Supplementary Table 1 from Carcinogen-Altered Genes in Rat Esophagus Positively Modulated to Normal Levels of Expression by Both Black Raspberries and Phenylethyl Isothiocyanate

Background and Objectives: Bronchopulmonary dysplasia (BPD) is a serious complication of preterm birth, resulting in significant morbidity and mortality. Recent studies have suggested that microRNA (miRNA) dysregulation is involved in the pathogenesis of BPD and may serve as biomarkers for early detection. We conducted a directed search for dysregulated miRNAs in lung and heart autopsy samples of infants with histologic BPD. Methods: We used archived lung and heart samples from BPD (13 lung, 6 heart) and control (24 lung, 5 heart) subjects. To measure miRNA expression, RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue specimens then reverse-transcribed, labeled, and hybridized to miRNA microarrays. The microarrays were scanned, and data were quantile normalized. Statistical analysis with a moderated t-test and control of the false discovery rate (5%) was used to compare normalized miRNA expression values between clinical categories. Results: With our set of 48 samples, 43 miRNAs had a significant difference in expression comparing BPD to non-BPD controls. Among the most statistically significant miRNAs, miR-378b, miRNA-184, miRNA-3667-5p, miRNA-3976, miRNA-4646-5p, and miRNA-7846-3p were all consistently upregulated in both the heart and lung tissues of BPD subjects. The cellular pathway predicted to be most affected by these miRNAs is the Hippo signaling pathway. Conclusions: This study identifies miRNAs that are similarly dysregulated in postmortem lung and heart samples in subjects with histologic BPD. These miRNAs may contribute to the pathogenesis of BPD, have potential as biomarkers, and may provide insight to novel approaches for diagnosis and treatment.

Exosomes are a class of small, secreted extracellular vesicles (EV) that have recently gained considerable attention for their role in normal cellular function, disease processes and potential as biomarkers. Exosomes serve as intercellular messengers and carry molecular cargo that can alter gene expression and the phenotype of recipient cells. Here, we investigated alterations of microRNA cargo in exosomes secreted by epileptogenic tissue in tuberous sclerosis complex (TSC), a multi-system genetic disorder that includes brain lesions known as tubers. Approximately 90% of TSC patients suffer from seizures that originate from tubers, and ~60% are resistant to antiseizure drugs. It is unknown why some tubers cause seizures while others do not, and the molecular basis of drug-resistant epilepsy is not well understood. It is believed that neuroinflammation is involved, and characterization of this mechanism may be key to disrupting the "vicious cycle" between seizures, neuroinflammation, and increased seizure susceptibility. We isolated exosomes from epileptogenic and non-epileptogenic TSC tubers, and we identified differences in their microRNA cargo using small RNA-seq. We identified 12 microRNAs (including miR-142-3p, miR-223-3p and miR-21-5p) that are significantly increased in epileptogenic tubers and contain nucleic acid motifs that activate toll-like receptors (TLR7/8), initiating a neuroinflammatory cascade. Exosomes from epileptogenic tissue caused induction of key pathways in cultured cells, including innate immune signaling (TLR), inflammatory response and key signaling nodes SQSTM1 (p62) and CDKN1A (p21). Genes induced in vitro were also significantly upregulated in epileptogenic tissue. These results provide new evidence on the role of exosomes and non-coding RNA cargo in the neuroinflammatory cascade of epilepsy and may help advance the development of novel biomarkers and therapeutic approaches for the treatment of drug-resistant epilepsy.

