RESUMO
This study aimed to shed light on the molecular and cellular mechanisms responsible for initiation and progression of liver malignancies by examining the role of phosphatase and tensin homolog on chromosome 10 (Pten) in liver tumor progression in miR-122a (Mir122a)-null mice. We generated and monitored liver tumor initiation in Mir122a-null Pten heterozygous (Mir122a-/-;Pten+/- and Mir122a-/-;Alb-Cre;Ptenfx/+) mice and compared the results with those in Mir122a-/- mice. Both Mir122a-/-;Pten+/- and Mir122a-/-;Alb-Cre;Ptenfx/+ mice developed visible liver tumor nodules at 6 months of age. In premalignant livers of Mir122a-/-;Pten+/- mice, decreased PTEN and increased phosphorylated AKT were specifically observed in periportal cells, associated with inflammatory and fibrotic microenvironments. Furthermore, IL-1ß and tumor necrosis factor-α levels significantly increased in Mir122a-/-;Pten+/- premalignant livers at 6 months of age. Oval cells expressing A6, epithelial cell adhesion molecule, keratin (K) 8, K19, and SRY (sex determining region Y)-box 9 (SOX9) were present in both Mir122a-/- and Mir122a-/-;Pten+/- livers. Interestingly, a hybrid hepatocyte-like population with intermediate levels of K8, HNF4α, and SOX9 was located proximally to the oval cells in Mir122a-/-;Pten+/- livers. Lineage-tracing experiments revealed that these intermediate levels of K8 hepatocyte-like cells may be the cells of origin for Mir122a-/-;Pten+/- liver tumors. These findings suggest that inflammatory microenvironments in the periportal area of Mir122a-null mice may locally cause Pten down-regulation and expand tumor-initiating cells, causing hepatocellular carcinoma.
Assuntos
Regulação Neoplásica da Expressão Gênica , Hepatócitos/patologia , Neoplasias Hepáticas Experimentais/patologia , MicroRNAs/fisiologia , Células-Tronco Neoplásicas/patologia , PTEN Fosfo-Hidrolase/fisiologia , Microambiente Tumoral/imunologia , Animais , Feminino , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
The liver is a vital organ involving in various major metabolic functions in human body. MicroRNA-122 (miR-122) plays an important role in the regulation of liver metabolism, but its intrinsic physiological functions require further clarification. This study integrated the genome-scale metabolic model of hepatocytes and mouse experimental data with germline deletion of Mir122a (Mir122a-/-) to infer Warburg-like effects. Elevated expression of MiR-122a target genes in Mir122a-/-mice, especially those encoding for metabolic enzymes, was applied to analyze the flux distributions of the genome-scale metabolic model in normal and deficient states. By definition of the similarity ratio, we compared the flux fold change of the genome-scale metabolic model computational results and metabolomic profiling data measured through a liquid-chromatography with mass spectrometer, respectively, for hepatocytes of 2-month-old mice in normal and deficient states. The Ddc gene demonstrated the highest similarity ratio of 95% to the biological hypothesis of the Warburg effect, and similarity of 75% to the experimental observation. We also used 2, 6, and 11 months of mir-122 knockout mice liver cell to examined the expression pattern of DDC in the knockout mice livers to show upregulated profiles of DDC from the data. Furthermore, through a bioinformatics (LINCS program) prediction, BTK inhibitors and withaferin A could downregulate DDC expression, suggesting that such drugs could potentially alter the early events of metabolomics of liver cancer cells.
