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1.
Cancers (Basel) ; 13(21)2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34771620

RESUMO

Hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (MET) signaling promotes tumorigenesis and tumor progression in various types of cancer, including uveal melanoma (UM). The roles of HGF/MET signaling have been studied in cell survival, proliferation, cell motility, and migration. Furthermore, HGF/MET signaling has emerged as a critical player not only in the tumor itself but also in the tumor microenvironment. Expression of MET is frequently observed in metastatic uveal melanoma and is associated with poor prognosis. It has been reported that HGF/MET signaling pathway activation is the major mechanism of treatment resistance in metastatic UM (MUM). To achieve maximal therapeutic benefit in MUM patients, it is important to understand how MET signaling drives cellular functions in uveal melanoma cells. Here, we review the HGF/MET signaling biology and the role of HGF/MET blockades in uveal melanoma.

2.
Noncoding RNA ; 7(4)2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34698264

RESUMO

Cutaneous melanoma (CM) is the most lethal tumor among skin cancers, and its incidence is constantly increasing. A deeper understanding of the molecular processes guiding melanoma pathogenesis could improve diagnosis, treatment and prognosis. MicroRNAs play a key role in melanoma biology. Recently, next generation sequencing (NGS) experiments, designed to assess small-RNA expression, revealed the existence of microRNA variants with different length and sequence. These microRNA isoforms are known as isomiRs and provide an additional layer to the complex non-coding RNA world. Here, we collected data from NGS experiments to provide a comprehensive characterization of miRNA and isomiR dysregulation in benign nevi (BN) and early-stage melanomas. We observed that melanoma and BN express different and specific isomiRs and have a different isomiR abundance distribution. Moreover, isomiRs from the same microRNA can have opposite expression trends between groups. Using The Cancer Genome Atlas (TCGA) dataset of skin cancers, we analyzed isomiR expression in primary melanoma and melanoma metastasis and tested their association with NF1, BRAF and NRAS mutations. IsomiRs differentially expressed were identified and catalogued with reference to the canonical form. The reported non-random dysregulation of specific isomiRs contributes to the understanding of the complex melanoma pathogenesis and serves as the basis for further functional studies.

3.
Cell Death Dis ; 12(5): 473, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980826

RESUMO

Malignant cutaneous melanoma (CM) is a potentially lethal form of skin cancer whose worldwide incidence has been constantly increasing over the past decades. During their lifetime, about 8% of CM patients will develop multiple primary melanomas (MPMs), usually at a young age and within 3 years from the first tumor/diagnosis. With the aim of improving our knowledge on MPM biology and pathogenesis, we explored the miRNome of 24 single and multiple primary melanomas, including multiple tumors from the same patient, using a small RNA-sequencing approach. From a supervised analysis, 22 miRNAs were differentially expressed in MPM compared to single CM, including key miRNAs involved in epithelial-mesenchymal transition. The first and second melanoma from the same patient presented a different miRNA profile. Ten miRNAs, including miR-25-3p, 149-5p, 92b-3p, 211-5p, 125a-5p, 125b-5p, 205-5p, 200b-3p, 21-5p, and 146a-5p, were further validated in 47 single and multiple melanoma samples. Pathway enrichment analysis of miRNA target genes revealed a more differentiated and less invasive status of MPMs compared to CMs. Bioinformatic analyses at the miRNA isoform (isomiR) level detected a panel of highly expressed isomiRs belonging to miRNA families implicated in human tumorigenesis, including miR-200, miR-30, and miR-10 family. Moreover, we identified hsa-miR-125a-5p|0|-2 isoform as tenfold over-represented in melanoma than the canonical form and differentially expressed in MPMs arising in the same patient. Target prediction analysis revealed that the miRNA shortening could change the pattern of target gene regulation, specifically in genes implicated in cell adhesion and neuronal differentiation. Overall, we provided a putative and comprehensive characterization of the miRNA/isomiR regulatory network of MPMs, highlighting mechanisms of tumor development and molecular features differentiating this subtype from single melanomas.


