Phenotype-Genotype Correlation Applying a Cocktail Approach and an Exome Chip Analysis Reveals Further Variants Contributing to Variation of Drug Metabolism.

Although great progress has been made in the fine-tuning of diplotypes, there is still a need to further improve the predictability of individual phenotypes of pharmacogenetically relevant enzymes. The aim of this study was to analyze the additional contribution of sex and variants identified by exome chip analysis to the metabolic ratio of five probe drugs. A cocktail study applying dextromethorphan, losartan, omeprazole, midazolam, and caffeine was conducted on 200 healthy volunteers. CYP2D6, 2C9, 2C19, 3A4/5, and 1A2 genotypes were analyzed and correlated with metabolic ratios. In addition, an exome chip analysis was performed. These SNPs correlating with metabolic ratios were confirmed by individual genotyping. The contribution of various factors to metabolic ratios was assessed by multiple regression analysis. Genotypically predicted phenotypes defined by CPIC discriminated very well the log metabolic ratios with the exception of caffeine. There were minor sex differences in the activity of CYP2C9, 2C19, 1A2, and CYP3A4/5. For dextromethorphan (CYP2D6), IP6K2 (rs61740999) and TCF20 (rs5758651) affected metabolic ratios, but only IP6K2 remained significant after multiple regression analysis. For losartan (CYP2C9), FBXW12 (rs17080138), ZNF703 (rs79707182), and SLC17A4 (rs11754288) together with CYP diplotypes, and sex explained 50% of interindividual variability. For omeprazole (CYP2C19), no significant influence of CYP2C:TG haplotypes was observed, but CYP2C19 rs12777823 improved the predictability. The comprehensive genetic analysis and inclusion of sex in a multiple regression model significantly improved the explanation of variability of metabolic ratios, resulting in further improvement of algorithms for the prediction of individual phenotypes of drug-metabolizing enzymes.

Genome­wide expression and methylation analyses reveal aberrant cell adhesion signaling in tyrosine kinase inhibitor­resistant CML cells.

Although chronic myeloid leukemia (CML) can be effectively treated using BCR­ABL1 kinase inhibitors, resistance due to kinase alterations or to BCR­ABL1 independent mechanisms remain a therapeutic challenge. For the latter, the underlying mechanisms are widely discussed; for instance, gene expression changes, epigenetic factors and alternative signaling pathway activation. In the present study, in vitro­CML cell models of resistance against the tyrosine kinase inhibitors (TKIs) imatinib (0.5 and 2 µM) and nilotinib (0.1 µM) with biological replicates were generated to identify novel mechanisms of resistance. Subsequently, genome­wide mRNA expression and DNA methylation were analyzed. While mRNA expression patterns differed largely between biological replicates, there was an overlap of 71 genes differentially expressed between cells resistant against imatinib or nilotinib. Moreover, all TKI resistant cell lines demonstrated a slight hypermethylation compared with native cells. In a combined analysis of 151 genes differentially expressed in the biological replicates of imatinib resistance, cell adhesion signaling, in particular the cellular matrix protein fibronectin 1 (FN1), was significantly dysregulated. This gene was also downregulated in nilotinib resistance. Further analyses showed significant FN1­downregulation in imatinib resistance on mRNA (P<0.001) and protein level (P<0.001). SiRNA­mediated FN1­knockdown in native cells reduced cell adhesion (P=0.02), decreased imatinib susceptibility visible by higher Ki­67 expression (1.5­fold, P=0.04) and increased cell number (1.5­fold, P=0.03). Vice versa, recovery of FN1­expression in imatinib resistant cells was sufficient to partially restore the response to imatinib. Overall, these results suggested a role of cell adhesion signaling and fibronectin 1 in TKI resistant CML and a potential target for novel strategies in treatment of resistant CML.

Interaction of Phytocompounds of Echinacea purpurea with ABCB1 and ABCG2 Efflux Transporters.

