RESUMO
OBJECTIVES: Single-cell level analysis of articular cartilage and meniscus tissues from human healthy and osteoarthritis (OA) knees. METHODS: Single-cell RNA sequencing (scRNA-seq) analyses were performed on articular cartilage and meniscus tissues from healthy (n=6, n=7) and OA (n=6, n=6) knees. Expression of genes of interest was validated using immunohistochemistry and RNA-seq and function was analysed by gene overexpression and depletion. RESULTS: scRNA-seq analyses of human knee articular cartilage (70 972 cells) and meniscus (78 017 cells) identified a pathogenic subset that is shared between both tissues. This cell population is expanded in OA and has strong OA and senescence gene signatures. Further, this subset has critical roles in extracellular matrix (ECM) and tenascin signalling and is the dominant sender of signals to all other cartilage and meniscus clusters and a receiver of TGFß signalling. Fibroblast activating protein (FAP) is also a dysregulated gene in this cluster and promotes ECM degradation. Regulons that are controlled by transcription factor ZEB1 are shared between the pathogenic subset in articular cartilage and meniscus. In meniscus and cartilage cells, FAP and ZEB1 promote expression of genes that contribute to OA pathogenesis, including senescence. CONCLUSIONS: These single-cell studies identified a senescent pathogenic cell cluster that is present in cartilage and meniscus and has FAP and ZEB1 as main regulators which are novel and promising therapeutic targets for OA-associated pathways in both tissues.
Assuntos
Cartilagem Articular , Menisco , Osteoartrite , Humanos , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Osteoartrite/patologia , Cartilagem Articular/metabolismo , Senescência Celular/genética , Condrócitos/metabolismoRESUMO
MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.
Assuntos
Linfócitos B/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Biossíntese de Proteínas/genética , Transcriptoma/genética , Regiões 5' não Traduzidas/genética , Animais , Linfócitos B/citologia , Sequência de Bases , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Células Cultivadas , Citometria de Fluxo , Perfilação da Expressão Gênica/métodos , Immunoblotting , Camundongos Knockout , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/genética , Ribossomos/metabolismoRESUMO
OBJECTIVES: Fibromyalgia (FM) is a common pain disorder characterized by nociceptive dysregulation. The basic biology of FM is poorly understood. Herein we have used agnostic gene expression as a potential probe for informing its underlying biology and the development of a proof-of-concept diagnostic gene expression signature. METHODS: We analyzed RNA expression in 70 FM patients and 70 healthy controls. The isolated RNA was amplified and hybridized to Affymetrix® Human Gene 1.1 ST Peg arrays. The data was analyzed using Partek Genomics Suite version 6.6. RESULTS: Fibromyalgia patients exhibited a differential expression of 421 genes (p<0.001), several relevant to pathways for pain processing, such as glutamine/glutamate signaling and axonal development. There was also an upregulation of several inflammatory pathways and downregulation of pathways related to hypersensitivity and allergy. Using rigorous diagnostic modeling strategies, we show "locked" gene signatures discovered on Training and Test cohorts, that have a mean Area Under the Curve (AUC) of 0.81 on randomized, independent external data cohorts. Lastly, we identified a subset of 10 probesets that provided a diagnostic sensitivity for FM of 95% and a specificity of 96%. We also show that the signatures for FM were very specific to FM rather than common FM comorbidities. CONCLUSIONS: These findings provide new insights relevant to the pathogenesis of FM, and provide several testable hypotheses that warrant further exploration and also establish the foundation for a first blood-based molecular signature in FM that needs to be validated in larger cohorts of patients.