Assuntos
Melanoma/genética , MicroRNAs/genética , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Melanoma/patologia , Pessoa de Meia-Idade , Neoplasias Cutâneas/patologia
4.
Bioinformatics ; 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33471076

RESUMO

MOTIVATION: MicroRNA (miRNA) precursor arms give rise to multiple isoforms simultaneously called "isomiRs." IsomiRs from the same arm typically differ by a few nucleotides at either their 5´ or 3´ termini, or both. In humans, the identities and abundances of isomiRs depend on a person's sex, population of origin, race/ethnicity, and on tissue type, tissue state, and disease type/subtype. Moreover, nearly half of the time the most abundant isomiR differs from the miRNA sequence found in public databases. Accurate mining of isomiRs from deep sequencing data is thus important. RESULTS: We developed isoMiRmap, a fast, standalone, user-friendly mining tool that identifies and quantifies all isomiRs by directly processing short RNA-seq datasets. IsoMiRmap is a portable "plug-and-play" tool, requires minimal setup, has modest computing and storage requirements, and can process an RNA-seq dataset with 50 million reads in just a few minutes on an average laptop. IsoMiRmap deterministically and exhaustively reports all isomiRs in a given deep sequencing dataset and quantifies them accurately (no double-counting). IsoMiRmap comprehensively reports all miRNA precursor locations from which an isomiR may be transcribed, tags as 'ambiguous' isomiRs whose sequences exist both inside and outside of the space of known miRNA sequences and reports the public identifiers of common single-nucleotide polymorphisms and documented somatic mutations that may be present in an isomiR. IsoMiRmap also identifies isomiRs with 3´ non-templated post-transcriptional additions. Compared to similar tools, isoMiRmap is the fastest, reports more bona fide isomiRs, and provides the most comprehensive information related to an isomiR's transcriptional origin. AVAILABILITY: The codes for isoMiRmap are freely available at https://cm.jefferson.edu/isoMiRmap/ and https://github.com/TJU-CMC-Org/isoMiRmap/. IsomiR profiles for the datasets of the 1000 Genomes Project, spanning five population groups, and The Cancer Genome Atlas (TCGA), spanning 33 cancer studies, are also available at https://cm.jefferson.edu/isoMiRmap/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

5.
J Appl Lab Med ; 6(4): 892-901, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-33319223

RESUMO

BACKGROUND: Control charting is routine in the quality assurance of traditional clinical laboratory testing. Genomic tests are not typically managed by control charting. We examined control charting to monitor the performance of a clinical next-generation sequencing (NGS) assay. METHODS: We retrospectively examined 3 years of control material (NA12878) data from clinical genomic epilepsy testing. Levey-Jennings plots were used to visualize changes in control material depth of sequencing coverage in genomic regions of an epilepsy genomic panel. Changes in depth of coverage were correlated with changes in the manufactured lot of capture probe reagent. Depth of coverage was also correlated between quality control material and clinical samples. RESULTS: Fifty-seven sequencing runs of NA12878 were analyzed for 1811 genomic regions targeting 108 genes. Manufactured probe lot changes were associated with significant changes in the average coverage of 537 genomic regions and the lowest coverage of 173 regions (using a critical cut-off of P < 5.52 x 10-6). Genomic regions with the highest sensitivity to lot-to-lot variation by average sequencing depth of coverage were not the same regions with the highest sensitivity by lowest sequencing depth of coverage. Levey-Jennings plots displayed differences in genomic depth of coverage across capture probe reagent lot changes. There was moderate correlation between the changes in depth of sequencing across lot changes for control material and clinical cases (r2 = 0.45). CONCLUSIONS: Genomic control charting can be used routinely by clinical laboratories to monitor assay performance and ensure the quality of testing.