Assuntos
Hepatócitos/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Análise do Fluxo Metabólico/métodos , MicroRNAs/genética , Animais , Glucose/metabolismo , Humanos , Neoplasias Hepáticas/genética , Metabolômica , Camundongos , Camundongos Knockout , MicroRNAs/metabolismoRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC), a primary liver malignancy, is the most common cancer in males and fourth common cancer in females in Taiwan. HCC patients usually have a poor prognosis due to late diagnosis. It has been classified as a complex disease because of the heterogeneous phenotypic and genetic traits of the patients and a wide range of risk factors. Micro (mi)RNAs regulate oncogenes and tumor suppressor genes that are known to be dysregulated in HCC. Several studies have found an association between downregulation of miR-122, a liver-specific miRNA, and upregulation of paternally expressed gene 10 (PEG10) in HCC; however, the correlation between low miR-122 and high PEG10 levels still remains to be defined and require more investigations to evaluate their performance as an effective prognostic biomarker for HCC. METHODS: An in silico approach was used to isolate PEG10, a potential miR-122 target implicated in HCC development. miR-122S binding sites in the PEG10 promoter were evaluated with a reporter assay. The regulation of PEG10 by miR-122S overexpression was examined by quantitative RT-PCR, western blotting, and immunohistochemistry in miR-122 knockout mice and liver tissue from HCC patients. The relationship between PEG10 expression and clinicopathologic features of HCC patients was also evaluated. RESULTS: miR-122 downregulated the expression of PEG10 protein through binding to 3'-untranslated region (UTR) of the PEG10 transcript. In miR-122 knockout mice and HCC patients, the deficiency of miR-122 was associated with HCC progression. The expression of PEG10 was increased in 57.3 % of HCC as compared to paired non-cancerous tissue samples. However, significant upregulation was detected in 56.5 % of patients and was correlated with Okuda stage (P = 0.05) and histological grade (P = 0.001). CONCLUSIONS: miR-122 suppresses PEG10 expression via direct binding to the 3'-UTR of the PEG10 transcript. Therefore, while PEG10 could not be an ideal diagnostic biomarker for HCC but its upregulation in HCC tissue still has predictive value for HCC prognosis.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/metabolismo , Biossíntese de Proteínas/genética , Proteínas/genética , Regiões 3' não Traduzidas/genética , Animais , Proteínas Reguladoras de Apoptose , Sequência de Bases , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos Knockout , MicroRNAs/genética , Pessoa de Meia-Idade , Modelos Biológicos , Gradação de Tumores , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Transcrição Gênica , Regulação para Cima/genética , alfa-Fetoproteínas/metabolismoRESUMO
BACKGROUND: MicroRNAs (miRNAs) simultaneously target many transcripts through partial complementarity binding, and have emerged as a key type of post-transcriptional regulator for gene expression. How miRNA accomplishes its pleiotropic effects largely depends on its expression and its target repertoire. Previous studies discovered thousands of miRNAs and numerous miRNA target genes mainly through computation and prediction methods which produced high rates of false positive prediction. The development of Argonaute cross-linked immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) provides a system to effectively determine miRNA target genes. Likewise, the accuracy of dissecting the transcriptional regulation of miRNA genes has been greatly improved by chromatin immunoprecipitation of the transcription factors coupled with sequencing (ChIP-Seq). Elucidation of the miRNA target repertoire will provide an in-depth understanding of the functional roles of microRNA pathways. To reliably reconstruct a miRNA-mediated regulatory network, we established a computational framework using publicly available, sequence-based transcription factor-miRNA databases, including ChIPBase and TransmiR for the TF-miRNA interactions, along with miRNA-target databases, including miRTarBase, TarBase and starBase, for the miRNA-target interactions. We applied the computational framework to elucidate the miRNA-mediated regulatory network in the Mir122aâ»/â» mouse model, which has an altered transcriptome and progressive liver disease. RESULTS: We applied our computational framework to the expression profiles of miRNA/mRNA of Mir122aâ»/â» mutant mice and wild-type mice. The miRNA-mediated network involves 40 curated TFs contributing to the aberrant expression of 65 miRNAs and 723 curated miRNA target genes, of which 56% was found in the differentially-expressed genes of Mir122a--mice. Hence, the regulatory network disclosed previously-known and also many previously-unidentified miRNA-mediated regulations in mutant mice. Moreover, we demonstrate that loss of imprinting at the chromosome 12qF1 region is associated with miRNA overexpression in human hepatocellular carcinoma and stem cells, suggesting initiation of precancerous changes in young mice deficient in miR-122. A group of 9 miRNAs was found to share miR-122 target genes, indicating synergy between miRNAs and target genes by way of multiplicity and cooperativity. CONCLUSIONS: The study provides significant insight into miRNA-mediated regulatory networks. Based on experimentally verified data, this network is highly reliable and effective in revealing previously-undetermined disease-associated molecular mechanisms. This computational framework can be applied to explore the significant TF-miRNA-miRNA target interactions in any complex biological systems with high degrees of confidence.
Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Fígado/metabolismo , MicroRNAs/genética , Animais , Carcinoma Hepatocelular/genética , Imunoprecipitação da Cromatina , Bases de Dados Genéticas , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Hepáticas/genética , Camundongos Knockout , Modelos Genéticos , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
MicroRNAs (miRNAs) are critical small non-coding RNAs that regulate gene expression by hybridizing to the 3'-untranslated regions (3'-UTR) of target mRNAs, subsequently controlling diverse biological processes at post-transcriptional level. How miRNA genes are regulated receives considerable attention because it directly affects miRNA-mediated gene regulatory networks. Although numerous prediction models were developed for identifying miRNA promoters or transcriptional start sites (TSSs), most of them lack experimental validation and are inadequate to elucidate relationships between miRNA genes and transcription factors (TFs). Here, we integrate three experimental datasets, including cap analysis of gene expression (CAGE) tags, TSS Seq libraries and H3K4me3 chromatin signature derived from high-throughput sequencing analysis of gene initiation, to provide direct evidence of miRNA TSSs, thus establishing an experimental-based resource of human miRNA TSSs, named miRStart. Moreover, a machine-learning-based Support Vector Machine (SVM) model is developed to systematically identify representative TSSs for each miRNA gene. Finally, to demonstrate the effectiveness of the proposed resource, an important human intergenic miRNA, hsa-miR-122, is selected to experimentally validate putative TSS owing to its high expression in a normal liver. In conclusion, this work successfully identified 847 human miRNA TSSs (292 of them are clustered to 70 TSSs of miRNA clusters) based on the utilization of high-throughput sequencing data from TSS-relevant experiments, and establish a valuable resource for biologists in advanced research in miRNA-mediated regulatory networks.
Assuntos
MicroRNAs/genética , Sítio de Iniciação de Transcrição , Linhagem Celular , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Humanos , MicroRNAs/química , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Análise de Sequência de RNA , Sitios de Sequências Rotuladas , Máquina de Vetores de SuporteRESUMO
MicroRNAs (miRNAs), i.e. small non-coding RNA molecules (â¼22 nt), can bind to one or more target sites on a gene transcript to negatively regulate protein expression, subsequently controlling many cellular mechanisms. A current and curated collection of miRNA-target interactions (MTIs) with experimental support is essential to thoroughly elucidating miRNA functions under different conditions and in different species. As a database, miRTarBase has accumulated more than 3500 MTIs by manually surveying pertinent literature after data mining of the text systematically to filter research articles related to functional studies of miRNAs. Generally, the collected MTIs are validated experimentally by reporter assays, western blot, or microarray experiments with overexpression or knockdown of miRNAs. miRTarBase curates 3576 experimentally verified MTIs between 657 miRNAs and 2297 target genes among 17 species. miRTarBase contains the largest amount of validated MTIs by comparing with other similar, previously developed databases. The MTIs collected in the miRTarBase can also provide a large amount of positive samples to develop computational methods capable of identifying miRNA-target interactions. miRTarBase is now available on http://miRTarBase.mbc.nctu.edu.tw/, and is updated frequently by continuously surveying research articles.
Assuntos
Bases de Dados de Ácidos Nucleicos , MicroRNAs/metabolismo , Regulação da Expressão Gênica , Interferência de RNA , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Integração de Sistemas , Interface Usuário-ComputadorRESUMO
UNLABELLED: MicroRNAs (miRNAs), which are inhibitors of gene expression, participate in diverse biological functions and in carcinogenesis. In this study, we show that liver-specific microRNA-122 (miR-122) is significantly down-regulated in liver cancers with intrahepatic metastasis and negatively regulates tumorigenesis. Restoration of miR-122 in metastatic Mahlavu and SK-HEP-1 cells significantly reduced in vitro migration, invasion, and anchorage-independent growth as well as in vivo tumorigenesis, angiogenesis, and intrahepatic metastasis in an orthotopic liver cancer model. Because an inverse expression pattern is often present between an miRNA and its target genes, we used a computational approach and identified multiple miR-122 candidate target genes from two independent expression microarray datasets. Thirty-two target genes were empirically verified, and this group of genes was enriched with genes regulating cell movement, cell morphology, cell-cell signaling, and transcription. We further showed that one of the miR-122 targets, ADAM17 (a disintegrin and metalloprotease 17) is involved in metastasis. Silencing of ADAM17 resulted in a dramatic reduction of in vitro migration, invasion, in vivo tumorigenesis, angiogenesis, and local invasion in the livers of nude mice, which is similar to that which occurs with the restoration of miR-122. CONCLUSION: Our study suggests that miR-122, a tumor suppressor microRNA affecting hepatocellular carcinoma intrahepatic metastasis by angiogenesis suppression, exerts some of its action via regulation of ADAM17. Restoration of miR-122 has a far-reaching effect on the cell. Using the concomitant down-regulation of its targets, including ADAM17, a rational therapeutic strategy based on miR-122 may prove to be beneficial for patients with hepatocellular carcinoma.