Asthma affects nearly 6 million children in the United States. Approximately 50% of children with asthma experience an exacerbation annually, 16.7% require an emergency department (ED) visit, and nearly 4.7% are hospitalized yearly, making asthma the third leading cause of pediatric inpatient admissions.1Zahran H.S. Bailey C.M. Damon S.A. Garbe P.L. Breysse P.N. Vital Signs: asthma in Children-United States, 2001-2016.MMWR Morb Mortal Wkly Rep. 2018; 67: 149-155Crossref PubMed Scopus (275) Google Scholar Asthma is a multifactorial disease that is often triggered by environmental factors in genetically predisposed individuals. Because the ED is frequently the first, if not the only, point of access to health care for many children with acute asthma, it provides a unique, yet largely unexplored, clinical setting to investigate novel molecular biomarkers for asthma severity and treatment response. MicroRNAs are a class of short, highly stable endogenous noncoding RNAs involved in regulating multiple cellular processes, either by preventing translation and/or inducing transcript degradation.2Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29561) Google Scholar Past research has primarily focused on identifying changes in microRNA expression associated with airway inflammation in acute asthma3Lu T.X. Munitz A. Rothenberg M.E. MicroRNA-21 is up-regulated in allergic airway inflammation and regulates IL-12p35 expression.J Immunol. 2009; 182: 4994-5002Crossref PubMed Scopus (492) Google Scholar; however, there is a paucity of pediatric data, as these studies were conducted primarily in adults, were set in subacute care settings, and utilized invasive methods of sample collection either via bronchoscopy or blood samples. We sought to evaluate microRNA expression patterns in a pediatric ED patient population with varying asthma severity by using tissue obtained via nasal swabs. Nasal and bronchial cells have identical morphology and similar responses to cytokines in vivo and in vitro4McDougall C.M. Blaylock M.G. Douglas J.G. Brooker R.J. Helms P.J. Walsh G.M. Nasal epithelial cells as surrogates for bronchial epithelial cells in airway inflammation studies.Am J Respir Cell Mol Biol. 2008; 39: 560-568Crossref PubMed Scopus (128) Google Scholar; therefore, nasal epithelia could potentially constitute an accessible surrogate for studying lower airway inflammation. The main objectives of our study were to demonstrate the feasibility of obtaining adequate amounts of high-quality microRNAs via nasal swabs and to compare microRNA expression patterns in pediatric patients with varying severity of asthma. We conducted a prospective, observational study on children 18 years of age or younger and presenting to an inner city, freestanding pediatric ED. This study was approved by the institutional review board, and patients were enrolled after informed consent had been obtained. We enrolled patients in 1 of the following 4 cohorts: patients with severe asthma exacerbation; patients with moderate asthma exacerbation; patients with a history of asthma with no exacerbation, and patients with no history of asthma or atopy (Table I). We defined acute asthma as wheezing or respiratory distress in a patient with a prior diagnosis of asthma or reactive airway disease. Asthma severity was determined by using the Pediatric Asthma Severity Score, a standardized asthma assessment tool. We excluded patients with a history of heart disease, those with lung disease other than asthma, and those who refused informed consent.Table IPatient demographicsCharacteristicTotal no. (N = 39)No history of asthma (n = 10)History of asthma with no exacerbation (n = 10)Moderate asthma exacerbation (n = 10)Severe asthma exacerbation (n = 9)Sex Male237583 Female163526Age <1 y11000 1-10 y224558 >10 y165551Ethnicity White43010 African American3571099 Hispanic00000Family history Asthma293899 Eczema233677Patient history of eczema190667 Open table in a new tab We enrolled a total of 50 participants and collected nasal swabs from each patient. RNA was successfully extracted and tested for quality from 48 specimens (see Methods in this article’s Online Repository at www.jacionline.org). MicroRNA expression microarrays were performed for approximately 10 patient samples having the best RNA quality in each group (N = 39 in total; see the Methods section and see also Table E1 in this article’s Online Repository at www.jacionline.org). Initial statistical analysis revealed that the expression values of some microRNAs were dependent on RNA quality. Therefore, for subsequent statistical analysis we excluded 11 samples with an RNA quality score less than 7 (see Methods). The remaining 28 microarrays included 8 samples each from the controls and the cohort with severe asthma exacerbation and 6 samples from both the cohort with moderate asthma exacerbation and the no-exacerbation cohort. With use of stringent statistical analysis in which the probability of a false positive was restricted to 1%, comparison of the group with severe asthma exacerbation and the control group revealed that microRNA miR-582-5p is significantly induced (∼24-fold) in the cohort of those with severe asthma exacerbation (Fig 1). The expression level of miR- 582-5p in the group with moderate exacerbation and the group with no exacerbation is intermediate compared with that in the control group and the group with severe asthma exacerbation (see Fig E1 in in this article’s Online Repository at www.jacionline.org). Interestingly, miR-582-5p has been found to be upregulated in sputum of subjects exposed to ozone, altering their respiratory immune response.5Fry R.C. Rager J.E. Bauer R. Sebastian E. Peden D.B. Jaspers I et al.Air toxics and epigenetic effects: ozone altered microRNAs in the sputum of human subjects.Am J Physiol Lung Cell Mol Physiol. 2014; 306: L1129-L1137Crossref PubMed Scopus (61) Google Scholar We investigated whether the increase in miR-582-5p expression might be due to alterations in cell populations. Using a set of microRNAs characteristic for lymphocytes, red blood cells, immune cells, and epithelial cells, we compared expression between the group with severe asthma exacerbation and the control group. No significant differences were observed (see Fig E2 and the Methods section in this article’s Online Repository at www.jacionline.org). Our study demonstrated that it is feasible to obtain adequate quantities of high-quality microRNAs by using a noninvasive approach and conduct complex genomic studies of pediatric asthma in the ED. Although the use of blood and/or bronchial epithelial cells has been a preferred method of obtaining the “ideal” tissue sample for biomarker studies in the past, this approach is not always practical, especially in the pediatric population. Here, we show that nasal epithelium can be used as a bronchial epithelium surrogate obtained via a substantially less invasive method compared with endobronchial lavage. There are several limitations to our study. The first is the small cohort size. Although prior research revealed that significant differences in gene expression are detectable in small sample sizes (N ≤ 30),6McGeachie M.J. Davis J.S. Kho A.T. Dahlin A. Sordillo J.E. Sun M. et al.Asthma remission: predicting future airways responsiveness using an miRNA network.J Allergy Clinical Immunol. 2017; 140: 598-600.