Assuntos
Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Laboratório Clínico , Humanos , Controle de Qualidade , Estudos Retrospectivos
6.
Sci Rep ; 10(1): 21782, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311640

RESUMO

Increasing evidence suggest that the glucose-lowering drug metformin exerts a valuable anti-senescence role. The ability of metformin to affect the biogenesis of selected microRNAs (miRNAs) was recently suggested. MicroRNA isoforms (isomiRs) are distinct variations of miRNA sequences, harboring addition or deletion of one or more nucleotides at the 5' and/or 3' ends of the canonical miRNA sequence. We performed a comprehensive analysis of miRNA and isomiR profile in human endothelial cells undergoing replicative senescence in presence of metformin. Metformin treatment was associated with the differential expression of 27 miRNAs (including miR-100-5p, -125b-5p, -654-3p, -217 and -216a-3p/5p). IsomiR analysis revealed that almost 40% of the total miRNA pool was composed by non-canonical sequences. Metformin significantly affects the relative abundance of 133 isomiRs, including the non-canonical forms of the aforementioned miRNAs. Pathway enrichment analysis suggested that pathways associated with proliferation and nutrient sensing are modulated by metformin-regulated miRNAs and that some of the regulated isomiRs (e.g. the 5' miR-217 isomiR) are endowed with alternative seed sequences and share less than half of the predicted targets with the canonical form. Our results show that metformin reshapes the senescence-associated miRNA/isomiR patterns of endothelial cells, thus expanding our insight into the cell senescence molecular machinery.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Metformina/farmacologia , MicroRNAs/biossíntese , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Fatores de Tempo
7.
Cancers (Basel) ; 12(2)2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32050636

RESUMO

Uveal melanoma (UM) is the most common primary eye malignancy in adults and up to 50% of patients subsequently develop systemic metastasis. Metastatic uveal melanoma (MUM) is highly resistant to immunotherapy. One of the mechanisms for resistance would be the immune-suppressive tumor microenvironment. Here, we have investigated the role of tryptophan 2,3-dioxygenase (TDO) in UM. Both TDO and indoleamine 2,3-dioxygenase (IDO) catalyze tryptophan and produce kynurenine, which could cause inhibition of T cell immune responses. We first studied the expression of TDO on tumor tissue specimens obtained from UM hepatic metastasis. High expression of TDO protein was confirmed in all hepatic metastasis. TDO was positive in both normal hepatocytes and the tumor cells with relatively higher expression in tumor cells. On the other hand, IDO protein remained undetectable in all of the MUM specimens. UM cell lines established from metastasis also expressed TDO protein and increasing kynurenine levels were detected in the supernatant of MUM cell culture. In TCGA database, higher TDO2 expression in primary UM significantly correlated to BAP1 mutation and monosomy 3. These results indicate that TDO might be one of the key mechanisms for resistance to immunotherapy in UM.

8.
Bioinformatics ; 36(3): 698-703, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504201

RESUMO

MOTIVATION: MicroRNAs (miRNAs) are small RNA molecules (∼22 nucleotide long) involved in post-transcriptional gene regulation. Advances in high-throughput sequencing technologies led to the discovery of isomiRs, which are miRNA sequence variants. While many miRNA-seq analysis tools exist, the diversity of output formats hinders accurate comparisons between tools and precludes data sharing and the development of common downstream analysis methods. RESULTS: To overcome this situation, we present here a community-based project, miRNA Transcriptomic Open Project (miRTOP) working towards the optimization of miRNA analyses. The aim of miRTOP is to promote the development of downstream isomiR analysis tools that are compatible with existing detection and quantification tools. Based on the existing GFF3 format, we first created a new standard format, mirGFF3, for the output of miRNA/isomiR detection and quantification results from small RNA-seq data. Additionally, we developed a command line Python tool, mirtop, to create and manage the mirGFF3 format. Currently, mirtop can convert into mirGFF3 the outputs of commonly used pipelines, such as seqbuster, isomiR-SEA, sRNAbench, Prost! as well as BAM files. Some tools have also incorporated the mirGFF3 format directly into their code, such as, miRge2.0, IsoMIRmap and OptimiR. Its open architecture enables any tool or pipeline to output or convert results into mirGFF3. Collectively, this isomiR categorization system, along with the accompanying mirGFF3 and mirtop API, provide a comprehensive solution for the standardization of miRNA and isomiR annotation, enabling data sharing, reporting, comparative analyses and benchmarking, while promoting the development of common miRNA methods focusing on downstream steps of miRNA detection, annotation and quantification. AVAILABILITY AND IMPLEMENTATION: https://github.com/miRTop/mirGFF3/ and https://github.com/miRTop/mirtop. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
MicroRNAs , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNA , Transcriptoma
9.
Pigment Cell Melanoma Res ; 33(1): 52-62, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31283110