Assuntos
Proteínas ADAM/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Proteína ADAM17 , Animais , Antagomirs , Carcinoma Hepatocelular/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fígado/patologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , OligonucleotídeosRESUMO
MicroRNAs (miRNAs) are small non-coding RNA molecules that can negatively regulate gene expression and thus control numerous cellular mechanisms. This work develops a resource, miRNAMap 2.0, for collecting experimentally verified microRNAs and experimentally verified miRNA target genes in human, mouse, rat and other metazoan genomes. Three computational tools, miRanda, RNAhybrid and TargetScan, were employed to identify miRNA targets in 3'-UTR of genes as well as the known miRNA targets. Various criteria for filtering the putative miRNA targets are applied to reduce the false positive prediction rate of miRNA target sites. Additionally, miRNA expression profiles can provide valuable clues on the characteristics of miRNAs, including tissue specificity and differential expression in cancer/normal cell. Therefore, quantitative polymerase chain reaction experiments were performed to monitor the expression profiles of 224 human miRNAs in 18 major normal tissues in human. The negative correlation between the miRNA expression profile and the expression profiles of its target genes typically helps to elucidate the regulatory functions of the miRNA. The interface is also redesigned and enhanced. The miRNAMap 2.0 is now available at http://miRNAMap.mbc.nctu.edu.tw/.
Assuntos
Bases de Dados Genéticas , Inativação Gênica , MicroRNAs/metabolismo , Animais , Sítios de Ligação , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Genômica , Humanos , Internet , Camundongos , MicroRNAs/genética , Ratos , Software , Interface Usuário-ComputadorRESUMO
OBJECTIVE: MicroRNA-122 (miR-122) is the most abundant miRNA in the liver and it plays an important role in regulating liver metabolism and tumor formation. Previous studies also reveal an anti-inflammatory function of miR-122; however, relatively little is known about the mechanisms by which miR-122 suppresses inflammation. This study aims to search the effect of miR-122 on proinflammatory chemokines/cytokines production in mice. METHODS: Quantitative real-time PCR, Western blot analysis, and ELISA were performed to examine gene expression. TargetScan, miRanda, and microT v3.0 were used to search for possible miR-122 target sites in the 3'-untranslated regions (3'-UTR) of candidate genes. Luciferase reporter assay and site-directed mutagenesis were applied to verify miR-122 target sequences. LPS was applied to peritoneal macrophages and mice to evaluate inflammatory response. RESULTS: The expression of proinflammatory chemokines, including Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10, and Relb in the livers of miR-122 knockout (KO) mice was increased. We identified Relb as a direct miR-122 target. Overexpressing RelB in the mouse liver increased the expression of Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10. Peritoneal macrophages from miR-122 KO mice had a higher level of RelB, and they showed a stronger NF-κB activation and more TNF-α and IL-6 secretion after LPS stimulation. Overexpression of RelB in a macrophage cell line augmented LPS-induced TNF-α and IL-6 production. miR-122 KO mice showed a greatly increased mortality rate and generated a stronger and lasting inflammatory response to LPS. CONCLUSIONS: Deletion of miR-122 caused an upregulation of proinflammatory chemokines and RelB in the liver. Increased RelB may contribute to increases in these chemokine in the liver. Intriguingly, deletion of miR-122 also enhanced the sensitivity of macrophages and mice to LPS. Our results reveal that reducing RelB expression is a new mechanism by which miR-122 regulates inflammation.
