RESUMO

The molecular processes linked to the development and progression of Crohn's disease (CD) and ulcerative colitis (UC) are not completely understood. MicroRNAs (miRNAs) regulate gene expression and are indicated as diagnostic, prognostic, and predictive biomarkers in chronic degenerative diseases. Our objectives included the identification of global miRNA expression in CD and UC, as well as miRNA target genes, miRNA-mRNA interaction networks, and biological functions associated with these different forms of inflammatory bowel disease (IBD). METHODS: By performing a comprehensive meta-analysis, we integrated miRNA expression data from nine studies in IBD. We obtained detailed information on significantly deregulated miRNAs (fold change, FC ≥ 2 and p < 0.05), sample type and number, and platform applied for analysis in the training and validation sets. Further bioinformatic analyses were performed to identify miRNA target genes, by using the microRNA Data Integration Portal tool. We also sought to identify statistically enriched pathways of genes regulated by miRNAs using ToppGene Suite. Additional analyses were performed to filter for genes expressed in intestinal tissue using the European Bioinformatics Institute (EBI) database. RESULTS: Our findings showed the upregulation of 15 miRNAs in CD and 33 in UC. Conversely, six miRNAs were downregulated in CD, while seven were downregulated in UC. These results indicate a greater deregulation of miRNAs in UC compared to CD. Of note, miRNA target genes were enriched for immune system regulation pathways. Among significantly deregulated miRNAs with a higher number of miRNA-target gene interactions, we identified miR-199a-5p and miR-362-3p altered in CD, while among UC case patients, miRNA-target gene interactions were higher for miR-155-5p. CONCLUSIONS: The identified miRNAs play roles in regulating genes associated with immune system regulation and inflammation in IBD. Such miRNAs and their target genes have the potential to serve as clinically relevant biomarkers. These findings hold promise for enhancing the accuracy of diagnoses and facilitating the development of personalized treatment strategies for individuals with various forms of IBD.

RESUMO

Herein, we aimed at identifying global transcriptome microRNA (miRNA) changes and miRNA target genes in lung adenocarcinoma. Samples were selected as training (N = 24) and independent validation (N = 34) sets. Tissues were microdissected to obtain >90% tumor or normal lung cells, subjected to miRNA transcriptome sequencing and TaqMan quantitative PCR validation. We further integrated our data with published miRNA and mRNA expression datasets across 1,491 lung adenocarcinoma and 455 normal lung samples. We identified known and novel, significantly over- and under-expressed (p ≤ 0.01 and FDR≤0.1) miRNAs in lung adenocarcinoma compared to normal lung tissue: let-7a, miR-10a, miR-15b, miR-23b, miR-26a, miR-26b, miR-29a, miR-30e, miR-99a, miR-146b, miR-181b, miR-181c, miR-421, miR-181a, miR-574 and miR-1247. Validated miRNAs included let-7a-2, let-7a-3, miR-15b, miR-21, miR-155 and miR-200b; higher levels of miR-21 expression were associated with lower patient survival (p = 0.042). We identified a regulatory network including miR-15b and miR-155, and transcription factors with prognostic value in lung cancer. Our findings may contribute to the development of treatment strategies in lung adenocarcinoma.

Assuntos

Adenocarcinoma/genética , Redes Reguladoras de Genes/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Transcriptoma , Adenocarcinoma/mortalidade , Adenocarcinoma de Pulmão , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Fatores de Transcrição/genética

RESUMO

BACKGROUND: Hemangioma is a common benign tumor in the childhood; however our knowledge about the molecular mechanisms of hemangioma development and progression are still limited. Currently, microRNAs (miRNAs) have been shown as gene expression regulators with an important role in disease pathogenesis. Our goals were to identify miRNA-mRNA expression networks associated with infantile hemangioma. METHODS: We performed a meta-analysis of previously published gene expression datasets including 98 hemangioma samples. Deregulated genes were further used to identify microRNAs as potential regulators of gene expression in infantile hemangioma. Data were integrated using bioinformatics methods, and genes were mapped in proteins, which were then used to construct protein-protein interaction networks. RESULTS: Deregulated genes play roles in cell growth and differentiation, cell signaling, angiogenesis and vasculogenesis. Regulatory networks identified included microRNAs miR-9, miR-939 and let-7 family; these microRNAs showed the most number of interactions with deregulated genes in infantile hemangioma, suggesting that they may have an important role in the molecular mechanisms of disease. Additionally, results were used to identify drug-gene interactions and druggable gene categories using Drug-Gene Interaction Database. We show that microRNAs and microRNA-target genes may be useful biomarkers for the development of novel therapeutic strategies for patients with infantile hemangioma. CONCLUSIONS: microRNA-regulated pathways may play a role in infantile hemangioma development and progression and may be potentially useful for future development of novel therapeutic strategies for patients with infantile hemangioma.