RESUMO

Uveal melanoma (UVM) is the most common primary intraocular malignancy in adults. With over 50% of patients developing metastatic disease, there is an unmet need for improved diagnostic and therapeutic options. Efforts to understand the molecular biology of the disease have revealed several markers that correlate with patient prognosis, including the copy number of chromosome 3, genetic alterations in the BAP1, EIF1AX and SF3B1 genes, and other transcriptional features. Here, we expand upon previous reports by comprehensively characterizing the short RNA-ome in 80 primary UVM tumor samples. In particular, we describe a previously unseen complex network involving numerous regulatory molecules that comprise microRNA (miRNAs), novel UVM-specific miRNA loci, miRNA isoforms (isomiRs), and tRNA-derived fragments (tRFs). Importantly, we show that the abundance profiles of isomiRs and tRFs associate with various molecular phenotypes, metastatic disease, and patient survival. Our findings suggest deep involvement of isomiRs and tRFs in the disease etiology of UVM. We posit that further study and characterization of these novel molecules will improve understanding of the mechanisms underlying UVM, and lead to the development of new diagnostic and therapeutic approaches.


Assuntos
Melanoma/genética , Melanoma/patologia , MicroRNAs/genética , RNA de Transferência/genética , Neoplasias Uveais/genética , Neoplasias Uveais/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Progressão da Doença , Feminino , Loci Gênicos , Humanos , Masculino , MicroRNAs/metabolismo , Metástase Neoplásica , RNA de Transferência/metabolismo , Análise de Sobrevida
10.
Sci Rep ; 9(1): 14238, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578372

RESUMO

The impermeability of the luminal endothelial cell monolayer is crucial for the normal performance of the vascular and lymphatic systems. A key to this function is the integrity of the monolayer's intercellular junctions. The known repertoire of junction-regulating genes is incomplete. Current permeability assays are incompatible with high-throughput genome-wide screens that could identify these genes. To overcome these limitations, we designed a new permeability assay that consists of cell monolayers grown on ~150 µm microcarriers (MCs). Each MC functions as a miniature individual assay of permeability (MAP). We demonstrate that false-positive results can be minimized, and that MAP sensitivity to thrombin-induced increase in monolayer permeability is similar to the sensitivity of impedance measurement. We validated the assay by showing that the expression of single guide RNAs (sgRNAs) that target genes encoding known thrombin signaling proteins blocks effectively thrombin-induced junction disassembly, and that MAPs carrying such cells can be separated effectively by fluorescence-assisted sorting from those that carry cells expressing non-targeting sgRNAs. These results indicate that MAPs are suitable for high-throughput experimentation and for genome-wide screens for genes that mediate the disruptive effect of thrombin on endothelial cell junctions.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células Endoteliais/citologia , Estudo de Associação Genômica Ampla/métodos , Ensaios de Triagem em Larga Escala/métodos , RNA Guia/genética , Junções Aderentes/fisiologia , Linhagem Celular Transformada , Células Clonais/metabolismo , Colágeno/metabolismo , Células Endoteliais/metabolismo , Fibronectinas/metabolismo , Citometria de Fluxo/métodos , Corantes Fluorescentes/análise , Gelatina , Biblioteca Gênica , Estudo de Associação Genômica Ampla/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Masculino , Miniaturização , Permeabilidade/efeitos dos fármacos , Interferência de RNA , Proteínas Repressoras/genética , Transdução de Sinais/genética , Trombina/metabolismo , Trombina/farmacologia , Junções Íntimas/fisiologia , Transcrição Genética , Fator A de Crescimento do Endotélio Vascular/farmacologia
11.
Parkinsonism Relat Disord ; 65: 203-209, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31402278

