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1.
Journal of Southern Medical University ; (12): 425-431, 2022.
Article in Chinese | WPRIM | ID: wpr-936333

ABSTRACT

OBJECTIVE@#To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene.@*METHODS@#The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription.@*RESULTS@#We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively.@*CONCLUSION@#pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.


Subject(s)
Humans , Core Binding Factor Alpha 1 Subunit/genetics , Lipopolysaccharides/pharmacology , Luciferases/genetics , Metformin/pharmacology , NFATC Transcription Factors/genetics , Promoter Regions, Genetic , T-Lymphocytes , Transcription, Genetic/drug effects , Transfection
2.
Protein & Cell ; (12): 29-38, 2021.
Article in English | WPRIM | ID: wpr-880916

ABSTRACT

Prostate cancer is the most commonly diagnosed non-cutaneous cancers in North American men. While androgen deprivation has remained as the cornerstone of prostate cancer treatment, resistance ensues leading to lethal disease. Forkhead box A1 (FOXA1) encodes a pioneer factor that induces open chromatin conformation to allow the binding of other transcription factors. Through direct interactions with the Androgen Receptor (AR), FOXA1 helps to shape AR signaling that drives the growth and survival of normal prostate and prostate cancer cells. FOXA1 also possesses an AR-independent role of regulating epithelial-to-mesenchymal transition (EMT). In prostate cancer, mutations converge onto the coding sequence and cis-regulatory elements (CREs) of FOXA1, leading to functional alterations. In addition, FOXA1 activity in prostate cancer can be modulated post-translationally through various mechanisms such as LSD1-mediated protein demethylation. In this review, we describe the latest discoveries related to the function and regulation of FOXA1 in prostate cancer, pointing to their relevance to guide future clinical interventions.


Subject(s)
Humans , Male , Amino Acid Sequence , Epigenesis, Genetic , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Hepatocyte Nuclear Factor 3-alpha/metabolism , Histone Demethylases/metabolism , Histones/metabolism , Mutation , Prostate/pathology , Prostatic Neoplasms/pathology , Protein Binding , Protein Processing, Post-Translational , Receptors, Androgen/metabolism , Signal Transduction , Transcription, Genetic
3.
China Journal of Chinese Materia Medica ; (24): 4367-4379, 2021.
Article in Chinese | WPRIM | ID: wpr-888135

ABSTRACT

The present study analyzed the effects of planting density on the development, quality, and gene transcription characte-ristics of Rehmannia glutinosa using 85-5 and J9 as materials with three planting densities of 5 000, 25 000, and 50 000 plants/Mu(1 Mu≈667 m~2). The agronomic characteristics of leaves and tuberous roots, the content of catalpol and acteoside, and the changes of gene expression were determined. The results showed that the leaf size, the diameter of tuberous root, leaf biomass, tuberous root number, and tuberous root biomass per plant at low density were significantly higher than those of medium and high densities. The content of catalpol and acteoside in leaves was higher at high density. The content of catalpol in tuberous roots was higher at low density, and the change trend was similar to that in leaves, while the content of acteoside in tuberous roots was higher at high density. Transcriptome analysis found that about 1/2 of the expansin genes could change regularly in response to density treatment, which was rela-ted to the development of tuberous roots. The change trend of the gene expression of multiple catalytic enzymes involved in the biosynthesis of catalpol and acteoside was consistent with that of their content, which was presumedly involved in the accumulation and regulation of density-responsive medicinal components. Based on the analysis of the development, medicinal components, and gene expression characteristics of R. glutinosa at different densities, this study is expected to provide an important basis for regulating the quality and yield of medicinal materials of R. glutinosa by managing the planting density.


Subject(s)
Gene Expression Profiling , Plant Leaves/genetics , Plant Roots/genetics , Rehmannia/genetics , Transcription, Genetic
4.
Electron. j. biotechnol ; 43: 55-61, Jan. 2020. tab, ilus, graf
Article in English | LILACS | ID: biblio-1087522

ABSTRACT

Background: Matrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology. Results: The HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells. Conclusions: This novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.