Assuntos
Fibromialgia/genética , Perfilação da Expressão Gênica , Transcriptoma/fisiologia , Adulto , Carboxipeptidases A/genética , Feminino , Fibromialgia/sangue , Fator de Transcrição GATA2/genética , Estudo de Associação Genômica Ampla , Humanos , Pessoa de Meia-Idade , Nociceptividade/fisiologia , Receptores de IgE/genética , Transdução de Sinais/genéticaRESUMO
Elucidating how cell populations promote onset and progression of intervertebral disc degeneration (IDD) has the potential to enable more precise therapeutic targeting of cells and mechanisms. Single-cell RNA-sequencing (scRNA-seq) is performed on surgically separated annulus fibrosus (AF) (19,978; 26,983 cells) and nucleus pulposus (NP) (20,884; 24,489 cells) from healthy and diseased human intervertebral discs (IVD). In both tissue types, depletion of cell subsets involved in maintenance of healthy IVD is observed, specifically the immature cell subsets - fibroblast progenitors and stem cells - indicative of an impairment of normal tissue self-renewal. Tissue-specific changes are also identified. In NP, several fibrotic populations are increased in degenerated IVD, indicating tissue-remodeling. In degenerated AF, a novel disease-associated subset is identified, which expresses disease-promoting genes. It is associated with pathogenic biological processes and the main gene regulatory networks include thrombospondin signaling and FOXO1 transcription factor. In NP and AF cells thrombospondin protein promoted expression of genes associated with TGFß/fibrosis signaling, angiogenesis, and nervous system development. The data reveal new insights of both shared and tissue-specific changes in specific cell populations in AF and NP during IVD degeneration. These identified mechanisms and molecules are novel and more precise targets for IDD prevention and treatment.
Assuntos
Anel Fibroso , Degeneração do Disco Intervertebral , Núcleo Pulposo , Humanos , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Anel Fibroso/metabolismo , Anel Fibroso/patologia , Masculino , Pessoa de Meia-Idade , Feminino , Adulto , Disco Intervertebral/metabolismo , Disco Intervertebral/patologiaRESUMO
The most common cause of kidney transplant failure is the poorly characterized histopathologic entity interstitial fibrosis and tubular atrophy (IFTA). There are no known unifying mechanisms, no effective therapy, and no proven preventive strategies. Possible mechanisms include chronic immune rejection, inflammation, drug toxicity, and chronic kidney injury from secondary factors. To gain further mechanistic insight, we conducted a large-scale proteogenomic study of kidney transplant biopsies with IFTA of varying severity. We acquired proteomic data using tandem mass spectrometry with subsequent quantification, analysis of differential protein expression, validation, and functional annotations to known molecular networks. We performed genome-wide expression profiling in parallel. More than 1400 proteins with unique expression profiles traced the progression from normal transplant biopsies to biopsies with mild to moderate and severe disease. Multiple sets of proteins were mapped to different functional pathways, many increasing with histologic severity, including immune responses, inflammatory cell activation, and apoptosis consistent with the chronic rejection hypothesis. Two examples include the extensive population of the alternative rather than the classical complement pathway, previously not appreciated for IFTA, and a comprehensive control network for the actin cytoskeleton and cell signaling of the acute-phase response. In summary, this proteomic effort using kidney tissue contributes mechanistic insight into several biologic processes associated with IFTA.
