RESUMO
Sex-specific discrepancies in bladder cancer (BCa) are reported, and new studies imply that microbiome may partially explain the diversity. We aim to provide characterization of the bladder microbiome in both sexes diagnosed with non-muscle-invasive BCa with specific insight into cancer grade. In our study, 16S rRNA next-generation sequencing was performed on midstream urine, bladder tumor sample, and healthy-appearing bladder mucosa. Bacterial DNA was isolated using QIAamp Viral RNA Mini Kit. Metagenomic analysis was performed using hypervariable fragments of the 16S rRNA gene on Ion Torrent Personal Genome Machine platform. Of 41 sample triplets, 2153 taxa were discovered: 1739 in tumor samples, 1801 in healthy-appearing bladder mucosa and 1370 in midstream urine. Women were found to have smaller taxa richness in Chao1 index than men (p = 0.03). In comparison to low-grade tumors, patients with high-grade lesions had lower bacterial diversity and richness in urine. Significant differences between sexes in relative abundance of communities at family level were only observed in high-grade tumors.
RESUMO
BMP6 is an iron-sensing cytokine whose transcription in liver sinusoidal endothelial cells (LSECs) is enhanced by high iron levels, a step that precedes the induction of the iron-regulatory hormone hepcidin. While several reports suggested a cell-autonomous induction of Bmp6 by iron-triggered signals, likely via sensing of oxidative stress by the transcription factor NRF2, other studies proposed the dominant role of a paracrine yet unidentified signal released by iron-loaded hepatocytes. To further explore the mechanisms of Bmp6 transcriptional regulation, we used female mice aged 10-11 months, which are characterized by hepatocytic but not LSEC iron accumulation, and no evidence of systemic iron overload. We found that LSECs of aged mice exhibit increased Bmp6 mRNA levels as compared to young controls, but do not show a transcriptional signature characteristic of activated NFR2-mediated signaling in FACS-sorted LSECs. We further observed that primary murine LSECs derived from both wild-type and NRF2 knock-out mice induce Bmp6 expression in response to iron exposure. By analyzing transcriptomic data of FACS-sorted LSECs from aged versus young mice, as well as early after iron citrate injections, we identified ETS1 as a candidate transcription factor involved in Bmp6 transcriptional regulation. By performing siRNA-mediated knockdown, small-molecule treatments, and chromatin immunoprecipitation in primary LSECs, we show that Bmp6 transcription is regulated by iron via ETS1 and p38/JNK MAP kinase-mediated signaling, at least in part independently of NRF2. Thereby, these findings identify the new components of LSEC iron sensing machinery broadly associated with cellular stress responses.
Assuntos
Células Endoteliais , Ferro , Feminino , Camundongos , Animais , Ferro/metabolismo , Células Endoteliais/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Hepcidinas/genética , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos Knockout , Proteína Morfogenética Óssea 6/genéticaRESUMO
The key association between gut dysbiosis and cancer is already known. Here, we used whole-genome shotgun sequencing (WGS) and gas chromatography/mass spectrometry (GC/MS) to conduct metagenomic and metabolomic analyses to identify common and distinct taxonomic configurations among 40, 45, 71, 34, 50, 60, and 40 patients with colorectal cancer, stomach cancer, breast cancer, lung cancer, melanoma, lymphoid neoplasms and acute myeloid leukemia (AML), respectively, and compared the data with those from sex- and age-matched healthy controls (HC). α-diversity differed only between the lymphoid neoplasm and AML groups and their respective HC, while ß-diversity differed between all groups and their HC. Of 203 unique species, 179 and 24 were under- and over-represented, respectively, in the case groups compared with HC. Of these, Faecalibacillus intestinalis was under-represented in each of the seven groups studied, Anaerostipes hadrus was under-represented in all but the stomach cancer group, and 22 species were under-represented in the remaining five case groups. There was a marked reduction in the gut microbiome cancer index in all case groups except the AML group. Of the short-chain fatty acids and amino acids tested, the relative concentration of formic acid was significantly higher in each of the case groups than in HC, and the abundance of seven species of Faecalibacterium correlated negatively with most amino acids and formic acid, and positively with the levels of acetic, propanoic, and butanoic acid. We found more differences than similarities between the studied malignancy groups, with large variations in diversity, taxonomic/metabolomic profiles, and functional assignments. While the results obtained may demonstrate trends rather than objective differences that correlate with different types of malignancy, the newly developed gut microbiota cancer index did distinguish most of the cancer cases from HC. We believe that these data are a promising step forward in the search for new diagnostic and predictive tests to assess intestinal dysbiosis among cancer patients.