Subject(s)
Humans , Matrix Metalloproteinase 12/genetics , CRISPR-Cas Systems , Luciferases/genetics , Transcription, Genetic , Cell Communication , Cell Line , Promoter Regions, Genetic/genetics , Cell Culture Techniques , Extracellular Matrix , Gene Knock-In Techniques , Clustered Regularly Interspaced Short Palindromic Repeats
5.
Chinese Journal of Contemporary Pediatrics ; (12): 24-30, 2020.
Article in Chinese | WPRIM | ID: wpr-781700

ABSTRACT

OBJECTIVE@#To study the expression of microRNA-495-5p (miRNA-495-5p) in the serum of preterm infants with bronchopulmonary dysplasia (BPD) based on a bioinformatics analysis, and to provide a theoretical basis for further research on the association between miRNA-495-5p and BPD.@*METHODS@#A total of 40 preterm infants who were admitted to the neonatal intensive care unit from January 2015 to December 2016 were enrolled. Among these infants, 20 with early clinical manifestations of BPD were enrolled as the BPD group, and 20 without such manifestations were enrolled as the control group. Peripheral blood samples were collected. The miRNA microarray technique was used to screen out differentially expressed miRNAs in serum between the two groups. RT-PCR was used for validation of results. TargetScan, miRDB, and miRWalk databases were used to predict the target genes of miRNA-495-5p. The DAVID database was used to perform gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the target genes.@*RESULTS@#Compared with the control group, the BPD group had a significant increase in the expression of miRNA-495-5p in serum (P<0.05). A total of 117 target genes of miRNA-495-5p were predicted by the above three databases and they were involved in several molecular functions (including transcriptional regulatory activity, transcriptional activation activity, and transcription cofactor activity), biological processes (such as metabolic regulation, DNA-dependent transcriptional regulation, and vascular pattern), and cell components (including nucleoplasm, membrane components, and insoluble components) (P<0.05). As for signaling pathways, these genes were significantly enriched in the mTOR signaling pathway (P<0.05).@*CONCLUSIONS@#MiRNA-495-5p may be involved in the development and progression of BPD by regulating angiogenesis, stem cell differentiation, apoptosis, and autophagy, which provides clues for further research on the role and functional mechanism of miRNA-495-5p in BPD.


Subject(s)
Humans , Infant, Newborn , Bronchopulmonary Dysplasia , Computational Biology , Infant, Premature , MicroRNAs , Genetics , Transcription, Genetic
7.
Chinese Journal of Hematology ; (12): 889-894, 2019.
Article in Chinese | WPRIM | ID: wpr-1012091

ABSTRACT

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.


Subject(s)
Humans , China , Core Binding Factor Alpha 2 Subunit , Leukemia, Myeloid, Acute , RUNX1 Translocation Partner 1 Protein , Real-Time Polymerase Chain Reaction , Transcription, Genetic , WT1 Proteins
8.
Journal of Experimental Hematology ; (6): 1463-1468, 2019.
Article in Chinese | WPRIM | ID: wpr-775698

ABSTRACT

OBJECTIVE@#To investigate the transcriptional regulation of transcription factor MZF-1 on acute monocytic leukemia-related gene MLAA-34.@*METHODS@#The effect of MZF-1 on the transcriptional activity of MLAA-34 gene promoter was analyzed by luciferase reporter gene detection system and site-directed mutation technique. The EMSA and ChIP assay were used to verify whether MZF-1 directly and specifically binds to the core region of MLAA-34 promoter. The over-expression vector and interference vector of MZF-1 were constructed to transfect U937 cells, and RT-PCR and Western blot were used to detect the transcription and expression changes of MLAA-34 gene.@*RESULTS@#The transcription factor MZF-1 had a regulatory effect on MLAA-34 gene expression, and the relative luciferase activity was decreased after MZF-1 binding point mutation (P<0.01). EMSA and ChIP experiments demonstrated that MZF-1 could directly bind to MLAA-34 promoter and play a regulatory role. In the over-expression test, the increase of MZF-1 could up-regulate the expression of MLAA-34 (P<0.05). In the interference test, the decrease of MZF-1 could down-regulate the expression of MLAA-34 (P<0.05).@*CONCLUSION@#Transcription factor MZF-1 can bind to the transcriptional regulatory region on the promoter of MLAA-34 gene and promote the transcription of MLAA-34 gene in acute monocytic leukemia.


Subject(s)
Humans , Antigens, Neoplasm , Genetics , Apoptosis Regulatory Proteins , Genetics , Gene Expression Regulation, Neoplastic , Genes, Reporter , Hepatocyte Nuclear Factor 1-alpha , Kruppel-Like Transcription Factors , Metabolism , Leukemia, Monocytic, Acute , Promoter Regions, Genetic , Transcription, Genetic
9.
Genomics, Proteomics & Bioinformatics ; (4): 140-153, 2019.
Article in English | WPRIM | ID: wpr-772940