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar our findings warrant further validation. However, our statistical analysis showed that significant changes in microRNA expression could be detected in nasal epithelia with a low false-positive rate, even in a limited cohort. We found that high-quality RNAs are important to obtain robust results. Further improvements in the collection protocol may provide improved consistency in RNA quality. Second, the majority of enrolled patients were African American, reflecting the demographics of our ED population. A multicenter study with a diverse population would be the optimal setting to validate our results. In conclusion, it is feasible to obtain high-quality microRNAs from nasal epithelium in pediatric patients presenting to the ED. In this proof-of-principle study, we demonstrated that miR-582-5p is robustly overexpressed in nasal epithelia of children with severe, acute asthma. Subsequent studies and validation are warranted to further investigate the relationship between miR-582-5p and asthma severity to explore the potential of this microRNA as a clinical biomarker and to help guide targeted therapies. We thank the patients and their families for their kind cooperation. We also thank Elizabeth Duffy for her research and editorial support. Patients were enrolled and samples were collected in the ED of Children’s Hospital of Michigan between June 2012 and June 2013. Nasal epithelia were collected by using a Rhino-Pro Curette (Arlington Scientific, Springville, Utah). Samples were stored at –80˚ C until they were analyzed for microRNA expression. Total RNA from nasal epithelia was isolated by using the All-in-One Purification Kit per the vendor’s protocol (Norgen Biotek Corp, Thorold, Ontario, Canada). The quantity, purity, and quality of the RNA were verified, and an RNA integrity number (RIN) was assigned to each specimen. RINs range from 1 to 10, with 10 representing the best quality. Samples with a RIN less than 7.0 (n = 11) were excluded from subsequent statistical analysis, as this reflected RNA degradation (Table E1).E1Schroeder A. Mueller O. Stocker S. Salowsky R. Leiber M. Gassmann M. et al.The RIN: an RNA integrity number for assigning integrity values to RNA measurements.BMC Molecular Biol. 2006; 7: 3Crossref PubMed Scopus (1817) Google Scholar Expression of microRNAs was measured in each sample by using microRNA expression arrays (G4872A-031181, Agilent, Santa Clara, Calif). For each sample, 100 ng of total RNA and Agilent microRNA spike-in controls were processed and fluorescently labeled per the vendor’s protocol. Hybridization was performed per the vendor protocol, and the slides were scanned with an Agilent dual-laser scanner. Tiff images were analyzed by using Agilent’s feature extraction software (version 11.0.1.1) to obtain fluorescent intensities for each spot on the arrays. Numerous quality control parameters were inspected. The data were imported into GeneSpring (Agilent) for analysis, and probe intensity values were quantile-normalized within each microarray. Low-intensity expression values are the least reliable; therefore, we first filtered out microRNA probes in the lower 20 percentile of expression of the overall microarray. This provided 1078 microRNAs and controls (of 1368) for subsequent analysis. A moderated t test (GeneSpring) was applied to the normalized expression data for the 1368 microRNAs and used to test for statistical significance between groups. The type I (false-positive) error rate was controlled by using the Westfall-Young family-wise multiple test correction. A corrected P value of .01 was considered significant. With this stringency, there is only a 1% chance of having 1 or more false positives among the microRNAs identified as significant. Fold change (FC) is often used in analysis of gene expression data to provide a measure of the change in the expression level of a gene when comparing 2 conditions. The FC for a given gene is derived from the ratio (R) of the expression level for the gene in the 2 conditions (A and B), where R equals A divided by B. Then, FC equals R when R is greater than or equal to 0, and FC equals –1 divided by R when R is less than 0. A log transformation of the expression ratio (eg, log2R) is also a convenient representation because positive values represent an increase in expression and negative values reflect a decrease.E2Wang Y. Yang L. Li P. Huang H. Liu T. He H. et al.Circulating microRNA Signatures associated with childhood asthma.Clin Lab. 2015; 61: 467-474PubMed Google Scholar Potential differences in cell type populations were investigated by examining the expression values of microRNAs that are characteristic markers of specific cell types.E3Teruel-Montoya R. Kong X. Abraham S. Ma L. Kunapuli S.P. Holinstat M. et al.MicroRNA expression differences in human hematopoietic cell lineages enable regulated transgene expression.PLoS One. 2014; 9: e102259Crossref PubMed Scopus (64) Google Scholar, E4Gilicze A.B. Wiener Z. Tóth S. Buzás E. Pállinger É. Falcone F.H. et al.Myeloid-derived microRNAs, miR-223, miR27a, and miR-652, are dominant players in myeloid regulation.Biomed Res Int. 2014; 2014: 870267Crossref PubMed Scopus (27) Google Scholar, E5Tserel L. Runnel T. Kisand K. Pihlap M. Bakhoff L. Kolde R. et al.MicroRNA expression profiles of human blood monocyte-derived dendritic cells and macrophages reveal miR-511 as putative positive regulator of Toll-like receptor.J Biol Chem. 2011; 286: 26487-26495Crossref PubMed Scopus (110) Google Scholar, E6Martinez-Anton A. Sokolowska M. Kern S. Davis A.S. Alsaaty S. Taubenberger J.K. et al.Changes in microRNA and mRNA expression with differentiation of human bronchial epithelial cells.Am J Respir Cell Mol Biol. 2013 Sep; 49 (PMID: 23590309): 384-395Crossref PubMed Scopus (43) Google Scholar, E7McCall M.N. Kim M.S. Adil M. Patil A.H. Lu Y. Mitchell C.J. et al.Toward the human cellular microRNAome.Genome Res. 2017; 27: 1769-1781Crossref PubMed Scopus (95) Google Scholar Expression in the group with severe asthma exacerbation and in the control group were compared (Table E2).Fig E2Cell types in collected epithelia are consistent between the control group and the group with severe asthma. Expression of microRNAs characteristic for specific cells types was compared between the controls with no history (Hx) of asthma and the subjects with severe asthma. Significant differences were not observed, suggesting consistency in cell type populations in the patient specimens. RBC, Red blood cell.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1RNA quality and groupSample IDGroupRIN1011Severe9.71020No Hx9.11042Severe9.11007No Hx8.91016Moderate8.71021Moderate8.71030No Hx8.71010Severe8.71001Hx8.61015Hx8.51036Moderate8.51031Severe8.51002Severe81027Hx7.91034Moderate7.91005No Hx7.91013No Hx7.91008Severe7.91014Moderate7.81040No Hx7.81023Severe7.81004Hx7.71037Hx7.71041Hx7.71006No Hx7.71022No Hx7.71048Severe7.71017Moderate71018Severe6.91026Hx6.81003No Hx6.81044Hx6.71043Hx6.51033Moderate6.31012Moderate6.21029Moderate6.21039No Hx6.21038Hx5.91045Moderate5.9ID, Identifier; Hx, history; RIN, RNA integrity number. Open table in a new tab Table E2MicroRNA expression by cell typemicroRNACell typemiR-200b-3pEpitheliallet-7f-5pPlateletsmiR-451aRed blood cellsmiR-148a-3pGranulocytesmiR-150-5pLymphocytesmiR-212-3pDendritic and macrophagesmiR-99b-5pDendritic and macrophagesmiR-125a-3pMucosal mast cells Open table in a new tab ID, Identifier; Hx, history; RIN, RNA integrity number.