Preparations of Echinacea purpurea (E. purpurea) are widely used for the management of upper respiratory infections, influenza, and common cold, often in combination with other conventional drugs. However, the potential of phytochemical constituents of E. purpurea to cause herb-drug interactions via ABCB1 and ABCG2 efflux transporters remains elusive. The purpose of this study was to investigate the impact of E. purpurea-derived caffeic acid derivatives (cichoric acid and echinacoside) and tetraenes on the mRNA and protein expression levels as well as on transport activity of ABCB1 and ABCG2 in intestinal (Caco-2) and liver (HepG2) cell line models. The safety of these compounds was investigated by estimating EC20 values of cell viability assays in both cell lines. Regulation of ABCB1 and ABCG2 protein in these cell lines were analyzed after 24 h exposure to the compounds at 1, 10, and 50 µg/mL. Bidirectional transport of 0.5 µg/mL Hoechst 33342 and 5 µM rhodamine across Caco-2 monolayer and profiling for intracellular concentrations of the fluorophores in both cell lines were conducted to ascertain inhibition effects of the compounds. Cichoric acid showed no cytotoxic effect, while the EC20 values of tetraenes and echinacoside were 45.0 ± 3.0 and 52.0 ± 4.0 µg/mL in Caco-2 cells and 28.0 ± 4.3 and 62.0 ± 9.9 µg/mL in HepG2 cells, respectively. In general, the compounds showed heterogeneous induction of ABCB1 with the strongest 3.6 ± 1.2-fold increase observed for 10 µg/mL tetraenes in Caco-2 cells (p < 0.001). However, the compounds did not induce ABCG2. None of the phytocompounds inhibited significantly net flux of the fluorophores across Caco-2 monolayers. Overall, tetraenes moderately induced ABCB1 but not ABCG2 in Caco-2 and HepG2 cells while no compound significantly inhibited activity of these transporters at clinically relevant concentration to cause herb-drug interactions.

ABCB1, ABCG2, ABCC1, ABCC2, and ABCC3 drug transporter polymorphisms and their impact on drug bioavailability: what is our current understanding?

INTRODUCTION: Interindividual differences in drug response are a frequent clinical challenge partly due to variation in pharmacokinetics. ATP-binding cassette (ABC) transporters are crucial determinants of drug disposition. They are subject of gene regulation and drug-interaction; however, it is still under debate to which extend genetic variants in these transporters contribute to interindividual variability of a wide range of drugs. AREAS COVERED: This review discusses the current literature on the impact of genetic variants in ABCB1, ABCG2 as well as ABCC1, ABCC2, and ABCC3 on pharmacokinetics and drug response. The aim was to evaluate if results from recent studies would increase the evidence for potential clinically relevant pharmacogenetic effects. EXPERT OPINION: Although enormous efforts have been made to investigate effects of ABC transporter genotypes on drug pharmacokinetics and response, the majority of studies showed only weak if any associations. Despite few unique results, studies mostly failed to confirm earlier findings or still remained inconsistent. The impact of genetic variants on drug bioavailability is only minor and other factors regulating the transporter expression and function seem to be more critical. In our opinion, the findings on the so far investigated genetic variants in ABC efflux transporters are not suitable as predictive biomarkers.

The serotonin receptor 2A (HTR2A) rs6313 variant is associated with higher ongoing pain and signs of central sensitization in neuropathic pain patients.