ABSTRACT

Enhancers activate transcription in a distance-, orientation-, and position-independent manner, which makes them difficult to be identified. Self-transcribing active regulatory region sequencing (STARR-seq) measures the enhancer activity of millions of DNA fragments in parallel. Here we used STARR-seq to generate a quantitative global map of rice enhancers. Most enhancers were mapped within genes, especially at the 5' untranslated regions (5'UTR) and in coding sequences. Enhancers were also frequently mapped proximal to silent and lowly-expressed genes in transposable element (TE)-rich regions. Analysis of the epigenetic features of enhancers at their endogenous loci revealed that most enhancers do not co-localize with DNase I hypersensitive sites (DHSs) and lack the enhancer mark of histone modification H3K4me1. Clustering analysis of enhancers according to their epigenetic marks revealed that about 40% of identified enhancers carried one or more epigenetic marks. Repressive H3K27me3 was frequently enriched with positive marks, H3K4me3 and/or H3K27ac, which together label enhancers. Intergenic enhancers were also predicted based on the location of DHS regions relative to genes, which overlap poorly with STARR-seq enhancers. In summary, we quantitatively identified enhancers by functional analysis in the genome of rice, an important model plant. This work provides a valuable resource for further mechanistic studies in different biological contexts.


Subject(s)
Acetylation , Base Sequence , Deoxyribonuclease I , Metabolism , Enhancer Elements, Genetic , Epigenesis, Genetic , Genes, Plant , Genomics , Methods , Histone Code , Genetics , Histones , Metabolism , Models, Genetic , Oryza , Genetics , Promoter Regions, Genetic , Genetics , Repetitive Sequences, Nucleic Acid , Genetics , Sequence Analysis, DNA , Transcription, Genetic
10.
Genomics, Proteomics & Bioinformatics ; (4): 129-139, 2019.
Article in English | WPRIM | ID: wpr-772938

ABSTRACT

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 helper T (T) cells and CD8 cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T1 and T2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent T1 or T2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T1/T2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T1/T2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.


Subject(s)
Humans , Antigens , Metabolism , CTLA-4 Antigen , Metabolism , Cell Differentiation , Cell Line , Cytokines , Metabolism , Cytotoxicity, Immunologic , Granulocyte-Macrophage Colony-Stimulating Factor , Pharmacology , Lymphocyte Activation , Allergy and Immunology , Lymphocyte Subsets , Metabolism , Phenotype , Proteomics , Receptors, Chimeric Antigen , Metabolism , Single-Cell Analysis , Methods , T-Lymphocytes, Regulatory , Metabolism , Th1 Cells , Cell Biology , Th2 Cells , Cell Biology , Transcription, Genetic , Up-Regulation
11.
Journal of Central South University(Medical Sciences) ; (12): 122-127, 2019.
Article in Chinese | WPRIM | ID: wpr-813101

ABSTRACT

To clone human mitogen-activated protein kinase kinase 6 (MKK6) gene promoter and explore its transcription activity by ubiquitin specific peptidase 22 (USP22).
 Methods: MKK6 gene promoter was amplified by PCR and two bases mutation within USP22 binding site was subsequently introduced. The wild type and mutant MKK6 promoter were inserted into the luciferase report vector pGL3-Basic, respectively. Recombinant plasmids were co-transfected with plasmid pRL-TK into HeLa cells, and the luciferase activities were measured by dual luciferase reporter system. Furthermore, the direct interaction between USP22 and MKK6 promoter was detected by chromatin immunoprecipitation (ChIP) assay. Finally, the MKK6 transcription activity was measured after knockdown of USP22.
 Results: The recombinant luciferase report vectors containing wild or mutant type of MKK6 promoter were successfully constructed. Mutation of USP22 binding site resulted in decrease of MKK6 promoter-driven luciferase activity in HeLa cells (P<0.05). USP22 could interact directly with MKK6 promoter. Down-regulation of USP22 led to the decreased MKK6 mRNA expression (P<0.05).
 Conclusion: USP22 could regulate the transcription activity of MKK6 gene in HeLa cells.


Subject(s)
Humans , HeLa Cells , Luciferases , MAP Kinase Kinase 6 , Promoter Regions, Genetic , Thiolester Hydrolases , Metabolism , Transcription, Genetic
12.
São Paulo; s.n; 2019. 105 p. ilust, tabelas.
Thesis in Portuguese | LILACS, Inca | ID: biblio-1179932