Assuntos
Fígado/fisiologia , Macrófagos/fisiologia , MicroRNAs/genética , Fator de Transcrição RelB/metabolismo , Animais , Quimiocinas/metabolismo , Citocinas/metabolismo , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Fator de Transcrição RelB/genética , Regulação para CimaRESUMO
Aurora-A, a mitotic serine/threonine kinase with oncogene characteristics, has recently drawn intense attention because of its association with the development of human cancers and its relationship with mitotic progression. Using the gene expression profiles of Aurora-A as a template to search for and compare transcriptome expression profiles in publicly accessible microarray data sets, we identified HURP (encodes hepatoma upregulated protein) as one of the best Aurora-A-correlated genes. Empirical validation indicates that HURP has several characteristics in common with Aurora-A. These two genes have similar expression patterns in hepatocellular carcinoma, liver regeneration after partial hepatectomy, and cell cycle progression and across a variety of tissues and cell lines. Moreover, Aurora-A phosphorylated HURP in vitro and in vivo. Ectopic expression of either the catalytically inactive form of Aurora-A or the HURP-4P mutant, in which the Aurora-A phosphorylation sites were replaced with Ala, resulted in HURP instability and complex disassembly. In addition, HURP-wild-type stable transfectants were capable of growing in low-serum environments whereas HURP-4P grew poorly under low-serum conditions and failed to proliferate. These studies together support the view that the ability to integrate evidence derived from microarray studies into biochemical analyses may ultimately augment our predictive power when analyzing the potential role of poorly characterized proteins. While this combined approach was simply an initial attempt to answer a range of complex biological questions, our findings do suggest that HURP is a potential oncogenic target of Aurora-A.
Assuntos
Carcinoma Hepatocelular/metabolismo , Perfilação da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Regeneração Hepática/fisiologia , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Sequência de Aminoácidos , Animais , Aurora Quinase A , Aurora Quinases , Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Regeneração Hepática/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Recent work has demonstrated that microRNAs (miRNAs) are involved in critical biological processes by suppressing the translation of coding genes. This work develops an integrated database, miRNAMap, to store the known miRNA genes, the putative miRNA genes, the known miRNA targets and the putative miRNA targets. The known miRNA genes in four mammalian genomes such as human, mouse, rat and dog are obtained from miRBase, and experimentally validated miRNA targets are identified in a survey of the literature. Putative miRNA precursors were identified by RNAz, which is a non-coding RNA prediction tool based on comparative sequence analysis. The mature miRNA of the putative miRNA genes is accurately determined using a machine learning approach, mmiRNA. Then, miRanda was applied to predict the miRNA targets within the conserved regions in 3'-UTR of the genes in the four mammalian genomes. The miRNAMap also provides the expression profiles of the known miRNAs, cross-species comparisons, gene annotations and cross-links to other biological databases. Both textual and graphical web interface are provided to facilitate the retrieval of data from the miRNAMap. The database is freely available at http://mirnamap.mbc.nctu.edu.tw/.
Assuntos
Bases de Dados de Ácidos Nucleicos , Regulação da Expressão Gênica , MicroRNAs/genética , MicroRNAs/fisiologia , Animais , Mapeamento Cromossômico , Bases de Dados de Ácidos Nucleicos/estatística & dados numéricos , Cães , Genoma , Genômica , Humanos , Internet , Camundongos , MicroRNAs/química , Precursores de RNA/química , Ratos , Interface Usuário-ComputadorRESUMO
DDX3 is a DEAD box RNA helicase with diverse biological functions. Using colony formation assay, our results revealed that DDX3 inhibited the colony formation ability of various tumor cells, and this inhibition might be due to a reduced growth rate caused by DDX3. Additionally, we identified p21(waf1/cip1), a cyclin-dependent kinase inhibitor, as a target gene of DDX3, and the up-regulation of p21(waf1/cip1) expression accounted for the colony-suppressing activity of DDX3. Moreover, DDX3 exerted its transactivation function on p21(waf1/cip1) promoter through an ATPase-dependent but helicase-independent mechanism, and the four Sp1 sites located within the -123 to -63 region, relative to the transcription start site of p21(waf1/cip1) promoter, were essential for the response to DDX3. Furthermore, DDX3 interacted and cooperated with Sp1 to up-regulate the