BACKGROUND: The serotonin receptor 2A (HTR2A) has been described as an important facilitation mediator of spinal nociceptive processing leading to central sensitization (CS) in animal models of chronic pain. However, whether HTR2A single nucleotide variants (SNVs) modulate neuropathic pain states in patients has not been investigated so far. The aim of this study was to elucidate the potential association of HTR2A variants with sensory abnormalities or ongoing pain in neuropathic pain patients. METHODS: At total of 240 neuropathic pain patients and 253 healthy volunteers were included. Patients were phenotypically characterized using standardized quantitative sensory testing (QST). Patients and controls were genotyped for HTR2A g.-1438G > A (rs6311) and c.102C > T (rs6313). Genotype-related differences in QST parameters were assessed considering QST profile clusters, principal somatosensory components and sex. RESULTS: There was an equal distribution of rs6313 and linked rs6311 between patients and controls. However, the rs6313 variant was significantly associated with a principal component of pinprick hyperalgesia and dynamic mechanical allodynia, indicating enhanced CS in patients with sensory loss (-0.34 ± 0.15 vs. +0.31 ± 0.11 vs., p < .001). In this cluster, the variant allele was also associated with single QST parameters of pinprick hyperalgesia (MPT, +0.64 ± 0.18 vs. -0.34 ± 0.23 p = .002; MPS, +0.66 ± 0.17 vs. -0.09 ± 0.23, p = .009) and ongoing pain was increased by 30%. CONCLUSIONS: The specific association of the rs6313 variant with pinprick hyperalgesia and increased levels of ongoing pain suggests that the HTR2A receptor might be an important modulator in the development of CS in neuropathic pain. SIGNIFICANCE: This article presents new insights into serotonin receptor 2A-mediating mechanisms of central sensitization in neuropathic pain patients. The rs6313 variant allele was associated with increased mechanical pinprick sensitivity and increased levels of ongoing pain supporting a contribution of central sensitization in the genesis of ongoing pain providing a possible route for mechanism-based therapies.

Expression differences of miR-142-5p between treatment-naïve chronic myeloid leukemia patients responding and non-responding to imatinib therapy suggest a link to oncogenic ABL2, SRI, cKIT and MCL1 signaling pathways critical for development of therapy resistance.

BACKGROUND: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by constitutive activity of the tyrosine kinase BCR-ABL1. Although the introduction of tyrosine kinase inhibitors (TKIs) has substantially improved patients' prognosis, drug resistance remains one of the major challenges in CML therapy. MicroRNAs (miRNAs), a class of short non-coding RNAs acting as post-transcriptional regulators, are implicated in CML progression and drug resistance. The aim of the present study was to analyze the miRNA expression profiles of 45 treatment-naïve CML patients in chronic phase (28 peripheral blood and 17 bone marrow samples) with respect to future response to imatinib therapy. METHODS: TaqMan low density arrays were used to analyze the miRNA expression pattern of the patient samples. For selected microRNAs, reporter gene assays were performed to study their ability to regulate CML associated target genes. RESULTS: Significant lower expression levels of miR-142-5p were identified in both, peripheral blood and bone marrow samples of future non-responders suggesting a potential tumor suppressor role of this miRNA. This was supported by reporter gene assays that identified the survival, proliferation and invasion promoting CML related genes ABL2, cKIT, MCL1 and SRI as targets of miR-142-5p and miR-365a-3p, the latter identified as potential biomarker in peripheral blood samples. CONCLUSION: MiR-142-5p and to a certain extend also miR-365a-3p were able to discriminate treatment-naïve CML patients not responding to imatinib in the course of their treatment from patients, who responded to therapy. However, further large-scale studies should clarify if the identified miRNAs have the potential as predictive biomarkers for TKI resistance.

Phosphorylation of steroid receptor coactivator-3 (SRC-3) at serine 857 is regulated by the p38MAPK-MK2 axis and affects NF-κB-mediated transcription.

Steroid receptor coactivator-3 (SRC-3) regulates the activity of both nuclear hormone receptors and a number of key transcription factors. It is implicated in the regulation of cell proliferation, inflammation and in the progression of several common cancers including breast, colorectal and lung tumors. Phosphorylation is an important regulatory event controlling the activities of SRC-3. Serine 857 is the most studied phospho-acceptor site, and its modification has been reported to be important for SRC-3-dependent tumor progression. In this study, we show that the stress-responsive p38MAPK-MK2 signaling pathway controls the phosphorylation of SRC-3 at S857 in a wide range of human cancer cells. Activation of the p38MAPK-MK2 pathway results in the nuclear translocation of SRC-3, where it contributes to the transactivation of NF-kB and thus regulation of IL-6 transcription. The identification of the p38MAPK-MK2 signaling axis as a key regulator of SRC-3 phosphorylation and activity opens up new possibilities for the development and testing of novel therapeutic strategies to control both proliferative and metastatic tumor growth.

Transcriptional and Post-Transcriptional Regulation of Duodenal P-Glycoprotein and MRP2 in Healthy Human Subjects after Chronic Treatment with Rifampin and Carbamazepine.