Assuntos

Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Hemangioma/genética , MicroRNAs/genética , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células , Biologia Computacional , Progressão da Doença , Humanos , Lactente , MicroRNAs/metabolismo , Mapas de Interação de Proteínas

RESUMO

Aquaporins have important roles in various physiological processes in plants, including growth, development and adaptation to stress. In this study, a gene encoding a root-specific tonoplast intrinsic aquaporin (TIP) from Eucalyptus grandis (named EgTIP2) was investigated. The root-specific expression of EgTIP2 was validated over a panel of five eucalyptus organ/tissues. In eucalyptus roots, EgTIP2 expression was significantly induced by osmotic stress imposed by PEG treatment. Histochemical analysis of transgenic tobacco lines (Nicotiana tabacum SR1) harboring an EgTIP2 promoter:GUS reporter cassette revealed major GUS staining in the vasculature and in root tips. Consistent with its osmotic-stress inducible expression in eucalyptus, EgTIP2 promoter activity was up-regulated by mannitol treatment, but was down-regulated by abscisic acid. Taken together, these results suggest that EgTIP2 might be involved in eucalyptus response to drought. Additional searches in the eucalyptus genome revealed the presence of four additional putative TIP coding genes, which could be individually assigned to the classical TIP1-5 groups.

Assuntos

Adaptação Fisiológica , Aquaporinas/genética , Eucalyptus/genética , Regulação da Expressão Gênica de Plantas , Estresse Fisiológico , Aquaporinas/metabolismo , Sequência de Bases , Eucalyptus/citologia , Eucalyptus/fisiologia , Genes Reporter , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Pressão Osmótica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Nicotiana/genética , Nicotiana/fisiologia

RESUMO

Class III peroxidases (Prxs) are enzymes involved in a multitude of physiological and stress-related processes in plants. Here, we report on the characterization of a putative peroxidase-encoding gene from Coffea arabica (CaPrx) that is expressed in early stages of root-knot nematode (RKN) infection. CaPrx showed enhanced expression in coffee roots inoculated with RKN (at 12 h post-inoculation), but no significant difference in expression was observed between susceptible and resistant plants. Assays using transgenic tobacco plants harboring a promoter-ß-glucuronidase (GUS) fusion revealed that the CaPrx promoter was exclusively active in the galls induced by RKN. In cross sections of galls, GUS staining was predominantly localized in giant cells. Up-regulation of GUS expression in roots of transgenic plants following RKN inoculation was observed within 16 h. Moreover, no increase in GUS expression after treatment with jasmonic acid was detected. Altogether, these results point to a putative role of this peroxidase in the general coffee response to RKN infection.

Assuntos

Coffea/enzimologia , Coffea/genética , Genes de Plantas/genética , Peroxidases/genética , Doenças das Plantas/parasitologia , Raízes de Plantas/parasitologia , Tylenchoidea/fisiologia , Animais , Sequência de Bases , Coffea/imunologia , Coffea/parasitologia , Ciclopentanos/farmacologia , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Resistência à Doença/efeitos dos fármacos , Resistência à Doença/genética , Etiquetas de Sequências Expressas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes Reporter/genética , Glucuronidase/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Oxilipinas/farmacologia , Peroxidases/metabolismo , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Nicotiana/efeitos dos fármacos , Nicotiana/genética , Tylenchoidea/efeitos dos fármacos

RESUMO

A cDNA clone (designated CaIRL) encoding an isoflavone reductase-like protein from coffee (Coffea arabica) was retrieved during a search for genes showing organ/tissue-specific expression among the expressed sequence tags (EST) of the Brazilian coffee EST database. The CaIRL cDNA contains a single open reading frame of 946 nucleotides (nt) encoding 314 amino acids (predicted molecular weight of 34 kDa). Several features identified the predicted CaIRL protein as a new member of the PIP family of NADPH-dependent reductases. Expression studies demonstrated that CaIRL is expressed exclusively in coffee leaves and its transcript level is markedly increased in response to fungal infection and mechanical injury. Analysis of transgenic tobacco plants harboring a CaIRL 5'-flanking region (862 nt) fused to uidA reporter gene (GUS) confirmed the responsiveness of the putative promoter to abiotic stress in wounded leaves. In turn, a 5' deletion to -404 completely abolished promoter activation by abiotic stimulus in transgenic plants. The lack of GUS expression in non-wounded leaf tissues in transgenic tobacco was in contrast to the basal level of CaIRL expression observed in non-stressed healthy coffee leaves.

Assuntos

Café/enzimologia , Regulação da Expressão Gênica de Plantas , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Regiões Promotoras Genéticas , Estresse Fisiológico , Região 5'-Flanqueadora , Sequência de Aminoácidos , Café/genética , Café/microbiologia , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Filogenia , Doenças das Plantas/microbiologia , Folhas de Planta/enzimologia , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologia

RESUMO

BACKGROUND: Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress. RESULTS: The expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), beta-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions. CONCLUSION: Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.

Assuntos

Coffea/genética , Perfilação da Expressão Gênica/normas , Regulação da Expressão Gênica de Plantas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Coffea/fisiologia , Perfilação da Expressão Gênica/métodos , Proteínas de Plantas/genética , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos

RESUMO

An alpha actin gene segment, isolated from Nile tilapia (Oreochromis niloticus), was characterized by nucleotide sequencing, predicted amino acid sequence and Southern blot hybridization. Genomic DNA amplification resulted in a 1063-bp fragment corresponding to a partial alpha-cardiac muscle actin gene containing exons 3 to 6. Southern blot analysis of the restriction-digested DNA revealed that the Nile tilapia genome contains multiple muscle actin isoforms. Although comparison of the nucleotide sequence, amino acid residues and exon-intron organization of the isolated actin gene with those of other vertebrates showed a high level of identity, diagnostic amino acid residues can still be correlated to distinct actin genes in fish species.