Fetoplacental neuroblastoma metastasis has been postulated as a mechanism accounting for concordant cases where one twin develops a primary tumour and the second twin manifests the disease without an identifiable primary site. These tumours may originate and spread concomitantly due to the same genetic background shared by monozygotic twins. This study investigated the molecular profile of stage MS neuroblastoma presenting concomitantly in monozygotic twins. Comparative genomic hybridisation (aCGH) was done for each of the twin liver tumour and peripheral blood samples at diagnosis. Comparison of copy-number variation (CNV) regions revealed a set of CNVs that were common to both tumour specimens and not apparent in the blood. The CNV signature in both twins' tumours was highly similar, suggesting a common clonal origin. Additional findings included large deletion of chromosome 10 and amplification of chromosome 17. Notably, both liver samples had amplification of a short region involving DEIN (chromosome 4q34.1). Similar CNVs strongly support a common clonal origin and metastatic spread from one twin to the other. DEIN is a long-coding RNA (IncRNA) that has been found highly expressed in stage MS neuroblastoma and is likely involved in biological processes such as cell migration and metastasis.

BACKGROUND:Neuroinflammation and toll-like receptors (TLR) of the innate immune system have been implicated in epilepsy. We previously reported high levels of microRNAs miR-142-3p and miR-223-3p in epileptogenic brain tissue resected for the treatment of intractable epilepsy in children with tuberous sclerosis complex (TSC). As miR-142-3p has recently been reported to be a ligand and activator of TLR7, a detector of exogenous and endogenous single-stranded RNA, we evaluated TLR7 expression and downstream IL23A activation in surgically resected TSC brain tissue.METHODS:Gene expression analysis was performed on cortical tissue obtained from surgery of TSC children with pharmacoresistent epilepsy. Expression of TLRs 2, 4 and 7 was measured using NanoString nCounter assays. Real-time quantitative PCR was used to confirm TLR7 expression and compare TLR7 activation, indicated by IL-23A levels, to levels of miR-142-3p. Protein markers characteristic for TLR7 activation were assessed using data from our existing quantitative proteomics dataset of TSC tissue. Capillary electrophoresis Western blots were used to confirm TLR7 protein expression in a subset of samples.RESULTS:TLR7 transcript expression was present in all TSC specimens. The signaling competent form of TLR7 protein was detected in the membrane fraction of each sample tested. Downstream activation of TLR7 was found in epileptogenic lesions having elevated neuroinflammation indicated by clinical neuroimaging. TLR7 activity was significantly associated with tissue levels of miR-142-3p.CONCLUSION:TLR7 activation by microRNAs may contribute to the neuroinflammatory cascade in epilepsy in TSC. Further characterization of this mechanism may enable the combined of use of neuroimaging and TLR7 inhibitors in a personalized approach towards the treatment of intractable epilepsy.