RESUMO

INTRODUCTION: Parkinson's Disease (PD) is diagnosed clinically. Reliable non-invasive PD biomarkers are actively sought. Transfer RNAs produce short non-coding RNAs, the tRNA-derived fragments (tRF). tRF have been shown to play diverse roles, including in amyotrophic lateral sclerosis, and the response to ischemic stroke. Rich tRF populations are being reported in biofluids. We explored the possibility that tRF can serve as non-invasive biomarkers for PD. METHODS: We collected existing RNA-seq samples and re-analyzed a total of 254 legacy datasets from 3 previous studies, from male and female PD patients and controls that belong to three categories: prefrontal cortex samples from 29 patients and 33 controls; cerebrospinal fluid (CSF) samples from 63 patients and 64 controls; and, serum samples from 34 patients and 31 controls. First, we identified tRF exhaustively and deterministically in every dataset. Second, we determined tRF that are differentially abundant (DA) between PD and control samples, using uncorrected t-tests. Lastly, we assessed all the DA tRF from the previous step with Partial Least Squares - Discriminant Analysis (PLS-DA) to stringently sub-select tRF that can distinguish PD patients from controls. RESULTS: We show that PLS-DA identified tRF from prefrontal cortex, CSF, and serum that can distinguish PD patients from controls. A handful of identified tRF were previously investigated in neurological contexts. Signatures built from relatively few tRF suffice to distinguish PD from control in each category of samples with high sensitivity (89-100%) and specificity (79-98%). CONCLUSION: tRF-based signatures are promising candidates that warrant further evaluation as non-invasive PD biomarkers.


Assuntos
Doença de Parkinson/diagnóstico , Doença de Parkinson/metabolismo , Córtex Pré-Frontal/metabolismo , RNA de Transferência/metabolismo , Análise de Sequência de RNA/normas , Bancos de Tecidos , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Conjuntos de Dados como Assunto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/sangue , Doença de Parkinson/líquido cefalorraquidiano , RNA de Transferência/sangue , RNA de Transferência/líquido cefalorraquidiano , Sensibilidade e Especificidade , Fatores Sexuais
12.
Curr Opin Biotechnol ; 58: 202-210, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31323485

RESUMO

We herein provide a brief review of the trajectory that the field of short RNA research followed in the last 25 years. We place emphasis on the unexpected discoveries and the ramifications of these discoveries for the field, as well as offer some thoughts about what the next 25 years may bring. Arguably, the uncovered dependence of different types of short RNAs on individual attributes such as a person's sex, population origin, race, and on tissue type, tissue state, and disease was most unexpected. This dependence has important ramifications in that it will provide a boost to our understanding of the molecular mechanisms of health disparities as well as pave the way for novel approaches to designing improved and personalized diagnostics and therapeutics.