Assuntos
Fezes , Formiatos , Microbioma Gastrointestinal , Humanos , Feminino , Fezes/microbiologia , Masculino , Formiatos/metabolismo , Pessoa de Meia-Idade , Idoso , Neoplasias/metabolismo , Neoplasias/microbiologia , Adulto , Disbiose/microbiologia , Metabolômica/métodos , Metaboloma , Cromatografia Gasosa-Espectrometria de Massas , Metagenômica/métodosRESUMO
BACKGROUND: Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient. RESULTS: To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis. CONCLUSIONS: RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.
Assuntos
Fenóis , RNA , Animais , Camundongos , Humanos , Células Cultivadas , Manejo de EspécimesRESUMO
BACKGROUND: Fetal blood group (BG) and platelet (HPA) antigens may trigger maternal immunization, causing a fetal disease. Noninvasive prenatal diagnostics (NIPT) predicts fetal genotype, identifying pregnancies with no risk. All current techniques detect fetal antigen alleles with unspecific background and without estimation of fetal fraction, thus new protocols for detection of fetal BG/HPA alleles with ultrahigh sensitivity still need to be tested to improve NIPT. AIM: To design NIPT of clinically important antigens using Ion AmpliSeq HD technology. METHODS: Plasma DNA from 36 pregnant women (9-33 week of gestation, 24 immunized with anti-HPA-1a,-3b,-15a, -K, or -D+C+S), with known BG/HPA genotypes of their neonates/partners, was tested on Ion S5 System using the Ion AmpliSeq HD designer custom gene panel. NGS contained 25 rs-targets encoding relevant BG/HPA antigens and 10 markers. RESULTS: Using the NGS protocol, 76 out of 85 differences in fetal/maternal BG/HPA genotypes were determined in concentration above 2% fetal paternally inherited allele chimerism. The level of unspecific reads for BG/HPA alleles was below 0.87%. In 24 immunized women NGS revealed feto-maternal incompatibility in 11 cases (from 2.44% to 7.41%) and excluded in 10 (<0.05%), three cases had inconclusive results (1.79%, 0.19%, 0.11%). The presence of fetal DNA was confirmed in each case by detecting markers with at least 2% chimerism. CONCLUSION: The use of Ion AmpliSeq HD technology improves the prediction of feto-maternal incompatibility, increasing the sensitivity of BG/HPA NIPT and serving confirmation of the fetal DNA at the same workflow.
Assuntos
Antígenos de Plaquetas Humanas , Antígenos de Grupos Sanguíneos , Trombocitopenia Neonatal Aloimune , Antígenos de Grupos Sanguíneos/genética , DNA/genética , Feminino , Humanos , Recém-Nascido , Gravidez , TecnologiaRESUMO
Several single nucleotide polymorphisms (SNPs) associated with susceptibility to Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL) have been identified. The aim of this study was to identify susceptibility loci for HL and DLBCL in Polish patients. Altogether, DLBCL (n = 218 and HL patients (n = 224) and healthy individuals (n = 1181) were recruited. Lymphoma diagnosis was based on standard criteria. Genome-wide association study (GWAS) was performed using pooled-DNA samples on llumina Infinium Omni2.5 Exome-8 v1.3, and selected loci were replicated by TaqMan SNP genotyping of individuals. GWAS detected thirteen and seven SNPs associated with DLBCL and HL, respectively. In the replication study, six and seven SNPs reached significance after correction for multiple testing in the DLBCL and HL cohorts, respectively. One and four SNPs associated with DLBCL and HL, respectively, were localized within, and two SNPs-near the major histocompatibility complex (MHC) region. In conclusion, the majority of loci associated with HL and DLBCL aetiology in previous studies have potential roles in immune function. Our pooled-DNA GWAS enabled the identification of several susceptibility loci for DLBCL and HL in the Polish population; some of them were mapped within or adjacent to the MHC, and other associated SNPs were located outside the MHC.