Assuntos
Rejeição de Enxerto/genética , Transplante de Rim , Rim/patologia , Proteômica , Adulto , Idoso , Atrofia , Biópsia , Citoesqueleto/fisiologia , Feminino , Fibrose , Rejeição de Enxerto/fisiopatologia , Humanos , Rim/fisiopatologia , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of microarrays. Recently, there have been significant improvements in whole blood RNA isolation techniques which, through stabilization of RNA at the time of sample collection, avoid bias and artifacts introduced during sample handling. Despite these improvements, current human whole blood RNA stabilization/isolation kits are limited by the requirement of a venous blood sample of at least 2.5 mL. While fingerstick blood collection has been used for many different assays, there has yet to be a kit developed to isolate high quality RNA for use in gene expression studies from such small human samples. The clinical and field testing advantages of obtaining reliable and reproducible gene expression data from a fingerstick are many; it is less invasive, time saving, more mobile, and eliminates the need of a trained phlebotomist. Furthermore, this method could also be employed in small animal studies, i.e. mice, where larger sample collections often require sacrificing the animal. In this study, we offer a rapid and simple method to extract sufficient amounts of high quality total RNA from approximately 70 microl of whole blood collected via a fingerstick using a modified protocol of the commercially available Qiagen PAXgene RNA Blood Kit. RESULTS: From two sets of fingerstick collections, about 70 uL whole blood collected via finger lancet and capillary tube, we recovered an average of 252.6 ng total RNA with an average RIN of 9.3. The post-amplification yields for 50 ng of total RNA averaged at 7.0 ug cDNA. The cDNA hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips had an average % Present call of 52.5%. Both fingerstick collections were highly correlated with r(2) values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r(2) values ranging from 0.88 to 0.96 for fingerstick collection 1 and 0.94 to 0.96 for fingerstick collection 2. CONCLUSIONS: Our comparisons of RNA quality and gene expression data of the fingerstick method with traditionally processed sample workflows demonstrate excellent RNA quality from the capillary collection as well as very high correlations of gene expression data.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Perfilação da Expressão Gênica/métodos , Genoma Humano/imunologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA/genéticaRESUMO
Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.
Assuntos
Melanoma/metabolismo , Melanoma/patologia , N-Acetilexosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Humanos , Melanoma/genética , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilexosaminiltransferases/genética , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
In this chapter, we describe numerous methods to extract RNA, DNA, and protein from tissue, represented by kidney transplant biopsies, and from peripheral blood cells collected at various clinical sites. Gene expression profiling and SNP-based genome-wide association studies are done using various microarray platforms. In addition, protocols that enable simultaneous protein purification from these clinical samples, enable additional strategies for understanding of the molecular processes involved in organ transplantation, immunosuppressive drug regimens, and the elements determining allograft success and failure. Successfully establishing a multicenter clinical study was essential to meet our objectives for subject enrollment and transplant outcomes. This chapter focuses on our experience setting up and coordinating clinical sample collection from multiple transplant centers for the purpose of microarray analysis.
Assuntos
Análise em Microsséries/métodos , Estudos Multicêntricos como Assunto , Manejo de Espécimes/métodos , Biópsia , DNA/análise , DNA/genética , DNA/isolamento & purificação , Genômica , Globinas/análise , Globinas/isolamento & purificação , Rim/química , Rim/patologia , Leucócitos Mononucleares/química , RNA/análise , RNA/genética , RNA/isolamento & purificaçãoRESUMO
RNA integrity is a critical factor in obtaining meaningful gene expression data. Current methodologies rely on maintaining samples in cold environments during collection, transport, processing, and storage procedures, which are also extremely time-sensitive. Several RNA storage products are commercially available to help prevent degradation during the handling and storage steps; however, samples must be kept cold for optimal protection. We have evaluated a novel RNA storage medium based on anhydrobiosis for stabilizing and protecting samples from degradation at room temperature that are intended for use in microarray analysis. Samples were stored dry at room temperature for various time periods to assess any degradation or loss of activity as compared with frozen control samples. Recovered samples were used directly for analysis without further purification and exhibited no interference or inhibition in downstream applications. Comparison of gene expression profiles indicate no significant differences between freezer-stored control samples and those kept at room temperature protected in the RNA storage medium. The quality of recovered RNA was confirmed using spectrophotometry and Bioanalyzer analysis and was identical to control samples. The ability to stabilize RNA samples at ambient temperatures for extended time periods will have tremendous use, particularly for sample shipment to core facilities.