Assuntos
Medicina de Precisão , RNA
13.
Mol Cancer Res ; 17(9): 1815-1827, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31164413

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited and, very often, ineffective medical and surgical therapeutic options. The treatment of patients with advanced unresectable PDAC is restricted to systemic chemotherapy, a therapeutic intervention to which most eventually develop resistance. Recently, nab-paclitaxel (n-PTX) has been added to the arsenal of first-line therapies, and the combination of gemcitabine and n-PTX has modestly prolonged median overall survival. However, patients almost invariably succumb to the disease, and little is known about the mechanisms underlying n-PTX resistance. Using the conditionally reprogrammed (CR) cell approach, we established and verified continuously growing cell cultures from treatment-naïve patients with PDAC. To study the mechanisms of primary drug resistance, nab-paclitaxel-resistant (n-PTX-R) cells were generated from primary cultures and drug resistance was verified in vivo, both in zebrafish and in athymic nude mouse xenograft models. Molecular analyses identified the sustained induction of c-MYC in the n-PTX-R cells. Depletion of c-MYC restored n-PTX sensitivity, as did treatment with either the MEK inhibitor, trametinib, or a small-molecule activator of protein phosphatase 2a. IMPLICATIONS: The strategies we have devised, including the patient-derived primary cells and the unique, drug-resistant isogenic cells, are rapid and easily applied in vitro and in vivo platforms to better understand the mechanisms of drug resistance and for defining effective therapeutic options on a patient by patient basis.


Assuntos
Albuminas/farmacologia , Carcinoma Ductal Pancreático/genética , Resistencia a Medicamentos Antineoplásicos , Paclitaxel/farmacologia , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Regulação para Cima , Idoso , Idoso de 80 Anos ou mais , Albuminas/uso terapêutico , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Cultura Primária de Células , Células Tumorais Cultivadas , Peixe-Zebra
14.
Cell ; 177(2): 446-462.e16, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30951671

RESUMO

Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.


Assuntos
Ácidos Nucleicos Livres/isolamento & purificação , MicroRNA Circulante/isolamento & purificação , RNA/isolamento & purificação , Adulto , Líquidos Corporais/química , Linhagem Celular , Vesículas Extracelulares/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Masculino , MicroRNAs/isolamento & purificação , MicroRNAs/metabolismo , RNA/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA/métodos
15.
Cancer Res ; 79(12): 3034-3049, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30996049

RESUMO

tRNA-derived fragments (tRF) are a class of potent regulatory RNAs. We mined the datasets from The Cancer Genome Atlas (TCGA) representing 32 cancer types with a deterministic and exhaustive pipeline for tRNA fragments. We found that mitochondrial tRNAs contribute disproportionally more tRFs than nuclear tRNAs. Through integrative analyses, we uncovered a multitude of statistically significant and context-dependent associations between the identified tRFs and mRNAs. In many of the 32 cancer types, these associations involve mRNAs from developmental processes, receptor tyrosine kinase signaling, the proteasome, and metabolic pathways that include glycolysis, oxidative phosphorylation, and ATP synthesis. Even though the pathways are common to multiple cancers, the association of specific mRNAs with tRFs depends on and differs from cancer to cancer. The associations between tRFs and mRNAs extend to genomic properties as well; specifically, tRFs are positively correlated with shorter genes that have a higher density in repeats, such as ALUs, MIRs, and ERVLs. Conversely, tRFs are negatively correlated with longer genes that have a lower repeat density, suggesting a possible dichotomy between cell proliferation and differentiation. Analyses of bladder, lung, and kidney cancer data indicate that the tRF-mRNA wiring can also depend on a patient's sex. Sex-dependent associations involve cyclin-dependent kinases in bladder cancer, the MAPK signaling pathway in lung cancer, and purine metabolism in kidney cancer. Taken together, these findings suggest diverse and wide-ranging roles for tRFs and highlight the extensive interconnections of tRFs with key cellular processes and human genomic architecture. SIGNIFICANCE: Across 32 TCGA cancer contexts, nuclear and mitochondrial tRNA fragments exhibit associations with mRNAs that belong to concrete pathways, encode proteins with particular destinations, have a biased repeat content, and are sex dependent.