Assuntos
Estudo de Associação Genômica Ampla , Linfoma , DNA , Predisposição Genética para Doença , Humanos , Linfoma/genética , Polônia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Clostridium difficile (C. difficile) is a major source of healthcare-associated infection with a high risk of recurrence, attributable to many factors such as usage of antibiotics, older age and immunocompromised status of the patients. C. difficile has also a highly diverse genome, which may contribute to its high virulence. Herein we examined whether the genome conservation, measured as non-synonymous to synonymous mutations ratio (dN/dS) in core genes, presence of single genes, plasmids and prophages increased the risk of reinfection in a subset of 134 C. difficile isolates from our previous study in a singly hemato-oncology ward. METHODS: C. difficile isolates were subjected to whole-genome sequencing (WGS) on Ion Torrent PGM sequencer. Genomes were assembled with MIRA5 and annotated with prokka and VRprofile. Logistic regression was used to asses the relationship between single gene presence and the odds of infection recurrence. DN/dS ratios were computed with codeml. Functional annotation was conducted with eggNOG-Mapper. RESULTS: We have found that the presence of certain genes, associated with carbon metabolism and oxidative phosphorylation, increased the odds of infection recurrence. More core genes were under positive selective pressure in recurrent disease isolates - they were mostly associated with the metabolism of aminoacids. Finally, prophage elements were more prevalent in single infection isolates and plasmids did not influence the odds of recurrence. CONCLUSIONS: Our findings suggest higher genetic plasticity in isolates causing recurrent infection, associated mainly with metabolism. On the other hand, the presence of prophages seems to reduce the isolates' virulence.
Assuntos
Clostridioides difficile/genética , Variação Genética , Genoma Bacteriano/genética , Redes e Vias Metabólicas/genética , Reinfecção/microbiologia , Aminoácidos/metabolismo , Carbono/metabolismo , Clostridioides difficile/classificação , Infecções por Clostridium/microbiologia , Infecção Hospitalar/microbiologia , Humanos , Fosforilação Oxidativa , Prófagos/genética , Estudos Retrospectivos , Virulência , Sequenciamento Completo do GenomaRESUMO
Obesity is considered a serious chronic disease, associated with an increased risk of developing cardiovascular diseases, non-alcoholic fatty liver disease and type 2 diabetes. Monocyte chemoattractant protein-1-induced protein-1 (MCPIP1) is an RNase decreasing stability of transcripts coding for inflammation-related proteins. In addition, MCPIP1 plays an important role in the regulation of adipogenesis in vitro by reducing the expression of key transcription factors, including C/EBPß. To elucidate the role of MCPIP1 in adipocyte biology, we performed RNA-Seq and proteome analysis in 3T3-L1 adipocytes overexpressing wild-type (WTMCPIP1) and the mutant form of MCPIP1 protein (D141NMCPIP1). Our RNA-Seq analysis followed by confirmatory Q-RT-PCR revealed that elevated MCPIP1 levels in 3T3-L1 adipocytes upregulated transcripts encoding proteins involved in signal transmission and cellular remodeling and downregulated transcripts of factors involved in metabolism. These data are consistent with our proteomic analysis, which showed that MCPIP1 expressing adipocytes exhibit upregulation of proteins involved in cellular organization and movement and decreased levels of proteins involved in lipid and carbohydrate metabolism. Moreover, MCPIP1 adipocytes are characterized by decreased level of insulin receptor, reduced insulin-induced Akt phosphorylation, as well as depleted Glut4 level and impaired glucose uptake. Overexpression of Glut4 in 3T3-L1 cells expressed WTMCPIP1 rescued adipogenesis. Interestingly, we found decreased level of MCPIP1 along with an increase in body mass index in subcutaneous adipose tissue. The presented data show a novel role of MCPIP1 in modulating insulin sensitivity in adipocytes. Overall, our findings demonstrate that MCPIP1 is an important regulator of adipogenesis and adipocyte metabolism.