ABSTRACT

Introdução: Linfoma de Hodgkin (LH) é um dos linfomas mais frequentes no mundo ocidental, com cerca de 3 a 4 novos casos a cada 100 mil pessoas. Os microRNAs são pequenas moléculas que regulam a transcrição gênica e estão associadas ao desenvolvimento e progressão das neoplasias. Em LH clássico (LHC), alguns microRNAs já foram identificados a partir de células infectadas pelo EBV. Além destes, outros microRNAs já foram descritos em LHC, tanto em estudos onde foram realizadas esta avaliação em culturas celulares como utilizando a massa tumoral total de linfonodos, mas com pouca correlação com o comportamento biológico. Objetivo: O presente estudo tem como objetivo principal avaliar o perfil de expressão de microRNA em LHC, comparando casos livres de doença após a terapia com casos que exibiram recidivas. Material e métodos: Foram avaliados 106 pacientes com o diagnóstico de LHC, sendo coletados dados clínicos e avaliados os marcadores imuno-histoquímicos CD30, CD15, CD20 e CD15 destes casos, além da presença de EBV por CISH. Vinte casos foram selecionados para a avaliação da expressão de 377 microRNAs, 10 casos com recidiva e 10 casos sem evidência de doença. Como grupos comparadores, foram selecionadas 8 amostras de linfonodos reativos, 4 com hiperplasia folicular e 4 com hiperplasia paracortical. Os resultados do perfil de expressão foram submetidos ao método de agrupamento hierárquico não supervisionado. Resultados e discussão: Os parâmetros clínico-patológicos desta coorte não diferiram daqueles encontrados na literatura. A sobrevida global e a sobrevida livre de progressão foram de 92,1% e 83,6% em 5 anos, respectivamente. Os fatores associados ao prognóstico em análise multivariada foram idade, níveis séricos de albumina e DHL e estadiamento. O perfil de expressão dos microRNAs de todos os casos de LHC possibilitaram a separação completa dos mesmos em relação aos linfonodos reativos, independentemente do tipo de hiperplasia. Na comparação entre os casos de LHC, foram identificados 3 microRNAs diferencialmente expressos: miR-502-3p e miR-363, ambos hipoexpressos no grupo que continha todos os casos com recidivas e miR-886-5p, hiperexpresso neste grupo. Os dois primeiros microRNAs estão associados a supressão tumoral, através da inibição da proliferação e migração celular. O miR-886-5p é associado ao aumento da proliferação celular e inibição da apoptose. Em linfomas, este já foi descrito como estando hiperexpresso em linfomas T, incluindo linfoma anaplásico de grandes células. Conclusão: Os LHC possuem um perfil distinto de expressão de microRNAs, com muitos deles envolvendo mecanismos fundamentais na oncogênese, como ciclo celular e apoptose. Os microRNAs encontrados diferencialmente expressos nos casos recidivados no presente estudo podem estar associados ao comportamento menos indolente destas neoplasias, podendo ser alvo de futuras investigações, incluindo seu uso como potenciais alvos de terapia


Introduction: Hodgkin lymphoma (HL) is one of the most common lymphomas in the western world, with about 3 to 4 new cases per 100,000 people. MicroRNAs are small molecules that regulate gene transcription and are associated with the development and progression of neoplasms. In classic HL (CHL), some microRNAs have already been identified from EBV-infected cells. In addition, other microRNAs have already been described in CHL, both in studies where this evaluation was performed in cell cultures and using whole lymph node tumor tissue, but with little correlation with biological behavior. Objective: The present study aims to evaluate the microRNA expression profile in CHL, comparing disease-free cases after therapy with cases that showed recurrences. A total of 106 patients diagnosed with CHL were evaluated. Clinical data were collected and the immunohistochemical markers CD30, CD15, CD20 and CD15 of these cases were evaluated, as well as the presence of EBV by CISH. Twenty cases were selected for the analysis of the expression of 377 microRNAs, 10 cases with recurrences and 10 cases without evidence of disease. For comparison, 8 samples of reactive lymph nodes were selected, 4 with follicular hyperplasia and 4 with paracortical hyperplasia. The expression profile results were submitted to the unsupervised hierarchical clustering method. Results and discussion: The clinico-pathological parameters of this cohort did not differ from those found in the literature. Overall survival and progression-free survival were 92.1% and 83.6% at 5 years, respectively. Factors associated with prognosis in multivariate analysis were age, serum albumin and DHL levels, and staging. The microRNA expression profile of all CHL cases allowed their complete separation from reactive lymph nodes, regardless of the type of hyperplasia. When comparing the cases of CHL, 3 differentially expressed microRNAs were identified: miR-502-3p and miR-363, both hypoexpressed in the group containing all relapsed cases and miR-886-5p, hyperexpressed in this group. The first two microRNAs are associated with tumor suppression through inhibition of cell proliferation and migration. The miR-886-5p is associated with increased cell proliferation and inhibition of apoptosis. In lymphomas, it has been described as being overexpressed in T-cell lymphomas, including anaplastic large cell lymphoma. Conclusion: CHLs have a distinct microRNA expression profile, with many involving key mechanisms in oncogenesis, such as cell cycle and apoptosis. The differentially expressed microRNAs found in the relapsed cases in the present study may be associated with the less indolent behavior of these neoplasms and may be the subject of future investigations, including their use as potential therapeutic targets