promoter activity of p21(waf1/cip1). To determine the relevance of DDX3 in clinical cancers, the expression profile of DDX3 in various tumors was also examined. A declined expression of DDX3 mRNA and protein was found in approximately 58% to 73% of hepatoma specimens, which led to the reduction of p21(waf1/cip1) expression in a manner independent of p53 status. Additionally, an alteration of subcellular localization from nuclei to cytoplasm was also observed in >70% of cutaneous squamous cell carcinoma samples. Because DDX3 exhibits tumor suppressor functions, such as a growth-suppressive property and transcriptional activation of the p21(waf1/cip1) promoter, and is inactivated through down-regulation of gene expression or alteration of subcellular localization in tumor cells, all these features together suggest that DDX3 might be a candidate tumor suppressor.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Genes Supressores de Tumor , RNA Helicases/fisiologia , Adenosina Trifosfatases/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , RNA Helicases DEAD-box , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas , RNA Helicases/genética , RNA Helicases/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
Glycine N-methyltransferase (GNMT) is a tumor suppressor for HCC. It is down-regulated in HCC, but the mechanism is not fully understood. MicroRNA-224 (miR-224) acts as an onco-miR in HCC. This study is the first to investigate miR-224 targeting the coding region of GNMT transcript. The GNMT-MT plasmid containing a miR-224 binding site silent mutation of the GNMT coding sequence can escape the suppression of miR-224 in HEK293T cells. Expression of both exogenous and endogenous GNMT was suppressed by miR-224, while miR-224 inhibitor enhanced GNMT expression. miR-224 counteracts the effects of GNMT on the reduction of cell proliferation and tumor growth. The levels of miR-224 and GNMT mRNA showed a significant inverse relationship in tumor specimens from HCC patients. Utilizing CCl4-treated hepatoma cells and mice as a cell damage of inflammatory or liver injury model, we observed that the decreased expression levels of GNMT were accompanied with the elevated expression levels of miR-224 in hepatoma cells and mouse liver. Finally, hepatic AAV-mediated GNMT also reduced CCl4-induced miR-224 expression and liver fibrosis. These results indicated that AAV-mediated GNMT has potential liver protection activity. miR-224 can target the GNMT mRNA coding sequence and plays an important role in GNMT suppression during liver tumorigenesis.
Assuntos
Aciltransferases/genética , Carcinoma Hepatocelular/enzimologia , Regulação para Baixo/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/enzimologia , MicroRNAs/fisiologia , Regiões 3' não Traduzidas , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Células HEK293 , Humanos , Neoplasias Hepáticas/genética , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , RNA Mensageiro/genéticaRESUMO
The aim of this study is to elucidate the effects of Scutellaria baicalensis Georgi (SbG) extract and its constituents on macrophage-hepatocyte interaction in primary cultures. By using trans-well primary Kupffer cell culture or conditioned medium (CM) from murine macrophage RAW264.7 cell line (RAW cells), effects of SbG on hepatocyte growth were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and trypan blue exclusion assay. Cytokine production, antibody-neutralization studies, and molecular mechanisms of transforming growth factor (TGF)-beta1 gene expression were elucidated on SbG-treated RAW264.7 cells. In addition, recombinant human TGF-beta1 (r-human TGF-beta1) was added to elucidate the mechanisms of SbG effects on cultured hepatocytes. Immunohistochemistry using anti-NF-kappaB antibody was used to determine the possible signal transduction pathways in primary hepatocyte culture. The results showed that SbG stimulated the proliferation of cultured hepatocytes, possibly through NF-kappaB, but not of Toll-like receptor 4 activation; whereas SbG-RAW-CM and SbG in trans-well significantly suppressed the proliferation of hepatocytes. Antibody-neutralization studies revealed that TGF-beta1 was the main antimitotic cytokine in SbG-treated RAW cells CM. The growth stimulation effect of SbG on cultured hepatocytes was inhibited by exogenous administration of r-human TGF-beta1. Furthermore, SbG induced NF-kB translocation into the nuclei of cultured cells. In the RAW264.7 line, SbG and baicalin stimulated TGF-beta1 gene expression via NF-kappaB and protein kinase C activation. We conclude that SbG stimulates hepatocyte growth via activation of the NF-kappaB pathway and induces TGF-beta1 gene expression through the Kupffer cell-hepatocyte interaction, which subsequently results in the inhibition of SbG-stimulated hepatocyte growth.