Assuntos
Adipócitos/metabolismo , Adipogenia , Genômica , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Adulto , Animais , Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Insulina/farmacologia , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Mutação/genética , Obesidade/metabolismo , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Insulina/metabolismo , Ribonucleases/genética , Transdução de Sinais/efeitos dos fármacos , Magreza/metabolismo , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
BACKGROUND: Thyroid carcinoma (TC) is the most common endocrine system malignancy, and papillary thyroid carcinoma (PTC) accounts for >80% of all TC cases. Nevertheless, PTC pathogenesis is still not fully understood. The aim of the study was to elucidate the role of the FRMD5 protein in the regulation of biological pathways associated with the development of PTC. We imply that the presence of certain genetic aberrations (e.g., BRAF V600E mutation) is associated with the activity of FRMD5. METHODS: The studies were conducted on TPC1 and BCPAP (BRAF V600E) model PTC-derived cells. Transfection with siRNA was used to deplete the expression of FRMD5. The mRNA expression and protein yield were evaluated using RT-qPCR and Western blot techniques. Proliferation, migration, invasiveness, adhesion, spheroid formation, and survival tests were performed. RNA sequencing and phospho-kinase proteome profiling were used to assess signaling pathways associated with the FRMD5 expressional status. RESULTS: The obtained data indicate that the expression of FRMD5 is significantly enhanced in BRAF V600E tumor specimens and cells. It was observed that a drop in intracellular yield of FRMD5 results in significant alternations in the migration, invasiveness, adhesion, and spheroid formation potential of PTC-derived cells. Importantly, significant divergences in the effect of FRMD5 depletion in both BRAF-wt and BRAF-mutated PTC cells were observed. It was also found that knockdown of FRMD5 significantly alters the expression of multidrug resistant genes. CONCLUSIONS: This is the first report highlighting the importance of the FRMD5 protein in the biology of PTCs. The results suggest that the FRMD5 protein can play an important role in controlling the metastatic potential and multidrug resistance of thyroid tumor cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Proteínas Supressoras de Tumor/genética , Apoptose/genética , Estudos de Casos e Controles , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Esferoides Celulares/patologia , Câncer Papilífero da Tireoide/tratamento farmacológico , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
TNF-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein capable of selectively inducing apoptosis in cancer cells by binding to its cognate receptors. Here, we examined the anticancer efficacy of a recently developed chimeric AD-O51.4 protein, a TRAIL fused to the VEGFA-originating peptide. We tested AD-O51.4 protein activity against human colorectal cancer (CRC) models and investigated the resistance mechanism in the non-responsive CRC models. The quantitative comparison of apoptotic activity between AD-O51.4 and the native TRAIL in nine human colorectal cancer cell lines revealed dose-dependent toxicity in seven of them; the immunofluorescence-captured receptor abundance correlated with the extent of apoptosis. AD-O51.4 reduced the growth of CRC patient-derived xenografts (PDXs) with good efficacy. Cell lines that acquired AD-O51.4 resistance showed a significant decrease in surface TRAIL receptor expression and apoptosis-related proteins, including Caspase-8, HSP60, and p53. These results demonstrate the effectiveness of AD-O51.4 protein in CRC preclinical models and identify the potential mechanism underlying acquired resistance. Progression of AD-O51.4 to clinical trials is expected.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Camundongos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/genética , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.