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Transcription, Genetic , Hodgkin Disease , Immunohistochemistry , Gene Expression Profiling , MicroRNAs , Lymphoma , Recurrence , Cell Cycle , Apoptosis
13.
São Paulo; s.n; 2019. 82 p. ilust, quadros.
Thesis in Portuguese | LILACS, Inca | ID: biblio-1179697

ABSTRACT

ERBB2/HER2 é um gene frequentemente amplificado em cânceres de mama, estômago e ovário e está diretamente associado ao mal prognóstico de pacientes portadores desta alteração genética. A proteína HER2, super-expressa nestes tumores, é um importante alvo de terapias dirigidas e tem um papel relevante no tratamento de tumores com esta característica molecular. Entretanto, o surgimento de resistência aos tratamentos ainda é um desafio a ser superado. Em 2011, foi identificado o miR-4728-3p dentro de uma região intrônica de ERBB2/HER2, cuja super-expressão acompanhava a amplificação do oncogene. Pouco ainda se sabe sobre a função e possíveis alvos deste microRNA, o que motivou os estudos desta tese, onde usamos técnicas como RNA-Seq e proteômica buscando a identificação de alvos, seguidos de comparação com bancos de dados, além de ensaios in vitro e in vivo. Genes como RNASEH1, PSMC3, TUFM e GNA11 mostraram-se fortes candidatos-alvo do miR-4728-3p, sendo regulados após super-expressão e knockdown do microRNA em diferentes linhagens celulares. O bloqueio de miR-4728-3p em tumores gástricos HER2+ levou à diminuição do volume e peso tumoral, sugerindo um importante papel terapêutico no controle de tumores com amplificação de ERBB2/HER2. Os resultados obtidos indicam uma possível participação do miR-4728-3p na carcinogênese de tumores gástricos HER2+ e ampliam o conhecimento sobre a biologia de ERBB2/HER2. A identificação de alvos deste microRNA pode adicionalmente elucidar os mecanismos pelo qual o bloqueio do miR-4728-3p interfere no crescimento tumoral, levantando informações importantes sobre o controle da expressão gênica mediada por RNAs não- codificantes no câncer


The ERBB2/HER2 gene is frequently amplified in breast, stomach and ovarian cancers and is directly associated with poor prognosis. The HER2 protein, overexpressed in these tumors, is an important therapeutic target and plays relevant roles in tumor biology and prognosis. However, the emergence of resistance to anti-HER2 treatment is still a challenge to be overcomed. In 2011, miR-4728-3p was identified within an intronic region of the ERBB2/HER2 gene; when this region is amplified, both transcripts are also amplified and overexpressed. Little is known about the function and possible targets of this microRNA, which motivated the studies of this thesis, where techniques such as RNA-Seq and proteomics were employed to identify targets, followed by database analyses, in vitro and in vivo assays. Genes such as RNASEH1, PSMC3, TUFM and GNA11 were found to be strong target-candidates of miR-4728-3p and were regulated after the overexpression or knockdown of this miRNA in different cell lines. miR-4728-3p blockade in HER2+ gastric tumors of mouse xenografts led to decreased tumor volume and weight, suggesting its important therapeutic potential in the control of ERBB2/HER2 amplified tumors. The results obtained here indicate a possible participation of miR-4728-3p in the carcinogenesis of HER2+ gastric tumors and expand the knowledge of ERBB2/HER2 biology. The identification of targets of this microRNA may further elucidate the mechanisms by which miR-4728-3p blockade interferes with tumor growth, raising important information about the control of gene expression mediated by noncoding RNAs in cancer


Subject(s)
Stomach Neoplasms , Transcription, Genetic , Breast Neoplasms , Biomarkers, Tumor , Receptor, ErbB-2 , MicroRNAs
14.
Braz. j. microbiol ; 49(4): 770-776, Oct.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-974307

ABSTRACT

ABSTRACT Anaerobic digestion is important for the management of livestock manure with high ammonia level. Although ammonia effects on anaerobic digestion have been comprehensively studied, the molecular mechanism underlying ammonia inhibition still remains elusive. In this study, based on metatranscriptomic analysis, the transcriptional profile of microbial community in anaerobic digestion under low (1500 mg L-1) and high NH4 + (5000 mg L-1) concentrations, respectively, were revealed. The results showed that high NH4 + concentrations significantly inhibited methane production but facilitated the accumulations of volatile fatty acids. The expression of methanogenic pathway was significantly inhibited by high NH4 + concentration but most of the other pathways were not significantly affected. Furthermore, the expressions of methanogenic genes which encode acetyl-CoA decarbonylase and methyl-coenzyme M reductase were significantly inhibited by high NH4 + concentration. The inhibition of the co-expressions of the genes which encode acetyl-CoA decarbonylase was observed. Some genes involved in the pathways of aminoacyl-tRNA biosynthesis and ribosome were highly expressed under high NH4 + concentration. Consequently, the ammonia inhibition on anaerobic digestion mainly focused on methanogenic process by suppressing the expressions of genes which encode acetyl-CoA decarbonylase and methyl-coenzyme M reductase. This study improved the accuracy and depth of understanding ammonia inhibition on anaerobic digestion.