Tuberous sclerosis complex (TSC) is characterized by hamartomatous lesions in various organs and arises due to mutations in the TSC1 or TSC2 genes. TSC mutations lead to a range of neurological manifestations including epilepsy, cognitive impairment, autism spectrum disorders (ASD), and brain lesions that include cortical tubers. There is evidence that seizures arise at or near cortical tubers, but it is unknown why some tubers are epileptogenic while others are not. We have previously reported increased tryptophan metabolism measured with α[11C]-methyl-l-tryptophan (AMT) positron emission tomography (PET) in epileptogenic tubers in approximately two-thirds of patients with tuberous sclerosis and intractable epilepsy. However, the underlying mechanisms leading to seizure onset in TSC remain poorly characterized. MicroRNAs are enriched in the brain and play important roles in neurodevelopment and brain function. Recent reports have shown aberrant microRNA expression in epilepsy and TSC. In this study, we performed microRNA expression profiling in brain specimens obtained from TSC patients undergoing epilepsy surgery for intractable epilepsy. Typically, in these resections several non-seizure onset tubers are resected together with the seizure-onset tubers because of their proximity. We directly compared seizure onset tubers, with and without increased tryptophan metabolism measured with PET, and non-onset tubers to assess the role of microRNAs in epileptogenesis associated with these lesions. Whether a particular tuber was epileptogenic or non-epileptogenic was determined with intracranial electrocorticography, and tryptophan metabolism was measured with AMT PET. We identified a set of five microRNAs (miR-142-3p, 142-5p, 223-3p, 200b-3p and 32-5p) that collectively distinguish among the three primary groups of tubers: non-onset/AMT-cold (NC), onset/AMT-cold (OC), and onset/AMT-hot (OH). These microRNAs were significantly upregulated in OH tubers compared to the other two groups, and microRNA expression was most significantly associated with AMT-PET uptake. The microRNAs target a group of genes enriched for synaptic signaling and epilepsy risk, including SLC12A5, SYT1, GRIN2A, GRIN2B, KCNB1, SCN2A, TSC1, and MEF2C. We confirmed the interaction between miR-32-5p and SLC12A5 using a luciferase reporter assay. Our findings provide a new avenue for subsequent mechanistic studies of tuber epileptogenesis in TSC.

Tuberous sclerosis complex (TSC) is a multisystem genetic disorder caused by mutations in the TSC1 and TSC2 genes. Over 80% of TSC patients are affected by epilepsy, but the molecular events contributing to seizures in TSC are not well understood. Recent reports have demonstrated that the brain is enriched with microRNA activity, and they are critical in neural development and function. However, little is known about the role of microRNAs in TSC. Here, we report the characterization of aberrant microRNA activity in cortical tubers resected from 5 TSC patients surgically treated for medically intractable epilepsy. By comparing epileptogenic tubers with adjacent nontuber tissue, we identified a set of 4 coordinately overexpressed microRNAs (miRs 23a, 34a, 34b*, 532-5p). We used quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic profiling to investigate the combined effect of the 4 microRNAs on target proteins. The proportion of repressed proteins among the predicted targets was significantly greater than in the overall proteome and was highly enriched for proteins involved in synaptic signal transmission. Among the combinatorial targets were TSC1, coding for the protein hamartin, and several epilepsy risk genes. We found decreased levels of hamartin in epileptogenic tubers and confirmed targeting of the TSC1 3' UTR by miRs-23a and 34a.

Squalene synthase inhibitors (SSIs), such as squalestatin 1 (SQ1), reduce cholesterol biosynthesis but cause the accumulation of isoprenoids derived from farnesyl pyrophosphate (FPP), which can modulate the activity of nuclear receptors, including the constitutive androstane receptor (CAR), farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs). In comparison, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (e.g., pravastatin) inhibit production of both cholesterol and nonsterol isoprenoids. To characterize the effects of isoprenoids on hepatocellular physiology, microarrays were used to compare orthologous gene expression from primary cultured mouse and rat hepatocytes that were treated with either SQ1 or pravastatin. Compared with controls, 47 orthologs were affected by both inhibitors, 90 were affected only by SQ1, and 51 were unique to pravastatin treatment (P < 0.05, ≥1.5-fold change). When the effects of SQ1 and pravastatin were compared directly, 162 orthologs were found to be differentially coregulated between the two treatments. Genes involved in cholesterol and unsaturated fatty acid biosynthesis were up-regulated by both inhibitors, consistent with cholesterol depletion; however, the extent of induction was greater in rat than in mouse hepatocytes. SQ1 induced several orthologs associated with microsomal, peroxisomal, and mitochondrial fatty acid oxidation and repressed orthologs involved in cell cycle regulation. By comparison, pravastatin repressed the expression of orthologs involved in retinol and xenobiotic metabolism. Several of the metabolic genes altered by isoprenoids were inducible by a PPARα agonist, whereas cytochrome P450 isoform 2B was inducible by activators of CAR. Our findings indicate that SSIs uniquely influence cellular lipid metabolism and cell cycle regulation, probably due to FPP catabolism through the farnesol pathway.

