MIRNA-DISTILLER: A Stand-Alone Application to Compile microRNA Data from Databases

MicroRNAs (miRNA) are small non-coding RNA molecules of ∼22 nucleotides which regulate large numbers of genes by binding to seed sequences at the 3′-untranslated region of target gene transcripts. The target mRNA is then usually degraded or translation is inhibited, although thus resulting in posttranscriptional down regulation of gene expression at the mRNA and/or protein level. Due to the bioinformatic difficulties in predicting functional miRNA binding sites, several publically available databases have been developed that predict miRNA binding sites based on different algorithms. The parallel use of different databases is currently indispensable, but highly uncomfortable and time consuming, especially when working with numerous genes of interest. We have therefore developed a new stand-alone program, termed MIRNA-DISTILLER, which allows to compile miRNA data for given target genes from public databases. Currently implemented are TargetScan, microCosm, and miRDB, which may be queried independently, pairwise, or together to calculate the respective intersections. Data are stored locally for application of further analysis tools including freely definable biological parameter filters, customized output-lists for both miRNAs and target genes, and various graphical facilities. The software, a data example file and a tutorial are freely available at http://www.ikp-stuttgart.de/content/language1/html/10415.as

Pathway-Targeted Pharmacogenomics of CYP1A2 in Human Liver

The human drug metabolizing cytochrome P450 (CYP) 1A2, is one of the major P450 isoforms contributing by about 5–20% to the hepatic P450 pool and catalyzing oxidative biotransformation of up to 10% of clinically relevant drugs including clozapine and caffeine. CYP1A2 activity is interindividually highly variable and although twin studies have suggested a high heritability, underlying genetic factors are still unknown. Here we adopted a pathway-oriented approach using a large human liver bank (n = 150) to elucidate whether variants in candidate genes of constitutive, ligand-inducible, and pathophysiological inhibitory regulatory pathways may explain different hepatic CYP1A2 phenotypes. Samples were phenotyped for phenacetin O-deethylase activity, and the expression of CYP1A2 protein and mRNA was determined. CYP1A2 expression and function was increased in smokers and decreased in patients with inflammation and cholestasis. Of 169 SNPs in 17 candidate genes including the CYP1A locus, 136 non-redundant SNPs with minor allele frequency >5% were analyzed by univariate and multivariate methods. A total of 13 strong significant associations were identified, of which 10 SNPs in the ARNT, AhRR, HNF1α, IL1β, SRC-1, and VDR genes showed consistent changes for at least two phenotypes by univariate analysis. Multivariate linear modeling indicated that the polymorphisms and non-genetic factors together explained 42, 38, and 33% of CYP1A2 variation at activity, protein and mRNA levels, respectively. In conclusion, we identified novel trans-associations between regulatory genes and hepatic CYP1A2 function and expression, but additional genetic factors must be assumed to explain the full extent of CYP1A2 heritability

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

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Special Section on Epigenetic Regulation of Drug Metabolizing Enzymes and Transporters Expression Variability of Absorption, Distribution, Metabolism, Excretion-Related MicroRNAs in Human Liver: Influence of Nongenetic Factors and Association with Gene Ex

ABSTRACT Genes that are important for the detoxification of drugs and other xenobiotics show a high degree of interindividual variation attributable to regulation by diverse genetic, nongenetic, and epigenetic mechanisms including microRNAs (miRNAs). We selected a set of 56 miRNAs predicted to target the 39-untranslated region of absorption, distribution, metabolism, excretion (ADME) genes to assess their hepatic expression levels and interindividual variability in a well-documented human liver tissue cohort (n = 92), together with the well-known hepatic miRNAs miR-122, miR-21, miR-27b, and miR-148a. Quantification by stem-loop real-time reversetranscription polymerase chain reaction confirmed high expression for these microRNAs and revealed particularly strong variability of expression (>1000-fold) for miR-539, miR-200c, miR-31, miR-15a, and miR-22. Association analysis revealed a high degree of correlation among various miRNAs, suggesting coregulation. Statistical analysis considering liver donor meta-data including correction for multiple testing revealed strongly elevated levels of miR-21, miR-34a, miR-130b, and miR-132 in cholestatic liver and of miR-21 and miR-130b during inflammation, as indicated by elevated C-reactive protein levels in serum. Although none of the miRNAs was strongly associated with sex, several miRNAs, including miR-34a and miR-200a/b, were positively correlated with age. Association analysis with ADME gene expression profiles and with cytochrome P450 gene expression phenotypes (mRNA, protein, enzymatic activity) revealed numerous significant correlations. Negatively affected protein and/or activity levels were observed for CYP1A1 (e.g., miR-132, miR-142-3p, miR-21), CYP2A6 (miR-142-3p, miR-21), CYP2C19 (e.g., miR-130b, miR-185, miR-34a), and CYP2E1 (miR-10a, let-7g, miR-200c). These data should be useful to further elucidate regulatory functions of miRNAs in liver pathophysiology and regulation of ADME gene expression

Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

Background/Aims:Sterol regulatory element-binding protein (SREBP) 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specific and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specific knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression. Copyright (C) 2015 S. Karger AG, Base

Paraoxonase (PON1 and PON3) Polymorphisms: Impact on Liver Expression and Atorvastatin-Lactone Hydrolysis

Atorvastatin δ-lactone, a major, pharmacologically inactive metabolite, has been associated with toxicity. In a previous study we showed that polymorphisms of UGT1A3 influence atorvastatin δ-lactone formation. Here we investigated the reverse reaction, atorvastatin δ-lactone hydrolysis, in a human liver bank. Screening of microarray data revealed paraoxonases PON1 and PON3 among 17 candidate esterases. Microsomal δ-lactone hydrolysis was significantly correlated to PON1 and PON3 protein (rs = 0.60; rs = 0.62, respectively; P < 0.0001). PON1 and PON3 were strongly correlated to each other (rs = 0.60) but PON1 was shown to be more extensively glycosylated than PON3. In addition a novel splice-variant of PON3 was identified. Genotyping of 40 polymorphisms within the PON-locus identified PON1 promoter polymorphisms (−108T > C, −832G > A, −1741G > A) and a tightly linked group of PON3 polymorphisms (−4984A > G, −4105G > A, −1091A > G, −746C > T, and F21F) to be associated with changes in atorvastatin δ-lactone hydrolysis and expression of PON1 but not PON3. However, carriers of the common PON1 polymorphisms L55M or Q192R showed no difference in δ-lactone hydrolysis or PON expression. Haplotype analysis revealed decreased δ-lactone hydrolysis in carriers of the most common haplotype *1 compared to carriers of haplotypes *2, *3, *4, and *7. Analysis of non-genetic factors showed association of hepatocellular and cholangiocellular carcinoma with decreased PON1 and PON3 expression, respectively. Increased C-reactive protein and γ-glutamyl transferase levels were associated with decreased protein expression of both enzymes, and increased bilirubin levels, cholestasis, and presurgical exposure to omeprazole or pantoprazole were related to decreased PON3 protein. In conclusion, PON-locus polymorphisms affect PON1 expression whereas non-genetic factors have an effect on PON1 and PON3 expression. This may influence response to therapy or adverse events in statin treatment

Targeted epigenome editing of an endogenouslocus with chromatin modifiers is not stably maintained

Background: DNA methylation and histone 3 lysine 9 (H3K9) methylation are considered as epigenetic marks that can be inherited through cell divisions. To explore the functional consequences and stability of these modifications, we employed targeted installment of DNA methylation and H3K9 methylation in the vascular endothelial growth factor A (VEGF-A) promoter using catalytic domains of DNA or H3K9 methyltransferases that are fused to a zinc finger protein which binds a site in the VEGF-A promoter. Results: Expression of the targeted DNA and H3K9 methyltransferases caused dense deposition of DNA methylation or H3K9 di- and trimethylation in the promoter of VEGF-A and downregulation of VEGF-A gene expression. We did not observe positive feedback between DNA methylation and H3K9 methylation. Upon loss of the targeted methyltransferases from the cells, the epigenetic marks, chromatin environment, and gene expression Levels returned to their original state, indicating that both methylation marks were not stably propagated after their installment. Conclusions: The clear anti-correlation between DNA or H3K9 methylation and gene expression suggests a direct role of these marks in transcriptional control. The lack of maintenance of the transiently induced silenced chromatin state suggests that the stability of epigenetic signaling is based on an epigenetic network consisting of several molecular marks. Therefore, for stable reprogramming, either multivalent deposition of functionally related epigenetic marks or longer-lasting trigger stimuli might be necessary

A systems biology approach to dynamic modeling and inter-subject variability of statin pharmacokinetics in human hepatocytes

A dynamic model for the biotransformation of atorvastatin has been developed using quantitative metabolite measurements in primary human hepatocytes. The model comprises kinetics for transport processes and metabolic enzymes as well as population liver expression data allowing us to assess the impact of inter-individual variability of concentrations of key proteins. Application of computational tools for parameter sensitivity analysis enabled us to considerably improve the validity of the model and to create a consistent framework for precise computer-aided simulations in toxicology