Subject(s)
Bacteria/genetics , Bacteria/metabolism , Ammonia/metabolism , Bacteria/isolation & purification , Bacteria/classification , Transcription, Genetic , Bioreactors/microbiology , Fatty Acids, Volatile/metabolism , Microbiota , Anaerobiosis , Methane/metabolism
15.
Arq. Asma, Alerg. Imunol ; 2(3): 324-334, jul.set.2018. ilus
Article in Portuguese | LILACS | ID: biblio-1380922

ABSTRACT

Glicocorticosteroides são fármacos efetivos no tratamento de doenças inflamatórias e imunes. Agem em praticamente todas as células do corpo, antagonizando os efeitos patogênicos de inúmeras doenças. A maior parte de seus efeitos parece ser produto de sua ligação a receptores específicos armazenados no interior das células. Suas ações moduladoras da transcrição genética iniciam-se com a ligação ao seu receptor e posterior conexão aos genes alvo, num processo que conta com a participação de outros fatores e envolve múltiplos mecanismos (ação genômica). Os genes alvo incluem aqueles responsáveis por mediadores inflamatórios, como quimiocinas, citocinas, fatores de crescimento e seus receptores. Além de seus efeitos sobre o DNA, estimulando a produção de produtos anti-inflamatórios ou inibindo a transcrição de genes pró-inflamatórios, via acetilação ou deacetilação das histonas, respectivamente, os glicocorticosteroides possuem outros mecanismos de ação que não envolvem regulação genética (ação não genômica). Aparentemente, por mecanismos ainda não esclarecidos, os efeitos da corticoterapia são produto da associação das ações genômicas com as não genômicas. Os glicocorticosteroides representam o grande pilar terapêutico da asma, com efeitos sobre as células estruturais e funcionais do trato respiratório. Nessa situação particular, na qual costumam ser empregados continuadamente por períodos prolongados, com risco potencial de efeitos indesejáveis relevantes, é fundamental desvendar os processos envolvidos em seus mecanismos de ação para tentar desenvolver meios de reduzir os riscos associados e potencializar os efeitos desejados.


Glucocorticosteroids are effective drugs in the treatment of inflammatory and immune diseases. They act on virtually every cell in the body by antagonizing the pathogenic effects of numerous diseases. Most of its effects appear to be the product of its binding to specific receptors stored within cells. Its modulatory actions on genetic transcription begin with a linkage to its receptor and later connection to target genes, in a process that counts on the participation of other factors and involves multiple mechanisms (genomic action). Target genes include those responsible for inflammatory mediators, such as chemokines, cytokines, growth factors, and their receptors. In addition to its effects on DNA, stimulating the production of anti-inflammatory products or inhibiting the transcription of pro-inflammatory genes, via acetylation or deacetylation of histones, respectively, glucocorticosteroids have other mechanisms of action that do not involve genetic regulation (non-genomic effect). Apparently, by mechanisms not yet clarified, the effects of corticotherapy are the product of the association of genomic and non-genomic actions. Glucocorticosteroids represent the great therapeutic pillar of asthma, with effects on structural and functional cells of the respiratory tract. In this particular situation, where they are often used for prolonged periods, with a potential risk of relevant undesirable effects, it is essential to uncover the processes involved in their mechanisms of action in order to develop ways to reduce the associated risks and potentiate the desired effects.


Subject(s)
Humans , Asthma , Glucocorticoids , Glucocorticoids/administration & dosage , Respiratory System , Therapeutics , Transcription, Genetic , DNA , Cytokines , Adrenal Cortex Hormones , Genomics
16.
Salud pública Méx ; 60(1): 48-55, Jan.-Feb. 2018. graf
Article in English | LILACS | ID: biblio-903862

ABSTRACT

Abstract: Objective: To analyze the transcription pattern of neuropeptides in the ontogeny of a malaria vector, the mosquito Anopheles albimanus. Materials and methods: The transcription pattern of Crustacean CardioActive peptide (CCAP), corazonin, Ecdysis Triggering Hormone (ETH), allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR on larvae (1st - 4th instar), pupae and adult mosquitoes. Results: Unlike in other insects, transcripts of CCAP (70.8%), ETH (60.2%) and corazonin (76.5%) were expressed in 4th instar larvae, probably because these three neuropeptides are associated with the beginning of ecdysis. The neuropeptide ILP2 showed higher transcription levels in other stages and orcokinin decreased during the development of the mosquito. Conclusion: The CCAP, corazonin and ETH neuropeptides are potential targets for the design of control strategies aimed at disrupting An. albiamnus larval development.