To predict the outcome of intestinal drug transporter induction on pharmacokinetics, signaling of the DNA message along with messenger RNA (mRNA) transcription and protein translation leading to transporter function must be understood. We quantified the gene expression of PXR and CAR, gene expression and protein abundance of P-glycoprotein (P-gp), multidrug-resistance-associated protein 2 (MRP2) and breast-cancer-resistance protein, the content of 754 microRNAs in human duodenal biopsy specimens, and pharmacokinetics of talinolol and ezetimibe before and after the treatment with rifampin and carbamazepine. Rifampin significantly induced the transcription of ABCB1 and ABCC2 and protein abundance of P-gp but not of MRP2. The abundance of P-gp was significantly correlated to the plasma exposure of ezetimibe and its glucuronide. Carbamazepine induced the mRNA expressions of CAR, ABCB1, and ABCC2 but did not elevate protein abundance. Using in silico prediction tools and luciferase reporter assays, microRNAs were identified that can contribute to ligand-specific regulation of intestinal drug transporters and different changes in drug disposition after induction with rifampin and carbamazepine.

Implications of genetic variation of common Drug Metabolizing Enzymes and ABC Transporters among the Pakistani Population.

Genetic polymorphism of drug metabolizing enzymes and transporters may influence drug response. The frequency varies substantially between ethnicities thus having implications on appropriate selection and dosage of various drugs in different populations. The distribution of genetic polymorphisms in healthy Pakistanis has so far not been described. In this study, 155 healthy adults (98 females) were included from all districts of Karachi. DNA was extracted from saliva and genotyped for relevant SNVs in CYP1A1, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A5 as well as ALDH3A1, GSTA1, ABCB1 and ABCC2. About 64% of the participants were born to parents who were unrelated to each other. There was generally a higher prevalence (p < 0.05) of variant alleles of CYP450 1A2, 2B6, 2C19, 3A5, ALDH3A1, GSTM1 as well as ABCB1 and ABCC2 in this study cohort than in other ethnicities reported in the HapMap database. In contrast, the prevalence of variant alleles was lower in GSTA1. Therefore, in the Pakistani population sample from Karachi a significantly different prevalence of variant drug metabolizing enzymes and ABC transporters was observed as compared to other ethnicities, which could have putative clinical consequences on drug efficacy and safety.

Interaction of herbal products with prescribed medications: A systematic review and meta-analysis.

Although several studies on pharmacokinetic and/or pharmacodynamic herb-drug interactions (HDI) have been conducted in healthy volunteers, there is large uncertainty on the validity of these studies. A qualitative review and a meta-analysis were performed to establish the clinical evidence of these interaction studies. Out of 4026 screened abstracts, 32 studies were included into the qualitative analysis. The meta-analysis was performed on eleven additional studies. St. John's wort (SJW) significantly decreased the AUC (p < 0.0001) and clearance (p = 0.007) of midazolam. Further subgroup analysis identified age to affect Cmax of midazolam (p < 0.01) in the presence of SJW. Echinacea purpurea (EP) significantly increased the clearance of midazolam (p = 0.01). Evidence of publication bias (p > 0.001) was shown on the effect of the herbal products o half-life of midazolam. Green tea (GT) showed significant 85% decrease in plasma concentration of nadolol. The study findings suggest that GT, SJW and EP perpetuate significant interactions with prescribed medications via CYP3A4 or OATP1A2. Our studies show that meta-analyses are important in the area of natural products to provide necessary information on their use in overall medication plans in order to avoid unintended interactions.

Retinoic acid-induced survival effects in SH-SY5Y neuroblastoma cells.