Assuntos
Comunicação Celular/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Células de Kupffer/efeitos dos fármacos , Scutellaria baicalensis/química , Animais , Bromodesoxiuridina/metabolismo , Contagem de Células , Linhagem Celular , Proliferação de Células , Células Cultivadas , Hepatócitos/citologia , Hepatócitos/metabolismo , Interleucina-6/metabolismo , Células de Kupffer/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
This work develops an integrated system which, after a set of genes are inputted, is able to predict transcriptional regulatory sites and to detect the co-occurrence of these regulatory sites. The system integrates several site detection methods such as known site matching, over-presented oligonucleotide detection and DNA motif discovery programs. User profiles and history pages enable users to trace the sequence analyses of these transcriptional regulatory sites. Two groups of co-regulated genes were used to test the proposed system. The results predicted by the proposed system consist of known site homologs and putative regulatory sites. By comparing these sites with previously published results, the proposed system is able to help biologists identify possible candidates for the regulatory sites from groups of co-regulated genes. The integrated system is now available at http://rgsminer. csie.ncu.edu.tw/.
Assuntos
Genoma Humano , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA/métodos , Software , Transcrição Gênica , Bases de Dados de Ácidos Nucleicos , Expressão Gênica , Humanos , Integração de SistemasRESUMO
Identification of transcriptional regulatory sites plays an important role in the investigation of gene regulation. For this propose, we designed and implemented a data warehouse to integrate multiple heterogeneous biological data sources with data types such as text-file, XML, image, MySQL database model, and Oracle database model. The utility of the biological data warehouse in predicting transcriptional regulatory sites of coregulated genes was explored using a synexpression group derived from a microarray study. Both of the binding sites of known transcription factors and predicted over-represented (OR) oligonucleotides were demonstrated for the gene group. The potential biological roles of both known nucleotides and one OR nucleotide were demonstrated using bioassays. Therefore, the results from the wet-lab experiments reinforce the power and utility of the data warehouse as an approach to the genome-wide search for important transcription regulatory elements that are the key to many complex biological systems.
Assuntos
Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Armazenamento e Recuperação da Informação/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Elementos Reguladores de Transcrição/genética , Análise de Sequência de DNA/métodos , Fatores de Transcrição/genética , Algoritmos , Sítios de Ligação , Sistemas de Gerenciamento de Base de Dados , Reconhecimento Automatizado de Padrão/métodos , Ligação Proteica , Alinhamento de Sequência/métodos , Biologia de Sistemas/métodosRESUMO
Viral infection poses a major problem for public health, horticulture, and animal husbandry, possibly causing severe health crises and economic losses. Viral infections can be identified by the specific detection of viral sequences in many ways. The microarray approach not only tolerates sequence variations of newly evolved virus strains, but can also simultaneously diagnose many viral sequences. Many chips have so far been designed for clinical use. Most are designed for special purposes, such as typing enterovirus infection, and compare fewer than 30 different viral sequences. None considers primer design, increasing the likelihood of cross hybridization to similar sequences from other viruses. To prevent this possibility, this work establishes a platform and database that provides users with specific probes of all known viral genome sequences to facilitate the design of diagnostic chips. This work develops a system for designing probes online. A user can select any number of different viruses and set the experimental conditions such as melting temperature and length of probe. The system then returns the optimal sequences from the database. We have also developed a heuristic algorithm to calculate the probe correctness and show the correctness of the algorithm. (The system that supports probe design for identifying viruses has been published on our web page http://bioinfo.csie.ncu.edu.tw/.)
Assuntos
Sondas de DNA/genética , DNA Viral/análise , DNA Viral/genética , Bases de Dados Genéticas , Genoma Viral/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Mapeamento Cromossômico/métodos , DNA Viral/classificaçãoRESUMO
An analytic strategy was followed to identify putative regulatory genes during the development of human hepatocellular carcinoma (HCC). This strategy employed a bioinformatics analysis that used a database search to identify genes, which are differentially expressed in human HCC and are also under cell cycle regulation. A novel cell cycle regulated gene (HURP) that is overexpressed in HCC was identified. Full-length cDNAs encoding the human and mouse HURP genes were isolated. They share 72 and 61% identity at the nucleotide level and amino-acid level, respectively. Endogenous levels of HURP mRNA were found to be tightly regulated during cell cycle progression as illustrated by its elevated expression in the G(2)/M phase of synchronized HeLa cells and in regenerating mouse liver after partial hepatectomy. Immunofluorescence studies revealed that hepatoma up-regulated protein (HURP) localizes to the spindle poles during mitosis. Overexpression of HURP in 293T cells resulted in an enhanced cell growth at low serum levels and at polyhema-based, anchorage-independent growth assay. Taken together, these results strongly suggest that HURP is a potential novel cell cycle regulator that may play a role in the carcinogenesis of human cancer cells.