Resumen: Objetivo: Describir la expresión de neuropéptidos durante la ontogenia del mosquito vector de la malaria Anopheles albimanus. Material y métodos: Se midió la expresión de CCAP, corazonina, ETH, allatostatina, orcokinina, ILP2, ILP5 y bursicon en larvas de primer (2mm), segundo (4mm), tercer (5mm) y cuarto (6mm) estadio, pupas y mosquitos adultos, mediante qPCR. Resultados. A diferencia de otros insectos en donde, CCAP, corazonina y ETH se expresan principalmente en estadios pupales, en An. albimanus se expresaron mayoritariamente en larvas de cuarto estadio, CCAP tuvo 70.8% de expresión relativa, corazonina 76.5% y ETH 60.2%. ILP2 fue el neuropéptido que más se expresó en el primer, segundo y tercer estadio y orcokinina disminuyó durante el desarrollo del mosquito. Conclusión. Los péptidos estudiados se expresaron en todos los estadios de desarrollo del mosquito. Sin embargo, su expresión varió en cada uno de ellos. Los neuropéptidos CCAP, corazonina y ETH, que son esenciales para la transformación de lavas a pupas, pueden ser blancos potenciales para el diseño de estrategias de control dirigidas a interrumpir el desarrollo larvario de An. albimanus.


Subject(s)
Animals , Neuropeptides/biosynthesis , Molting/genetics , Insect Proteins/biosynthesis , Anopheles/genetics , Transcription, Genetic , Neuropeptides/genetics , Gene Expression Regulation, Developmental , Insect Proteins/genetics , Real-Time Polymerase Chain Reaction , Larva , Malaria , Anopheles/growth & development
17.
Journal of Zhejiang University. Science. B ; (12): 581-595, 2018.
Article in English | WPRIM | ID: wpr-1010396

ABSTRACT

Under different red (R):blue (B) photon flux ratios, the growth performance of rapeseed (Brassica napus L.) is significantly different. Rapeseed under high R ratios shows shade response, while under high B ratios it shows sun-type morphology. Rapeseed under monochromatic red or blue light is seriously stressed. Transcriptomic and proteomic methods were used to analyze the metabolic pathway change of rapeseed (cv. "Zhongshuang 11") leaves under different R:B photon flux ratios (including 100R:0B%, 75R:25B%, 25R:75B%, and 0R:100B%), based on digital gene expression (DGE) and two-dimensional gel electrophoresis (2-DE). For DGE analysis, 2054 differentially expressed transcripts (|log2(fold change)|≥1, q<0.005) were detected among the treatments. High R ratios (100R:0B% and 75R:25B%) enhanced the expression of cellular structural components, mainly the cell wall and cell membrane. These components participated in plant epidermis development and anatomical structure morphogenesis. This might be related to the shade response induced by red light. High B ratios (25R:75B% and 0R:100B%) promoted the expression of chloroplast-related components, which might be involved in the formation of sun-type chloroplast induced by blue light. For 2-DE analysis, 37 protein spots showed more than a 2-fold difference in expression among the treatments. Monochromatic light (ML; 100R:0B% and 0R:100B%) stimulated accumulation of proteins associated with antioxidation, photosystem II (PSII), DNA and ribosome repairs, while compound light (CL; 75R:25B% and 25R:75B%) accelerated accumulation of proteins associated with carbohydrate, nucleic acid, amino acid, vitamin, and xanthophyll metabolisms. These findings can be useful in understanding the response mechanisms of rapeseed leaves to different R:B photon flux ratios.


Subject(s)
Brassica napus/radiation effects , Brassica rapa/radiation effects , Carbon/chemistry , Chloroplasts/radiation effects , Computational Biology , Electrophoresis, Gel, Two-Dimensional , Gene Expression Regulation, Plant/radiation effects , Image Processing, Computer-Assisted , Light , Mass Spectrometry , Metabolic Networks and Pathways , Nitrogen/chemistry , Photons , Photosystem II Protein Complex/genetics , Plant Leaves/radiation effects , Plant Proteins/genetics , Proteome , Ribosomes , Transcription, Genetic , Transcriptome
18.
Asian Journal of Andrology ; (6): 284-289, 2018.
Article in English | WPRIM | ID: wpr-1009554

ABSTRACT

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.