Assuntos
Anti-Inflamatórios/farmacologia , Endotoxemia/tratamento farmacológico , Indazóis/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Piperazinas/farmacologia , Piridinas/farmacologia , Pirrolidinas/farmacologia , Triazóis/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Linhagem Celular , Quimiocina CCL2/sangue , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Reposicionamento de Medicamentos , Endotoxemia/mortalidade , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ativação Plaquetária/efeitos dos fármacos , Células RAW 264.7 , Transcriptoma/genéticaRESUMO
INTRODUCTION: Ewing sarcoma (ES) is a highly aggressive malignancy of bone and soft tissues characterized by the presence of a genetic fusion involving the EWSR1 gene. More than one-third of patients develop distant metastases, which are associated with unfavorable prognosis. Knowledge about the disease's genetic landscape may help foster progress in using targeted therapies in the treatment of ES. AIM OF THE STUDY: The objective is to assess the mutational landscape of ES in pretreatment samples, tumor samples after neoadjuvant chemotherapy, and in metastatic/recurrent tumors in children and adults. MATERIAL AND METHODS: DNA from 39 formalin-fixed paraffin-embedded tumor samples of 22 patients (17 adults, 5 children) were analyzed by targeted next generation sequencing (NGS) using the Oncomine Comprehensive Assay v3gene panel. Additional functional analyses were performed between patient subgroups. RESULTS: All samples were characterized by low tumor mutation burden (< 10 mut/Mb). The most commonly mutated genes were PIK3R1 (59%) and POLE (50%). The most widely detected variants in biopsy samples were PIK3R1 T369I (50%), FGFR1 E159K, and TP53 at codon 72 (both in 27.3%). Additionally, the ATR,BRCA1, RAD50,ATM,CHEK1, and NBN genes showed a significantly higher number of mutations in ES. Mutations in PIK3R1 were significantly more frequent in adults, while mutations in the pathways responsible for cell cycle control, DNA repair, and transcriptional regulation were more frequent in children. CONCLUSIONS: Besides EWSR1 fusion, ES is characterized by numerous point mutations that are potential targets for precision medicine. There is high genomic heterogeneity that may explain differences in outcomes between patient subgroups.
RESUMO
The mitochondrial amidoxime-reducing component (MARC) is a mammalian molybdenum-containing enzyme. All annotated mammalian genomes harbor two MARC genes, MARC1 and MARC2, which share a high degree of sequence similarity. Both molybdoenzymes reduce a variety of N-hydroxylated compounds. Besides their role in N-reductive drug metabolism, only little is known about their physiological functions. In this study, we characterized an existing KO mouse model lacking the functional MARC2 gene and fed a high-fat diet and also performed in vivo and in vitro experiments to characterize reductase activity toward known MARC substrates. MARC2 KO significantly decreased reductase activity toward several N-oxygenated substrates, and for typical MARC substrates, only small residual reductive activity was still detectable in MARC2 KO mice. The residual detected reductase activity in MARC2 KO mice could be explained by MARC1 expression that was hardly unaffected by KO, and we found no evidence of significant activity of other reductase enzymes. These results clearly indicate that MARC2 is mainly responsible for N-reductive biotransformation in mice. Striking phenotypical features of MARC2 KO mice were lower body weight, increased body temperature, decreased levels of total cholesterol, and increased glucose levels, supporting previous findings that MARC2 affects energy pathways. Of note, the MARC2 KO mice were resistant to high-fat diet-induced obesity. We propose that the MARC2 KO mouse model could be a powerful tool for predicting MARC-mediated drug metabolism and further investigating MARC's roles in energy homeostasis.