Neuroblastoma is a malignant childhood cancer arising from the embryonic sympathoadrenal lineage of the neural crest. Retinoic acid (RA) is included in the multimodal therapy of patients with high-risk neuroblastoma to eliminate minimal residual disease. However, the formation of RA-resistant cells substantially lowers 5-year overall survival rates. To examine mechanisms that lead to treatment failure, we chose human SH-SY5Y cells, which are known to tolerate incubation with RA by activating the survival kinases Akt and extracellular signal-regulated kinase 1/2. Characterization of downstream pathways showed that both kinases increased the phosphorylation of the ubiquitin ligase mouse double minute homolog 2 (Mdm2) and thereby enhanced p53 degradation. When p53 signaling was sustained by blocking complex formation with Mdm2 or enhancing c-Jun N-terminal kinase (JNK) activation, cell viability was significantly reduced. In addition, Akt-mediated phosphorylation of the cell-cycle regulator p21 stimulated complex formation with caspase-3, which also contributed to cell protection. Thus, treatment with RA augmented survival signaling and attenuated basal apoptotic pathways in SH-SY5Y cells, which increased cell viability.

SIGMA-1 Receptor Gene Variants Affect the Somatosensory Phenotype in Neuropathic Pain Patients.

Pain sensitivity is characterized by interindividual variability, determined by factors including genetic variation of nociceptive receptors and pathways. The sigma-1 receptor (SIGMAR1) is involved in pain modulation especially under pre-sensitized conditions. However, the contribution of SIGMAR1 genetic variants to pain generation and sensitivity is unknown yet. This study aimed to identify effects of 5 SIGMAR1 variants on the somatosensory phenotype of neuropathic pain patients (n = 228) characterized by standardized quantitative sensory testing. Principal component analysis revealed that the SIGMAR1 variants -297G>T (rs10814130) and 5A>C (rs1800866) significantly lowered thermal detection and heat/pressure nociception in particular in neuropathic pain patients with mainly preserved somatosensory function. Compared to wild-type, the variant allele -297T was associated with loss of warm detection (P = .049), lower heat-pain sensitivity (P = .027) and wind-up ratio (P = .023) as well as increased paradoxical heat sensation (P = .020). Likewise for 5A>C the strongest genotype-associated differences observed were reduced peripheral (less heat hyperalgesia; P = .026) and central sensitization (lower mechanical pain sensitivity; P = .026) in variant compared to wild-type carriers. This study indicates lack of association of SIGMAR1 -297G>T and 5A>C genetic variants to susceptibility to develop chronic pain, but significant modulation of somatosensory function in neuropathic pain patients. PERSPECTIVE: This article presents the first study indicating a modulation of somatosensory function in neuropathic pain patients by selected genetic variants in SIGMAR1. As our findings could contribute to the explanation of interindividual differences in drug response they might help to improve the treatment of neuropathic pain.

Dysregulation of Mucosal Membrane Transporters and Drug-Metabolizing Enzymes in Ulcerative Colitis.

Intestinal transporters and metabolizing enzymes are the important factors of the intestinal absorption barrier. Because there is evidence that their expression and function may be affected during inflammatory conditions, we investigated gene expression, protein abundance, and regulation of relevant intestinal transporters and metabolizing enzymes in the intestinal mucosa of patients with ulcerative colitis (UC). Specimens from inflamed and noninflamed tissues of 10 patients with UC as well as colonic control tissues of 10 patients without inflammation were subjected to gene (9 enzymes, 15 transporters, 9 cytokines) and microRNA (N = 54) expression analysis. Protein abundance was quantified by liquid chromatography-tandem mass spectrometry-based targeted proteomics. Gene expression of several metabolizing enzymes (e.g., CYP2C9, UGT1A1) and transporters such as ABCB1 (ABCB1), ABCG2 (ABCG2), and monocarboxylate transporter 1 (MCT1, SLC16A1) were significantly decreased during inflammation and negatively correlated to microRNAs. On contrary, multidrug resistance-protein 4 (MRP4, ABCC4), organic anion-transporting polypeptide 2B1 (OATP2B1, SLCO2B1), and organic cation transporter-like 2 (ORCTL2, SLC22A18) were significantly elevated in inflamed tissue. However, at protein level, these findings could only be confirmed for MCT1. UC is associated with complex changes in the intestinal expression of enzymes, transporters, cytokines, and microRNAs, which may affect efficacy of anti-inflammatory drug therapy or the disease state itself.

MicroRNA-655-3p regulates Echinacea purpurea mediated activation of ABCG2.