During cholestasis, the bile acid–conjugating enzymes, SULT2A1 and UGT2B4, work in concert to prevent the accumulation of toxic bile acids. To understand the impact of sulfotransferase deficiency on human hepatic gene expression, we knocked down 3′-phosphoadenosine-5′-phosphosulfate synthases (PAPSS) 1 and 2, which catalyze synthesis of the obligate sulfotransferase cofactor, in HepG2 cells. PAPSS knockdown caused no change in SULT2A1 expression; however, UGT2B4 expression increased markedly (∼41-fold increase in UGT2B4 mRNA content). Knockdown of SULT2A1 in HepG2 cells also increased UGT2B4 expression. To investigate the underlying mechanism, we transfected PAPSS-deficient HepG2 cells with a luciferase reporter plasmid containing ∼2 Kb of the UGT2B4 5′-flanking region, which included a response element for the bile acid–sensing nuclear receptor, farnesoid X receptor (FXR). FXR activation or overexpression increased UGT2B4 promoter activity; however, knocking down FXR or mutating or deleting the FXR response element did not significantly decrease UGT2B4 promoter activity. Further evaluation of the UGT2B4 5′-flanking region indicated the presence of distal regulatory elements between nucleotides −10090 and −10037 that negatively and positively regulated UGT2B4 transcription. Pulse-chase analysis showed that increased UGT2B4 expression in PAPSS-deficient cells was attributable to both increased mRNA synthesis and stability. Transfection analysis demonstrated that the UGT2B4 3′-untranslated region decreased luciferase reporter expression less in PAPSS-deficient cells than in control cells. These data indicate that knocking down PAPSS increases UGT2B4 transcription and mRNA stability as a compensatory response to the loss of SULT2A1 activity, presumably to maintain bile acid–conjugating activity.

During cholestasis, the bile acid–conjugating enzymes, SULT2A1 and UGT2B4, work in concert to prevent the accumulation of toxic bile acids. To understand the impact of sulfotransferase deficiency on human hepatic gene expression, we knocked down 39-phosphoadenosine-59-phosphosulfate synthases (PAPSS) 1 and 2, which catalyze synthesis of the obligate sulfotransferase cofactor, in HepG2 cells. PAPSS knockdown caused no change in SULT2A1 expression; however, UGT2B4 expression increased markedly (∼41-fold increase in UGT2B4 mRNA content). Knockdown of SULT2A1 in HepG2 cells also increased UGT2B4 expression. To investigate the underlying mechanism, we transfected PAPSS-deficient HepG2 cells with a luciferase reporter plasmid containing ∼2 Kb of the UGT2B4 59-flanking region, which included a response element for the bile acid–sensing nuclear receptor, farnesoid X receptor (FXR). FXR activation or overexpression increased UGT2B4 promoter activity; however, knocking down FXR or mutating or deleting the FXR response element did not significantly decrease UGT2B4 promoter activity. Further evaluation of the UGT2B4 59-flanking region indicated the presence of distal regulatory elements between nucleotides 210090 and 210037 that negatively and positively regulated UGT2B4 transcription. Pulse-chase analysis showed that increased UGT2B4 expression in PAPSS-deficient cells was attributable to both increased mRNA synthesis and stability. Transfection analysis demonstrated that the UGT2B4 39-untranslated region decreased luciferase reporter expression less in PAPSS-deficient cells than in control cells. These data indicate that knocking down PAPSS increases UGT2B4 transcription and mRNA stability as a compensatory response to the loss of SULT2A1 activity, presumably to maintain bile acid–conjugating activity.

Toxic equivalency factors (TEFs) for dioxin-like compounds are largely based on relative potency (REP) values derived from biochemical endpoints such as enzyme activity. As of yet, REPs based on gene expression changes have not been accounted for in the TEF values. In this study, primary rat hepatocytes were treated for 24h with 11 concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), or 2,3,7,8-tetrachlorodibenzofuran (TCDF) ranging from 0.00001 to 100nM. Differential changes in gene expression were analyzed using analysis of variance to assess the relative contributions of concentration, congener, and the interaction between concentration and congener for each gene. A total of 3283 genes showed significant changes with concentration (false discovery rate < .05 and fold-change ± 1.5 in at least 1 concentration for 1 congener). Among these genes, 399 were significant for both concentration and congener effects indicating parallel concentration-response curves with significant differences in potency. Only 8 genes showed a significant concentration and congener interaction term indicating a minority of genes show nonparallel dose-response curves among the 3 congeners. Benchmark dose (BMD) modeling was used to derive BMD values for induced individual genes and signaling pathways. The REP values for 4-PeCDF and TCDF were generally 3- to 5-fold lower than the World Health Organization (WHO) TEF values on both a gene and pathway basis. These findings suggest that the WHO TEF values may possibly overpredict the potency of these polychlorinated dibenzofuran congeners and demonstrate the importance of identifying functional pathways relevant to the toxicological modes of action for establishing pertinent REPs.