Assuntos
Metabolismo Energético , Proteínas Mitocondriais/metabolismo , Obesidade/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/genética , Obesidade/etiologia , Obesidade/genética , OxirreduçãoRESUMO
BACKGROUND: Inflammatory bowel diseases are classic polygenic disorders, with genetic loads that reflect immunopathological processes in response to the intestinal microbiota. Herein we performed the multiomics analysis by combining the large scale surveys of gut bacterial community, stool microRNA (miRNA) and short chain fatty acid (SCFA) signatures to correlate their association with the activity of Crohn's disease (CD). METHODS: DNA, miRNA, and metabolites were extracted from stool samples of 15 CD patients, eight with active disease and seven in remission, and nine healthy individuals. Microbial, miRNA and SCFA profiles were assessed using datasets from 16S rRNA sequencing, Nanostring miRNA and GC-MS targeted analysis, respectively. RESULTS: Pairwise comparisons showed that 9 and 23 taxa differed between controls and CD patients with active and inactive disease, respectively. Six taxa were common to both comparisons, whereas four taxa differed in CD patients. α-Diversity was lower in both CD groups than in controls. The levels of 13 miRNAs differed (p-value < 0.05; FC > 1.5) in CD patients and controls before FDR correction and 4 after. Of six SCFAs, the levels of two differed significantly (p-value < 0.05, FC > 1.5) in CD patients and controls, and the levels of four differed in patients with active and inactive CD. PLS-DA revealed models with smallest error rate for controls in bacterial component and inactive disease in metabolites. CONCLUSION: A complex interrelationship may exist between gut dysbiosis, miRNA profiling and SCFA level in response to intestinal inflammation.
Assuntos
Doença de Crohn , MicroRNAs , Microbiota , Doença de Crohn/genética , Ácidos Graxos Voláteis , Fezes , Humanos , RNA Ribossômico 16S/genéticaRESUMO
Most pancreatic neuroendocrine tumors (PNETs) are indolent, while pancreatic ductal adenocarcinomas (PDACs) are particularly aggressive. To elucidate the basis for this difference and to establish the biomarkers, by using the deep sequencing, we analyzed somatic variants across coding regions of 409 cancer genes and measured mRNA/miRNA expression in nine PNETs, eight PDACs, and four intestinal neuroendocrine tumors (INETs). There were 153 unique somatic variants considered pathogenic or likely pathogenic, found in 50, 57, and 24 genes in PDACs, PNETs, and INETs, respectively. Ten and 11 genes contained a pathogenic mutation in at least one sample of all tumor types and in PDACs and PNETs, respectively, while 28, 34, and 11 genes were found to be mutated exclusively in PDACs, PNETs, and INETs, respectively. The mRNA and miRNA transcriptomes of PDACs and NETs were distinct: from 54 to 1659 differentially expressed mRNAs and from 117 to 250 differentially expressed miRNAs exhibited high discrimination ability and resulted in models with an area under the receiver operating characteristics curve (AUC-ROC) >0.9 for both miRNA and mRNA. Given the miRNAs high stability, we proposed exploring that class of RNA as new pancreatic tumor biomarkers.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/patologia , Neoplasias Hepáticas/secundário , MicroRNAs/genética , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/patologia , RNA Mensageiro/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Diferenciação Celular , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Prognóstico , RNA Mensageiro/genética , Neoplasias PancreáticasRESUMO
Adipogenesis is a process of preadipocyte differentiation that requires action of numerous factors. Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) possesses the N-terminus of the PilT protein (PilT N-terminus or PIN domain) that has RNase properties. This protein degrades transcripts coding for inflammation and differentiation - related proteins. Moreover, MCPIP1 is a broad suppressor of the miRNA biogenesis. We previously found that MCPIP1 degrades transcript encoding CCAAT/Enhancer Binding Protein Beta (C/EBPß) and influences adipogenesis. Subsequently, we aimed to determine adipocyte miRNA expression profile in differentiating mouse preadipocytes, 3T3-L1, by overexpressing MCPIP1. Using Next-Generation Sequencing (NSG) we showed that MCPIP1 overexpression results in modulated levels of 58 miRNAs in adipocytes on day 2 of differentiation. Among them, 30 miRNAs showed significantly reduced levels and 28 showed increased levels in comparison to control. Approximately one third of the modulated miRNAs were not previously reported to be involved in adipocytes differentiation. Our analysis revealed that 24 down-regulated and 23 up-regulated miRNAs (at least 1.5-fold) influence 19 signaling pathways that are important for adipogenesis. Furthermore, reduced miRNA levels result in the up-regulation of their targets. By using luciferase reporter assay, we demonstrated that miR-32-5p and miR-9-3p directly target the 3'UTR region of Mapk8 and Tiam1, respectively. In addition, activation of MAP kinases pathway (JNK and p38), proposed as being regulated by down-regulated miRNAs, was higher in WTMCPIP1 than in D141NMCPIP1 or control 3T3-L1 adipocytes. Our results indicate a considerable impact of MCPIP1 on miRNAs levels and its significance in adipogenesis.