Assuntos
Armazenamento de Medicamentos/métodos , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , RNA/química , RNA/isolamento & purificação , Manejo de Espécimes/métodos , Humanos , RNA/genéticaRESUMO
BACKGROUND: Despite significant improvements in life expectancy of kidney transplant patients due to advances in surgery and immunosuppression, Chronic Allograft Nephropathy (CAN) remains a daunting problem. A complex network of cellular mechanisms in both graft and peripheral immune compartments complicates the non-invasive diagnosis of CAN, which still requires biopsy histology. This is compounded by non-immunological factors contributing to graft injury. There is a pressing need to identify and validate minimally invasive biomarkers for CAN to serve as early predictors of graft loss and as metrics for managing long-term immunosuppression. METHODS: We used DNA microarrays, tandem mass spectroscopy proteomics and bioinformatics to identify genomic and proteomic markers of mild and moderate/severe CAN in peripheral blood of two distinct cohorts (n = 77 total) of kidney transplant patients with biopsy-documented histology. FINDINGS: Gene expression profiles reveal over 2400 genes for mild CAN, and over 700 for moderate/severe CAN. A consensus analysis reveals 393 (mild) and 63 (moderate/severe) final candidates as CAN markers with predictive accuracy of 80% (mild) and 92% (moderate/severe). Proteomic profiles show over 500 candidates each, for both stages of CAN including 302 proteins unique to mild and 509 unique to moderate/severe CAN. CONCLUSIONS: This study identifies several unique signatures of transcript and protein biomarkers with high predictive accuracies for mild and moderate/severe CAN, the most common cause of late allograft failure. These biomarkers are the necessary first step to a proteogenomic classification of CAN based on peripheral blood profiling and will be the targets of a prospective clinical validation study.
Assuntos
Biomarcadores/sangue , Perfilação da Expressão Gênica , Genômica , Nefropatias/sangue , Transplante de Rim , Proteômica , Transplante Homólogo , Cromatografia Líquida , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Espectrometria de Massas em TandemRESUMO
Activity-based protein profiling (ABPP) is a chemical method that utilizes active-site-directed probes to determine the functional state of enzymes in complex proteomes. Probe-labeled enzymes are typically detected by in-gel fluorescence scanning, a robust technique that nonetheless exhibits some key deficiencies, including limited sensitivity and resolution, as well as ambiguity regarding the molecular identity of the enzymes under investigation. Here, we report a microarray platform for ABPP that addresses these limitations. In this platform, proteomes are treated with ABPP probes in solution, after which labeled enzymes are captured and visualized on glass slides displaying an array of anti-enzyme antibodies. We show that ABPP microarrays exhibit superior sensitivity and resolution compared to gel-based methods, permitting the parallel analysis of several enzyme activities in proteomes, including cancer-associated proteases such as urokinase, matrix metalloproteinase-9, and prostate-specific antigen.
Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Análise Serial de Proteínas/métodos , Proteoma/metabolismo , Serina Endopeptidases/metabolismo , Anticorpos/química , Benzofenonas/química , Sítios de Ligação , Metaloproteinase 9 da Matriz/imunologia , Proteoma/análise , Rodaminas/química , Sensibilidade e Especificidade , Serina Endopeptidases/imunologiaRESUMO
von Willebrand disease (VWD) type 1 is difficult to diagnose because of bleeding variability and low heritability of von Willebrand factor (VWF) levels. We compared a bleeding severity score and bleeding times to candidate gene haplotypes within pedigrees of 14 index cases, using a covariance components model for multivariate traits (Mendel: QTL Association). These pedigrees included 13 affected and 40 unaffected relatives, as defined by plasma ristocetin cofactor (VWF:RCo) levels. The bleeding severity score was derived from a detailed history. Donors were genotyped using a primer extension method, and 9 candidate genes were selected for analysis. VWF:RCo levels had the strongest influence on bleeding severity score and bleeding time. ITGA2 haplotype 2 (807C) and ITGA2B haplotype 1 (Ile(843)) were each associated with increased bleeding severity scores (P < .01 and P < .01, respectively). GP6 haplotype b (Pro(219)) was also associated with increased scores (P = .03) after adjustment for donor age. No association was observed with 6 other candidate genes, GP1BA, ITGB3, VWF, FGB, IL6, or TXA2R. Increased plasma VWF:Ag levels were associated with VWF haplotype 1 (-1793G; P = .02). These results establish that genetic differences in the adhesion receptor subunits alpha(2), alpha(IIb,) and GPVI can influence the phenotype of VWD type 1.