A toxicogenomics approach was used to qualitatively and quantitatively compare the gene expression changes in human and rat primary hepatocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes from five individual rats and five individual humans were exposed for 24 h to 11 concentrations of TCDD ranging from 0.00001 to 100nM and a vehicle control. Gene expression changes were analyzed using whole-genome microarrays containing 13,002 orthologs. Significant changes in expression of individual orthologs at any concentration (fold change [FC] ± 1.5 and false discovery rate < 0.05) were higher in the rat (1547) compared with human hepatocytes (475). Only 158 differentially expressed orthologs were common between rats and humans. Enrichment analysis was performed on the differentially expressed orthologs in each species with 49 and 34 enriched human and rat pathways, respectively. Only 12 enriched pathways were shared between the two species. The results demonstrate significant cross-species differences in expression at both the gene and pathway level. Benchmark dose analysis of gene expression changes showed an average 18-fold cross-species difference in potency among differentially expressed orthologs with the rat more sensitive than the human. Similar cross-species differences in potency were observed for signaling pathways. Using the maximum FC in gene expression as a measure of efficacy, the human hepatocytes showed on average a 20% lower efficacy among the individual orthologs showing differential expression. The results provide evidence for divergent cross-species gene expression changes in response to TCDD and are consistent with epidemiological and clinical evidence showing humans to be less sensitive to TCDD-induced hepatotoxicity.

Background In Saccharomyces cerevisiae, the G1 cyclin/cyclin-dependent kinase (CDK) complexes Cln1,-2,-3/Cdk1 promote S phase entry during the mitotic cell cycle but do not function during meiosis. It has been proposed that the meiosis-specific protein kinase Ime2, which is required for normal timing of pre-meiotic DNA replication, is equivalent to Cln1,-2/Cdk1. These two CDK complexes directly catalyze phosphorylation of the B-type cyclin/CDK inhibitor Sic1 during the cell cycle to enable its destruction. As a result, Clb5,-6/Cdk1 become activated and facilitate initiation of DNA replication. While Ime2 is required for Sic1 destruction during meiosis, evidence now suggests that Ime2 does not directly catalyze Sic1 phosphorylation to target it for destabilization as Cln1,-2/Cdk1 do during the cell cycle. Methodology/Principal Findings We demonstrated that Sic1 is eventually degraded in meiotic cells lacking the IME2 gene (ime2Δ), supporting an indirect role of Ime2 in Sic1 destruction. We further examined global RNA expression comparing wild type and ime2Δ cells. Analysis of these expression data has provided evidence that Ime2 is required early in meiosis for normal transcription of many genes that are also periodically expressed during late G1 of the cell cycle. Conclusions/Significance Our results place Ime2 at a position in the early meiotic pathway that lies upstream of the position occupied by Cln1,-2/Cdk1 in the analogous cell cycle pathway. Thus, Ime2 may functionally resemble Cln3/Cdk1 in promoting S phase entry, or it could play a role even further upstream in the corresponding meiotic cascade.

The present study used a postinitiation protocol to investigate molecular mechanisms by which black raspberries (BRBs) influence the late stages of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in rats. F344 rats were injected with NMBA and then fed either control diet or a diet containing 5% BRB powder. Control rats were injected with DMSO/water (20:80), the vehicle for NMBA. Esophagi from control, NMBA- and NMBA_BRB-treated rats were collected at 35 wk for histopathological, molecular, and immunohistochemical analyses. Treatment with 5% BRBs reduced the number of dysplastic lesions and the number and size of esophageal papillomas in NMBA-treated rats. When compared to esophagi from control rats, NMBA treatment led to the differential expression of 4807 genes in preneoplastic esophagus (PE) and 17 846 genes in esophageal papillomas. Dietary BRBs modulated 626 of the 4807 differentially expressed genes in PE and 625 of the 17 846 differentially expressed genes in esophageal papillomas towards normal levels of expression. In both PE and in papillomas, BRBs modulated the mRNA expression of genes associated with carbohydrate and lipid metabolism, cell proliferation and death, and inflammation. In these same tissues, BRBs modulated the expression of proteins associated with proliferation, apoptosis, inflammation, angiogenesis, and both cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Interestingly, matrix metalloproteinases involved in tissue invasion and metastasis, and proteins associated with cell-cell adhesion, were also modulated by BRBs. This is the first report of the effects of berries on the expression of genes associated with the late stages of rat esophageal carcinogenesis. (C) 2011 Wiley-Liss, Inc.

Toxicogenomics was used to examine mRNA expression profiles obtained from primary rat hepatocytes treated for 24h with 0.01 or 1.0nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 0.02 or 2.0nM 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF) and 0.1 or 10nM 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF). The concentrations of 2,3,4,7,8-PeCDF and 2,3,7,8-TCDF were chosen to be equivalent to 2,3,7,8-TCDD’s concentration based on the toxic equivalency factor/toxic equivalent (TEF/TEQ) method for estimating biological potency. 2,3,7,8-TCDD at 1.0nM altered the expression of 533 genes; 2,3,4,7,8-PeCDF at 2.0nM altered 182 genes, and 2,3,7,8-TCDF at 10nM altered 154 genes. Of these, 57 genes were affected by all three congeners. Agglomerative hierarchical clustering revealed distinct congener-dependent gene subclusters. Principal components analyses of the microarray data revealed that these congeners cluster independently of one another. Data presented here demonstrate that equivalent TEQ concentrations of 2,3,7,8-TCDD, 2,3,4,7,8-PeCDF and 2,3,7,8-TCDF, while altering the expression of a small battery of genes in common, also produce substantial congener specific alterations in gene expression.