Assuntos
Carcinoma Hepatocelular/genética , Ciclo Celular/genética , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Bases de Dados de Ácidos Nucleicos , Etiquetas de Sequências Expressas , Fígado/embriologia , Fígado/fisiologia , Regeneração Hepática/genética , Camundongos , Camundongos Endogâmicos BALB C , Mitose , Dados de Sequência Molecular , Especificidade de Órgãos , Homologia de Sequência de Aminoácidos , Fuso Acromático/genéticaRESUMO
The aim of this study was to demonstrate that regenerating liver responses to a herbal remedy could be presented by gene expression profiling. Compositions of the ingredients in the remedy containing Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium Wild (S/B remedy) were analyzed and quantified by high performance liquid chromatography. By using a 70% partial hepatectomy in BALB/c mice as an in vivo model, the effects of high dose (50 mg/kg) and low dose (1 mg/kg) S/B remedy were evaluated by cDNA microarray, followed by RT-PCR and real-time PCR confirmation. Factors affecting proliferative activities of mouse hepatocytes were measured by DNA flow cytometry, BrdU incorporation assay and serum interleukin-6 (IL-6) level. Based on global gene expression profiles, the results showed that the low dose S/B remedy down-regulated expression of immediate early genes and cell cycle-related genes, whereas the high dose had opposite effects. The gene expression was further verified by real-time RT-PCR. Proliferative activities, in terms of synthetic phase fractions and G2/M phase fractions, in vehicle, low dose, and high dose groups were 18.45+/-2.56%, 14.65+/-1.06%; 9.27+/-0.85%, 7.80+/-0.11%; and 18.90+/-2.17%, 22.95+/-0.25%, respectively. The serum IL-6 level was also dose-dependent in both low and high dose S/B remedy-treated mice. We conclude that in vivo gene expression profiling correlates with liver responses to a herbal remedy, which provides a new direction for pharmaceutical studies on human diseases.
Assuntos
Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Bromodesoxiuridina/metabolismo , Bupleurum/química , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , DNA/análise , DNA/genética , Relação Dose-Resposta a Droga , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatectomia/métodos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Interleucina-6/sangue , Fígado/metabolismo , Fígado/cirurgia , Regeneração Hepática/efeitos dos fármacos , Regeneração Hepática/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Extratos Vegetais/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Scutellaria baicalensis/química , Fatores de TempoRESUMO
PGC-1α, a major metabolic regulator of gluconeogenesis and lipogenesis, is strongly induced to coactivate Hepatitis B virus (HBV) gene expression in the liver of fasting mice. We found that 8-Br-cAMP and glucocorticoids synergistically induce PGC-1α and its downstream targets, including PEPCK and G6Pase. Also, HBV core promoter activity was synergistically enhanced by 8-Br-cAMP and glucocorticoids. Graptopetalum paraguayense (GP), a herbal medicine, is commonly used in Taiwan to treat liver disorders. Partially purified fraction of GP (named HH-F3) suppressed 8-Br-cAMP/glucocorticoid-induced G6Pase, PEPCK and PGC-1α expression and suppressed HBV core promoter activity. HH-F3 blocked HBV core promoter activity via inhibition of PGC-1α expression. Ectopically expressed PGC-1α rescued HH-F3-inhibited HBV surface antigen expression, HBV mRNA production, core protein levels, and HBV replication. HH-F3 also inhibited fatty acid synthase (FASN) expression and decreased lipid accumulation by down-regulating PGC-1α. Thus, HH-F3 can inhibit HBV replication, gluconeogenesis and lipogenesis by down-regulating PGC-1α. Our study indicates that targeting PGC-1α may be a therapeutic strategy for treatment of HBV infections. HH-F3 may have potential use for the treatment of chronic hepatitis B patients with associated metabolic syndrome.