Assuntos
Redes Reguladoras de Genes , Genoma Humano , Disparidades nos Níveis de Saúde , Neoplasias/genética , Neoplasias/patologia , RNA Mensageiro/genética , RNA de Transferência/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neoplasias/classificação , Neoplasias/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Transcriptoma
16.
Clin Chem ; 65(8): 972-985, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30872376

RESUMO

BACKGROUND: Single-cell genomics is an approach to investigate cell heterogeneity and to identify new molecular features correlated with clinical outcomes. This approach allows identification of the complexity of cell diversity in a sample without the loss of information that occurs when multicellular or bulk tissue samples are analyzed. CONTENT: The first single-cell RNA-sequencing study was published in 2009, and since then many more studies and single-cell sequencing methods have been published. These studies have had a major impact on several fields, including microbiology, neurobiology, cancer, and developmental biology. Recently, improvements in reliability and the development of commercial single-cell isolation platforms are opening the potential of this technology to the clinical laboratory. SUMMARY: In this review we provide an overview of the current state of single-cell genomics. We describe opportunities in clinical research and medical applications.


Assuntos
Genômica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Separação Celular/métodos , Epigenômica , Amplificação de Genes , História do Século XX , História do Século XXI , Humanos , Imunidade/genética , Microbiota/genética , Neoplasias/genética , Reprodutibilidade dos Testes , Análise de Célula Única/história , Análise de Célula Única/tendências , Transcriptoma/genética
17.
Hum Mutat ; 40(3): 288-298, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30578701

RESUMO

Autosomal recessive congenital ichthyosis (ARCI), a phenotypically heterogeneous group of non-syndromic Mendelian disorders of keratinization, is caused by mutations in as many as 13 distinct genes. We examined a cohort of 125 consanguineous families with ARCI for underlying genetic mutations. The patients' DNA was analyzed with a gene-targeted next generation sequencing panel comprising 38 ichthyosis associated genes. The interpretations of results of genomic data were assisted by genome-wide homozygosity mapping and transcriptome sequencing. Sequence data analysis identified biallelic mutations in 106 families out of a total of 125 (85%), most of them (102, 96.2%) being homozygous, reflecting consanguinity in these families. Among the 85 distinct mutations in 10 different genes, 45 (53%) were previously unreported. Phenotype-genotype correlations allowed assignment of specific genes in the majority of the families to a specific subtype of ARCI, lamellar ichthyosis (LI) versus congenital ichthyosiform erythroderma (CIE). Interestingly, mutations in several genes could give rise to an overlapping phenotype consistent with either LI or CIE. Also, this is the third report for SDR9C7 and SULT2B1, and fourth report for CERS3 mutations. Direct comparison of our results with previously published regional cohorts highlights the global mutation landscape of ARCI, however, population specific differences were noted.


Assuntos
Consanguinidade , Genes Recessivos , Genoma Humano , Ictiose Lamelar/genética , Sequência de Bases , Estudos de Coortes , Família , Feminino , Homozigoto , Humanos , Ictiose Lamelar/diagnóstico , Masculino , Mutação , Linhagem , Fenótipo , Sítios de Splice de RNA/genética
18.
Clin Cancer Res ; 25(2): 724-734, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30266763

RESUMO

PURPOSE: Heritable genetic variations can affect the inflammatory tumor microenvironment, which can ultimately affect cancer susceptibility and clinical outcomes. Recent evidence indicates that IDO2, a positive modifier in inflammatory disease models, is frequently upregulated in pancreatic ductal adenocarcinoma (PDAC). A unique feature of IDO2 in humans is the high prevalence of two inactivating single-nucleotide polymorphisms (SNP), which affords the opportunity to carry out loss-of-function studies directly in humans. In this study, we sought to address whether genetic loss of IDO2 may influence PDAC development and responsiveness to treatment.Experimental Design: Transgenic Ido2 +/+ and Ido2 -/- mice in which oncogenic KRAS is activated in pancreatic epithelial cells were evaluated for PDAC. Two patient data sets (N = 200) were evaluated for the two IDO2-inactivating SNPs together with histologic, RNA expression, and clinical survival data. RESULTS: PDAC development was notably decreased in the Ido2 -/- mice (30% vs. 10%, P < 0.05), with a female predominance similar to the association observed for one of the human SNPs. In patients, the biallelic occurrence of either of the two IDO2-inactivating SNPs was significantly associated with markedly improved disease-free survival in response to adjuvant radiotherapy (P < 0.01), a treatment modality that has been highly debated due to its variable efficacy. CONCLUSIONS: The results of this study provide genetic support for IDO2 as a contributing factor in PDAC development and argue that IDO2 genotype analysis has the immediate potential to influence the PDAC care decision-making process through stratification of those patients who stand to benefit from adjuvant radiotherapy.