Subject(s)
Animals , Cricetinae , Humans , Male , Connectin/genetics , Eukaryotic Initiation Factor-4G/genetics , Gene Expression Regulation/genetics , Gene Silencing , Growth Hormone/genetics , Hepatitis B Surface Antigens/genetics , Hepatitis B virus/genetics , Hepatitis B, Chronic/virology , Hydro-Lyases/metabolism , Pregnancy-Specific beta 1-Glycoproteins/genetics , RNA, Viral/analysis , Spermatozoa/virology , Trans-Activators/genetics , Transcription, Genetic , Transfection , Viral Regulatory and Accessory Proteins
19.
Neuroscience Bulletin ; (6): 747-758, 2018.
Article in English | WPRIM | ID: wpr-777020

ABSTRACT

Huntington's disease (HD) is a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin (Htt) protein. Mutant Htt causes synaptic transmission dysfunctions by interfering in the expression of synaptic proteins, leading to early HD symptoms. Synaptic vesicle proteins 2 (SV2s), a family of synaptic vesicle proteins including 3 members, SV2A, SV2B, and SV2C, plays important roles in synaptic physiology. Here, we investigated whether the expression of SV2s is affected by mutant Htt in the brains of HD transgenic (TG) mice and Neuro2a mouse neuroblastoma cells (N2a cells) expressing mutant Htt. Western blot analysis showed that the protein levels of SV2A and SV2B were not significantly changed in the brains of HD TG mice expressing mutant Htt with 82 glutamine repeats. However, in the TG mouse brain there was a dramatic decrease in the protein level of SV2C, which has a restricted distribution pattern in regions particularly vulnerable in HD. Immunostaining revealed that the immunoreactivity of SV2C was progressively weakened in the basal ganglia and hippocampus of TG mice. RT-PCR demonstrated that the mRNA level of SV2C progressively declined in the TG mouse brain without detectable changes in the mRNA levels of SV2A and SV2B, indicating that mutant Htt selectively inhibits the transcriptional expression of SV2C. Furthermore, we found that only SV2C expression was progressively inhibited in N2a cells expressing a mutant Htt containing 120 glutamine repeats. These findings suggest that the synaptic dysfunction in HD results from the mutant Htt-mediated inhibition of SV2C transcriptional expression. These data also imply that the restricted distribution and decreased expression of SV2C contribute to the brain region-selective pathology of HD.


Subject(s)
Animals , Mice , Aging , Metabolism , Brain , Metabolism , Pathology , Cell Line, Tumor , Gene Expression , Physiology , Huntingtin Protein , Genetics , Metabolism , Membrane Glycoproteins , Metabolism , Mice, Transgenic , Mutation , Nerve Tissue Proteins , Metabolism , RNA, Messenger , Metabolism , Transcription, Genetic , Physiology
20.
Genomics, Proteomics & Bioinformatics ; (4): 342-353, 2018.
Article in English | WPRIM | ID: wpr-772969

ABSTRACT

Transcriptional regulation is critical to cellular processes of all organisms. Regulatory mechanisms often involve more than one transcription factor (TF) from different families, binding together and attaching to the DNA as a single complex. However, only a fraction of the regulatory partners of each TF is currently known. In this paper, we present the Transcriptional Interaction and Coregulation Analyzer (TICA), a novel methodology for predicting heterotypic physical interaction of TFs. TICA employs a data-driven approach to infer interaction phenomena from chromatin immunoprecipitation and sequencing (ChIP-seq) data. Its prediction rules are based on the distribution of minimal distance couples of paired binding sites belonging to different TFs which are located closest to each other in promoter regions. Notably, TICA uses only binding site information from input ChIP-seq experiments, bypassing the need to do motif calling on sequencing data. We present our method and test it on ENCODE ChIP-seq datasets, using three cell lines as reference including HepG2, GM12878, and K562. TICA positive predictions on ENCODE ChIP-seq data are strongly enriched when compared to protein complex (CORUM) and functional interaction (BioGRID) databases. We also compare TICA against both motif/ChIP-seq based methods for physical TF-TF interaction prediction and published literature. Based on our results, TICA offers significant specificity (average 0.902) while maintaining a good recall (average 0.284) with respect to CORUM, providing a novel technique for fast analysis of regulatory effect in cell lines. Furthermore, predictions by TICA are complementary to other methods for TF-TF interaction prediction (in particular, TACO and CENTDIST). Thus, combined application of these prediction tools results in much improved sensitivity in detecting TF-TF interactions compared to TICA alone (sensitivity of 0.526 when combining TICA with TACO and 0.585 when combining with CENTDIST) with little compromise in specificity (specificity 0.760 when combining with TACO and 0.643 with CENTDIST). TICA is publicly available at http://geco.deib.polimi.it/tica/.


Subject(s)
Humans , Binding Sites , Chromatin Immunoprecipitation , Gene Expression Regulation , Hep G2 Cells , K562 Cells , Promoter Regions, Genetic , Sequence Analysis, DNA , Transcription Factors , Metabolism , Transcription, Genetic
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