The MCF10A cell line represents a model of the normal human breast epithelial cell. Estrogen sulfotransferase (SULT1E1), a major estrogen‐inactivating enzyme, is up‐regulated in confluent relative to preconfluent MCF10A cells. The mechanism responsible for SULT1E1 up‐regulation in confluent MCF10A cells was investigated. Transient or stable tranfection of reporter constructs containing up to 7 kb of the SULT1E1 5′‐flanking region failed to recapitulate confluency‐induced SULT1E1 up‐regulation. The half‐life of SULT1E1 mRNA in preconfluent MCF10A cells was estimated to be ~4h based on analysis of SULT1E1 mRNA levels in MCF10A cells that were replated after reaching confluency. Microarray analysis revealed that 85 microRNAs were significantly up‐regulated and 24 microRNAs were down‐regulated in confluent relative to preconfluent MCF10A cells. Computational analysis of the SULT1E1 3′‐untranslated region identified 4 putative candidate binding sites for confluency‐regulated microRNAs. The confluency‐related decrease in two of these microRNAs (miR‐221 and miR‐100*) corresponded with the confluency‐induced increase in SULT1E1 mRNA observed in MCF10A cells. These results support a role for the post‐transcriptional up‐regulation of SULT1E1 mRNA expression in confluent relative to preconfluent MCF10A cells. Supported by NIH grant (ARRA) ES016373 (MRM) and EHS Center grant ES06636.

Abstracts: Frontiers in Cancer Prevention Research 2008 A131 We reported that the feeding of a diet containing 5 or 10% freeze-dried black raspberries (BRB) to N -nitrosomethylbenzylamine (NMBA)-treated rats results in a 39-64% reduction in the number of esophageal papillomas when using either anti-initiation or anti-promotion/progression protocols. The molecular events associated with the effects of BRB on NMBA-induced preneoplastic and papillomatous esophageal lesions however, have not been fully elucidated. In the present study, 4-5 week-old male F344 rats were injected s.c. with NMBA (0.3 mg/kg b.w., 3x/wk for 5 wks) after which they were fed either control diet or diet containing 5% BRB until the end of the study (35 wks). Control rats were injected s.c. with a solution of DMSO/water (20:80), the vehicle for NMBA. Esophagi from vehicle control, NMBA- and NMBA + BRB-treated rats were collected at 35 weeks for histologic grading, and for microarray and Real-Time PCR analyses. Treatment with 5% BRB reduced the number of preneoplastic lesions (dysplasias) and the number and size of papillomas in the esophagus of NMBA-treated rats. When compared to esophagi from vehicle control rats, NMBA treatment alone led to the differential expression of 4,807 genes in preneoplastic esophagus and 17,846 genes in esophageal papillomas. Treatment with 5% BRB resulted in a modulation towards control levels of expression of 626 genes in preneoplastic esophagus and 627 genes in papillomas. In both preneoplastic esophagus and in papillomas, the berry-modulated genes were associated with regulation of cell proliferation, inflammation and receptor-mediated pathways. Twenty-five genes were commonly modulated (down- or up-regulated) by BRB in both preneoplastic lesions and in papillomas. Interestingly, several of these genes are associated with matrix metalloproteinases involved in tissue invasion and metastasis, cell-cell adhesion and with calcium signaling. This is the first report suggesting that berries might influence genes involved in tissue invasion and metastasis in the rat esophagus. (Supported by NCI grant No. CA103180) Citation Information: Cancer Prev Res 2008;1(7 Suppl):A131.

The goal of the present study is to unveil the gene expression profile specific to the biological processes of human breast epithelial cell invasion and migration using an MCF10A model in which the H‐Ras or N‐Ras signaling pathway is constitutively activated. H‐Ras, but not N‐Ras, induces invasion/migration. H‐Ras‐mediated MCF10A invasion involves increased expression of MMP‐2 and ‐9. Analysis of whole human genome microarray revealed that 412 genes were differentially expressed among MCF10A, N‐Ras MCF10A and H‐Ras MCF10A cells. Two calcium‐binding proteins, S100A8 and S100A9, were prominently upregulated in an H‐Ras‐specific manner. Importantly, siRNA‐mediated knockdown of S100A8 or S100A9 expression significantly reduced H‐Ras‐mediated MCF10A cell invasion/migration. We further demonstrated that S100A8 was more closely associated with MMP‐9 expression mediated by ERKs pathway while S100A9 might play a major role in MMP‐2 upregulation which is dependent on p38MAPK pathway. Taken together, this study reveals S100A8 and S100A9 as candidate markers for metastatic potential of breast epithelial cells. Our gene profile data provide invaluable information which might be useful for identification of additional potential targets for prognosis and/or therapy of metastatic breast cancer [Supported by the NIEHS, NCI R41, USA and the Fostering Project of the Lab of Excellency, Korea].