Assuntos
Biomarcadores Tumorais , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Alelos , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Genótipo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias Pancreáticas/radioterapia , Polimorfismo de Nucleotídeo Único , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
19.
EMBO Mol Med ; 11(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30518636

RESUMO

Though congenital hydrocephalus is heritable, it has been linked only to eight genes, one of which is MPDZ Humans and mice that carry a truncated version of MPDZ incur severe hydrocephalus resulting in acute morbidity and lethality. We show by magnetic resonance imaging that contrast medium penetrates into the brain ventricles of mice carrying a Mpdz loss-of-function mutation, whereas none is detected in the ventricles of normal mice, implying that the permeability of the choroid plexus epithelial cell monolayer is abnormally high. Comparative proteomic analysis of the cerebrospinal fluid of normal and hydrocephalic mice revealed up to a 53-fold increase in protein concentration, suggesting that transcytosis through the choroid plexus epithelial cells of Mpdz KO mice is substantially higher than in normal mice. These conclusions are supported by ultrastructural evidence, and by immunohistochemistry and cytology data. Our results provide a straightforward and concise explanation for the pathophysiology of Mpdz-linked hydrocephalus.


Assuntos
Permeabilidade Capilar , Proteínas de Transporte/genética , Plexo Corióideo/patologia , Plexo Corióideo/fisiopatologia , Hidrocefalia/patologia , Hidrocefalia/fisiopatologia , Animais , Meios de Contraste/análise , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Imageamento por Ressonância Magnética , Proteínas de Membrana , Camundongos
20.
Methods Mol Biol ; 1882: 239-252, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30378060

RESUMO

Post-transcriptional regulation of gene expression through interaction between RNA binding proteins (RBPs) and target mRNAs have gained considerable interest over the last decade. Altered expression of RBPs as detected in pancreatic ductal adenocarcinoma (PDAC) cells alters mRNA processing, and in turn, the entire transcriptome and proteome. Thus, this gene regulatory mechanism can regulate important pro-oncogenic signaling pathways (e.g., TP53, WEE1, and c-MYC) in PDAC cells. Ribonucleoprotein immunoprecipitation assays (RNP-IP or RIP) are a modified immunoprecipitation method to study physical interactions between RBPs and their mRNA targets. As a first step to explore RBP interactomes and define novel therapeutic targets and dysregulated pathways in disease, RIPs are a sensitive and established molecular biology technique used to isolate and differentiate bound transcripts to RBPs in a variety of experimental conditions. This chapter describes an up-to-date, detailed protocol for performing this assay in mammalian cytoplasmic extracts (i.e., PDAC cells), and reviews current methods to validate target binding sites such as electrophoretic mobility shift assay (EMSA) and cross-linking immunoprecipitation polymerase chain reaction (CLIP-PCR).


Assuntos
Carcinoma Ductal Pancreático/genética , Perfilação da Expressão Gênica/métodos , Neoplasias Pancreáticas/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Reagentes para Ligações Cruzadas/química , Ensaio de Desvio de Mobilidade Eletroforética/instrumentação , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Perfilação da Expressão Gênica/instrumentação , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunoprecipitação/instrumentação , Imunoprecipitação/métodos , Neoplasias Pancreáticas/patologia , Ligação Proteica , RNA Mensageiro/química , Proteínas de Ligação a RNA/química
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