Assuntos
Adipogenia/genética , MicroRNAs/genética , Ribonucleases/genética , Fatores de Transcrição/genética , Células 3T3-L1 , Adipócitos/fisiologia , Animais , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Análise em Microsséries , TransfecçãoRESUMO
BACKGROUND & AIMS: Wilson's disease (WD) is an autosomal recessive disorder associated with disease-causing alterations across the ATP7B gene, with highly variable symptoms and age of onset. We aimed to assess whether the clinical variability of WD relates to modifier genes. METHODS: A total of 248 WD patients were included, of whom 148 were diagnosed after age of 17. Human exome libraries were constructed using AmpliSeq technology and sequenced using the IonProton platform. RESULTS: ATP7B p.His1069Gln mutation was present in 215 patients, with 112 homozygotes and 103 heterozygotes. Three other mutations: p.Gln1351Ter, p.Trp779Ter and c.3402delC were identified in >10 patients. Among patients, 117 had a homozygous mutation, 101 were compound heterozygotes, 27 had one heterozygous mutation, and 3 other patients had no identifiable pathogenic variant of ATP7B. Sixteen mutations were novel, found as part of a compound mutation or as a sole, homozygous mutation. For disease phenotype prediction, age at diagnosis was a deciding factor, while frameshift allelic variants of ATP7B and being male increased the odds of developing a neurological phenotype. Rare allelic variants in ESD and INO80 increased and decreased chances for the neurological phenotype, respectively, while rare variants in APOE and MBD6 decreased the chances of WD early manifestation. Compound mutations contributed to earlier age of onset. CONCLUSIONS: In a Polish population, genetic screening for WD may help genotype for four variants (p.His1069Gln, p.Gln1351Ter, p.Trp779Ter and c.3402delC), with direct sequencing of all ATP7B amplicons as a second diagnostic step. We also identified some allelic variants that may modify a WD phenotype.
Assuntos
ATPases Transportadoras de Cobre/genética , Sequenciamento do Exoma , Degeneração Hepatolenticular/genética , Mutação , Adolescente , Adulto , Idade de Início , Alelos , Criança , Pré-Escolar , Feminino , Frequência do Gene , Testes Genéticos , Heterozigoto , Homozigoto , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Fenótipo , Polônia , Adulto JovemRESUMO
BACKGROUND: While histopathological evaluation remains the gold standard for diagnosis of prostate cancer (PCa), sampling errors remain a frequent problem; therefore, use of tissue biomarkers that can distinguish between benign and malignant prostate disease is a potentially beneficial diagnostic strategy. METHODS: Deep sequencing of the miRNA transcriptome of 14 benign prostatic hyperplasia (BPH) and 60 cancerous and non-cancerous prostate samples extracted from 34 cancer-bearing prostates removed by prostatectomy was performed; of the latter 60 samples, 16, 21, and 23 samples contained <10%, >30%, and no dysplastic cells, respectively. The predictive value of selected miRNAs was then tested by quantitative reverse-transcribed PCR (qRT-PCR), using two separate chemistries, Exiqon and Taqman, to evaluate the tissue samples obtained by prostatectomy. Validation experiments were also performed for a subset of miRNAs by qRT-PCR of 87 prostate core biopsies. RESULTS: We identified 123 miRNAs significantly dysregulated in PCa (adjusted P-values <0.05); 110 and 13 miRNAs were dysregulated only in cancerous samples and non-cancerous samples extracted from cancer-bearing prostates, respectively, while 31 were dysregulated regardless of the dysplastic cell content of the studied specimens. The clinical utility of eight selected miRNAs was analyzed using the same sample set with two qRT-PCR chemistries. Measurable qRT-PCR signals were obtained for seven and six miRNAs using the Exiqon and Taqman chemistries, respectively, and expression levels of six and four of these miRNAs differed significantly between BPH and PCa samples, regardless of dysplastic cell content. Validation experiments on core biopsies using qRT-PCR confirmed differential expression between BPH and PCa of four miRNAs (miR-187-3p, miR-183-5p, miR-32-5p, and miR-141-5p) using the Exiqon and one miRNA (miR-187-3p) with the Taqman chemistry. CONCLUSIONS: Our sequencing analyses identified several candidate diagnostic miRNAs and confirmed some which have previously been reported as diagnostic in prostate malignancy. The results of this study suggest also that some of selected miRNAs can differentiate between non-malignant and malignant prostates even when neoplastic cells are missing from the studied specimen.