1. The aim of this study was to investigate the regulatory effect of Echinacea purpurea (EP) on efflux transporters ABCB1 and ABCG2 and to identify specific microRNAs contributing to their post-transcriptional regulation. 2. ABCB1 and ABCG2 levels were assessed in human hepatoblastoma HepG2 cells treated with 50 µg/mL methanolic extract of commercial EP capsules for different durations. The microRNA expression profile of HepG2 cells after EP treatment was evaluated and in silico target prediction was subsequently conducted to identify specific microRNAs with binding sites in the 3'-UTR of ABCB1 and ABCG2. Luciferase reporter gene assays and site-directed mutagenesis were used to confirm the binding site of identified microRNA within the 3'-UTR of the target gene. 3. EP increased ABCB1 (10-fold ± 3.4, p < 0.001) and ABCG2 (2.7-fold ± 0.5, p < 0.01) mRNA levels after 12 h exposure. Twenty-four microRNAs showed significant expression differences at all durations of exposure to EP. MiR-655-3p showed a 6.79-fold decrease in expression after 12 h exposure compared to 0 h, was predicted in silico to bind ABCG2 3'-UTR and showed a significant negative correlation (p = 0.01) to ABCG2 expression level. The binding of miR-655-3p to ABCG2 3'-UTR was confirmed by reporter gene assays (reduction of reporter gene activity to 60%; p = 0.0001). 4. These results suggest that EP regulates ABCG2 expression via downregulation of miR-655-3p in the liver cells. Thus, miR-655-3p downregulation could be applied to predict EP mediated drug interactions.

MicroRNA-212/ABCG2-axis contributes to development of imatinib-resistance in leukemic cells.

BCR-ABL-independent resistance against tyrosine kinase inhibitor is an emerging problem in therapy of chronic myeloid leukemia. Such drug resistance can be linked to dysregulation of ATP-binding cassette (ABC)-transporters leading to increased tyrosine kinase inhibitor efflux, potentially caused by changes in microRNA expression or DNA-methylation. In an in vitro-imatinib-resistance model using K-562 cells, microRNA-212 was found to be dysregulated and inversely correlated to ABC-transporter ABCG2 expression, targeting its 3'-UTR. However, the functional impact on drug sensitivity remained unknown. Therefore, we performed transfection experiments using microRNA-mimics and -inhibitors and investigated their effect on imatinib-susceptibility in sensitive and resistant leukemic cell lines. Under imatinib-treatment, miR-212 inhibition led to enhanced cell viability (p = 0.01), reduced apoptosis (p = 0.01) and cytotoxicity (p = 0.03). These effects were limited to treatment-naïve cells and were not observed in cells, which were resistant to various imatinib-concentrations (0.1 µM to 2 µM). Further analysis in treatment-naïve cells revealed that miR-212 inhibition resulted in ABCG2 upregulation and increased ABCG2-dependent efflux. Furthermore, we observed miR-212 promoter hypermethylation in 0.5 and 2 µM IM-resistant sublines, whereas ABCG2 methylation status was not altered. Taken together, the miR-212/ABCG2-axis influences imatinib-susceptibility contributing to development of imatinib-resistance. Our data reveal new insights into mechanisms initiating imatinib-resistance in leukemic cells.

Hematopoietic stem cell involvement in BCR-ABL1-positive ALL as a potential mechanism of resistance to blinatumomab therapy.

The bispecific T-cell engager blinatumomab targeting CD19 can induce complete remission in relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, some patients ultimately relapse with loss of CD19 antigen on leukemic cells, which has been established as a novel mechanism to escape CD19-specific immunotherapies. Here, we provide evidence that CD19-negative (CD19-) relapse after CD19-directed therapy in BCP-ALL may be a result of the selection of preexisting CD19- malignant progenitor cells. We present 2 BCR-ABL1 fusion-positive BCP-ALL patients with CD19- myeloid lineage relapse after blinatumomab therapy and show BCR-ABL1 positivity in their hematopoietic stem cell (HSC)/progenitor/myeloid compartments at initial diagnosis by fluorescence in situ hybridization after cell sorting. By using the same approach with 25 additional diagnostic samples from patients with BCR-ABL1-positive BCP-ALL, we identified HSC involvement in 40% of the patients. Patients (6 of 8) with major BCR-ABL1 transcript encoding P210BCR-ABL1 mainly showed HSC involvement, whereas in most of the patients (9 of 12) with minor BCR-ABL1 transcript encoding P190BCR-ABL1, only the CD19+ leukemia compartments were BCR-ABL1 positive (P = .02). Our data are of clinical importance, because they indicate that both CD19+ cells and CD19- precursors should be targeted to avoid CD19- relapses in patients with BCR-ABL1-positive ALL.

