n≥13) and hyperplastic (n≥10) polyps plus controls (n≥16) identified unique expression patterns for VSIG1 and MUC17 in SSA/Ps. CONCLUSION: A subset of genes and pathways are uniquely increased in SSA/Ps, compared to adenomatous polyps, thus supporting the concept that cancer develops by different pathways in these phenotypically distinct polyps with markedly different gene expression profiles. Immunostaining for a subset of these genes differentiates both syndromic and sporadic SSA/Ps from adenomatous and hyperplastic polyps.
Assuntos
Adenoma/genética , Adenoma/patologia , Pólipos do Colo/genética , Pólipos do Colo/patologia , Análise de Sequência de RNA/métodos , Adenoma/metabolismo , Idoso , Antígenos de Neoplasias/metabolismo , Análise por Conglomerados , Colo/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Colonoscopia , Conexinas/metabolismo , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Lectinas Tipo C/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Mucinas/metabolismo , Proteínas Associadas a Pancreatite , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
We describe the first report of RNA sequencing of 5' capped (Pol II) RNAs isolated from acutely hepatitis C virus (HCV) infected Huh 7.5 cells that provides a general approach to identifying differentially expressed annotated and unannotated genes that participate in viral-host interactions. We identified 100, 684, and 1,844 significantly differentially expressed annotated genes in acutely infected proliferative Huh 7.5 cells at 6, 48, and 72 hours, respectively (fold change ≥ 1.5 and Bonferroni adjusted p-values < 0.05). Most of the differentially expressed genes (>80%) and biological pathways (such as adipocytokine, Notch, Hedgehog and NOD-like receptor signaling) were not identified by previous gene array studies. These genes are critical components of host immune, inflammatory and oncogenic pathways and provide new information regarding changes that may benefit the virus or mediate HCV induced pathology. RNAi knockdown studies of newly identified highly upregulated FUT1 and KLHDC7B genes provide evidence that their gene products regulate and facilitate HCV replication in hepatocytes. Our approach also identified novel Pol II unannotated transcripts that were upregulated. Results further identify new pathways that regulate HCV replication in hepatocytes and suggest that our approach will have general applications in studying viral-host interactions in model systems and clinical biospecimens.
Assuntos
Perfilação da Expressão Gênica , Hepacivirus/patogenicidade , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Capuzes de RNA/química , Linhagem Celular , Humanos , Análise de Sequência de RNA , Fatores de Tempo , Replicação ViralRESUMO
In a previously developed mouse model, arsenic exposure in utero induces tumors at multiple sites in the offspring as adults, often duplicating human targets. However, human environmental inorganic arsenic exposure occurs during the entire life span, not just part of gestation. Thus, "whole-life" inorganic arsenic carcinogenesis in mice was studied. CD1 mice were exposed to 0, 6, 12, or 24 ppm arsenic in the drinking water 2 weeks prior to breeding, during pregnancy, lactation, and after weaning through adulthood. Tumors were assessed in offspring until 2 years of age. Arsenic induced dose-related increases in lung adenocarcinoma (both sexes), hepatocellular carcinoma (both sexes), gallbladder tumors (males), and uterine carcinomas. Arsenic induced dose-related increases in ovarian tumors (including carcinomas) starting with the lowest dose. Adrenal tumors increased at all doses (both sexes). Arsenic-induced lung and liver cancers were highly enriched for cancer stem cells, consistent with prior work with skin cancers stimulated by prenatal arsenic. Reproductive tract tumors overexpressed cyclooxygenase-2 and estrogen receptor-α. Arsenic target sites were remarkably similar to prior transplacental studies, although tumors from whole-life exposure were generally more aggressive and frequent. This may indicate that arsenic-induced events in utero dictate target site in some tissues, whereas other exposure periods of arsenic enhance incidence or progression, though other factors could be at play, like cumulative dose. Whole-life arsenic exposure induced tumors at dramatically lower external doses than in utero arsenic only while more realistically duplicating human exposure.
Assuntos
Arsenitos/toxicidade , Carcinógenos Ambientais/toxicidade , Exposição Materna/efeitos adversos , Neoplasias Experimentais/induzido quimicamente , Compostos de Sódio/toxicidade , Animais , Testes de Carcinogenicidade , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Especificidade de Órgãos , Gravidez , Fatores de TempoRESUMO
Microarray studies of chronic hepatitis C infection have provided valuable information regarding the host response to viral infection. However, recent studies of the human transcriptome indicate pervasive transcription in previously unannotated regions of the genome and that many RNA transcripts have short or lack 3' poly(A) ends. We hypothesized that using ENCODE tiling arrays (1% of the genome) in combination with affinity purifying Pol II RNAs by their unique 5' m7GpppN cap would identify previously undescribed annotated and unannotated genes that are differentially expressed in liver during hepatitis C virus (HCV) infection. Both 5'-capped and poly(A)+ populations of RNA were analyzed using ENCODE tiling arrays. Sixty-four annotated genes were significantly increased in HCV cirrhotic as compared to control liver; twenty-seven (42%) of these genes were identified only by analyzing 5' capped RNA. Thirty-one annotated genes were significantly decreased; sixteen (50%) of these were identified only by analyzing 5' capped RNA. Bioinformatic analysis showed that capped RNA produced more consistent results, provided a more extensive expression profile of intronic regions and identified upregulated Pol II transcriptionally active regions in unannotated areas of the genome in HCV cirrhotic liver. Two of these regions were verified by PCR and RACE analysis. qPCR analysis of liver biopsy specimens demonstrated that these unannotated transcripts, as well as IRF1, TRIM22 and MET, were also upregulated in hepatitis C with mild inflammation and no fibrosis. The analysis of 5' capped RNA in combination with ENCODE tiling arrays provides additional gene expression information and identifies novel upregulated Pol II transcripts not previously described in HCV infected liver. This approach, particularly when combined with new RNA sequencing technologies, should also be useful in further defining Pol II transcripts differentially regulated in specific disease states and in studying RNAs regulated by changes in pre-mRNA splicing or 3' polyadenylation status.