Assuntos
MicroRNAs/metabolismo , Próstata/patologia , Neoplasias da Próstata/diagnóstico , Biomarcadores Tumorais , Biópsia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , MicroRNAs/genética , Próstata/metabolismo , Prostatectomia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , TranscriptomaRESUMO
BACKGROUND: Approximately 90% of colorectal cancer (CRC) deaths are caused by tumors ability to migrate into the adjacent tissues and metastase into distant organs. More than 40 genes have been causally linked to the development of CRC but no mutations have been associated with metastasis yet. To identify molecular basis of CRC metastasis we performed whole-exome and genome-scale transcriptome sequencing of 7 liver metastases along with their matched primary tumours and normal tissue. Multiple, spatially separated fragments of primary tumours were analyzed in each case. Uniformly malignant tissue specimen were selected with macrodissection, for three samples followed with laser microdissection. RESULTS: > 100 sequencing coverage allowed for detection of genetic alterations in subpopulation of tumour cells. Mutations in KRAS, APC, POLE, and PTPRT, previously associated with CRC development, were detected in most patients. Several new associations were identified, including PLXND1, CELSR3, BAHD1 and PNPLA6. CONCLUSIONS: We confirm the essential role of inflammation in CRC progression but question the mechanism of matrix metalloproteinases activation described in other work. Comprehensive sequencing data made it possible to associate genome-scale mutation distribution with gene expression patterns. To our knowledge, this is the first work to report such link in CRC metastasis context.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Hepáticas/genética , Mutação , Metástase Neoplásica/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Exoma , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/secundário , Análise de Sequência de RNARESUMO
BACKGROUND & AIMS: Macro-aspartate aminotransferase (macro-AST) manifests as a persistent elevation of AST levels, because of association of the protein with immunoglobulins in the circulation. Macro-AST is a rare, benign condition without a previously confirmed genetic basis. METHODS: Whole exome sequencing (WES)-based screening was performed on 32 participants with suspected familial macro-AST, while validation of variants was performed on an extended cohort of 92 probands and 1,644 healthy controls using Taqman genotyping. RESULTS: A missense variant (p.Gln208Glu, rs374966349) in glutamate oxaloacetate transaminase 1 (GOT1) was found, as a putative causal variant predisposing to familial macro-AST. The GOT1 p.Gln208Glu mutation was detected in 50 (54.3%) of 92 probands from 20 of 29 (69%) families, while its prevalence in healthy controls was only 0.18%. In silico analysis demonstrated that the amino acid at this position is not conserved among different species and that, functionally, a negatively charged glutamate on the GOT1 surface could strongly anchor serum immunoglobulins. CONCLUSIONS: Our data highlight that testing for the p.Gln208Glu genetic variant may be useful in diagnosis of macro-AST. LAY SUMMARY: Higher than normal levels of aspartate aminotransferase (AST) in the bloodstream may be a sign of a health problem. Individuals with macro-AST have elevated blood AST levels, without ongoing disease and often undergo unnecessary medical tests before the diagnosis of macro-AST is established. We found a genetic variant in the GOT1 gene associated with macro-AST. Genetic testing for this variant may aid diagnosis of macro-AST.