Clinically Relevant Multidrug Transporters Are Regulated by microRNAs along the Human Intestine.

Intestinal drug transporters are crucial determinants for absorption and oral bioavailability of drugs. In healthy tissue donors, a recent study revealed profound discrepancies between mRNA expression and protein abundance as well as differences in the protein content between small and large intestine for clinically relevant multidrug transporters as the ATP binding cassette transporter subfamily B member 1 (ABCB1) and subfamily C member 3 (ABCC3) and the solute carrier family 15 member 1 (SLC15A1, PEPT1). As the mechanisms underlying these observations remained unclear, the aim of the present study was to elucidate the intestinal regiospecific microRNA profile under physiological conditions and identify specific microRNAs contributing to the post-transcriptional regulation of major drug transporters. For this purpose, tissue samples were collected from six intestinal sites obtained from six healthy tissue donors. The expression of 754 microRNAs was determined using qRT-PCR based low density arrays, and microRNA expression levels were correlated with transporter protein abundance quantified by targeted proteomics. A total of 241 microRNA-transporter pairs were identified, showing significant negative correlations to protein abundance (p < 0.05). Out of these, for nine pairs, the binding of the microRNA to the respective transporter 3'-UTR was predicted in silico. Besides the already known interactions of miR-27a-3p-ABCB1 and miR-193a-3p-PEPT1, reporter gene assays confirmed binding of miR-192-5p to the ABCC3 3'-UTR (reduction of reporter gene activity by 31%; p = 0.0012), miR-409-3p to the ABCB1 3'-UTR (reduction by 38%; p = 0.0006), and miR-193b-3p as well as miR-27a-3p to PEPT1 3'-UTR (reduction by 49% (p = 0.0012) and 20% (p = 0.0043), respectively). These results suggest that mucosal microRNA expression contributes to the explanation of discrepancies between mRNA expression and protein abundance as well as site-dependent differences in protein content along the human intestine under physiological conditions, as exemplified for ABCB1, ABCC3, and PEPT1.

Correction: A European Spectrum of Pharmacogenomic Biomarkers: Implications for Clinical Pharmacogenomics.

[This corrects the article DOI: 10.1371/journal.pone.0162866.].

miRNA-187-3p-Mediated Regulation of the KCNK10/TREK-2 Potassium Channel in a Rat Epilepsy Model.

Regulatory RNAs play a key role in the regulation of protein expression patterns in neurological diseases. Here we studied the regulation of miRNAs in a chronic rat model of temporal lobe epilepsy. The analysis was focused on a putative link with pharmacoresponsiveness as well as the functional implications of the regulation of a selected miRNA. The findings did not reveal a difference in hippocampal miRNA expression between phenobarbital responders and nonresponders. However, when comparing rats following status epilepticus with control rats we identified 13 differentially expressed miRNAs with miRNA-187-3p being most strongly regulated. mRNAs encoding KCNK10/TREK-2 as well as DYRK2 were confirmed as targets of miRNA-187-3p. Expression of the potassium channel protein KCNK10/TREK-2 negatively correlated with hippocampal miRNA-187-3p expression and proved to be upregulated in the chronic phase of the epilepsy model. In conclusion, our data do not suggest a relevant impact of miRNA expression patterns on pharmacoresponsiveness. However, we confirmed regulation of miRNA-187-3p and demonstrated that it impacts the expression of the two-pore domain potassium channel protein KCNK10/TREK-2. Considering evidence from brain ischemia models, KCNK10/TREK-2 upregulation might serve a protective function with a beneficial impact on astrocytic potassium and glutamate homeostasis.