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Physiological genomics [journal]
- Human Aldosterone Synthase Gene Polymorphism Promotes miRNA binding and Regulates Gene Expression. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Oct 28.:physiolgenomics.00084.2014.
Hypertension is a serious risk factor for myocardial infarction, heart failure, vascular disease, stroke, and renal failure. Like other complex disease, hypertension is caused by a combination of genetic and environmental factors. The renin-angiotensin-aldosterone system (RAAS) plays an important role in the regulation of blood pressure. The octapeptide, angiotensin-II (Ang-II), is one of the most active vasopressor agents and is obtained from precursor molecule, angiotensinogen (AGT), by the combined proteolytic action of renin and angiotensin converting enzyme (ACE). Ang-II increases the expression of aldosterone synthase (coded by Cyp11B2 gene) which is the rate limiting enzyme in the biosynthesis of aldosterone. Previous studies have shown that increased expression of aldosterone synthase increases blood pressure and cardiac hypertrophy in transgenic mice. Human Cyp11B2 gene has a T/C polymorphism at -344 positions in its 5'-UTR and -344T allele is associated with hypertension. Human Cyp11B2 gene also has an A/G polymorphism at 735 position in its 3'-UTR (rs28491316) which is in linkage disequilibrium with SNP at -344. We show here that (a) miR-766 binds to the 735G-allele and not the 735A-allele of the hCyp11B2 gene and (b) transfection of miR-766 reduces the human aldosterone synthase mRNA and protein level in human adrenocortical cells H295R. These studies suggest that miR766 may down-regulate the expression of human aldosterone synthase gene and reduce blood pressure in human subjects containing -344T allele.
- In Utero Exposure to Pre-Pregnancy Maternal Obesity and Post-weaning High Fat Diet Impairs Regulators of Mitochondrial Dynamics in Rat Placenta and Offspring. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Oct 21.:physiolgenomics.00059.2014.
The proportion of pregnant women who are obese at conception continues to rise. Compelling evidence suggests the intrauterine environment is an important determinant of offspring health. Maternal obesity and unhealthy diets are shown to promote metabolic programming in the offspring. Mitochondria are maternally inherited and we have previously shown impaired mitochondrial function in rat offspring exposed to maternal obesity in utero. Mitochondrial health is maintained by mitochondrial dynamics, or the processes of fusion and fission, which serve to repair damaged mitochondria, remove irreparable mitochondria, and maintain mitochondrial morphology. An imbalance between fusion and fission has been associated with obesity, insulin resistance, and reproduction complications. In the present study, we examined the influence of maternal obesity and post-weaning high fat diet (HFD) on key regulators of mitochondrial fusion and fission in rat offspring at important developmental milestones which included post-natal day (PND)35 (2 wk HFD) and PND130 (∼16 wk HFD). Our results indicate HFD-fed offspring had reduced mRNA expression of presenilin associated rhomboid-like (PARL), optic atrophy (OPA)1, mitofusin (Mfn)1, Mfn2, fission (Fis)1, and nuclear respiratory factor (Nrf)1 at PND35, while OPA1 and Mfn2 remained decreased at PND130. Putative transcriptional regulators of mitochondrial dynamics were reduced in rat placenta and offspring liver and skeletal muscle (peroxisome proliferator-activated receptor gamma co-activator (PGC1)α, PGC1β, and estrogen-related receptor (ERR)α), consistent with indirect calorimetry findings revealing reduced energy expenditure and impaired fat utilization. Overall, maternal obesity detrimentally alters mitochondrial targets that may contribute to impaired mitochondrial health and increased obesity susceptibility in later life.
- TNF-α alters the release and transfer of microparticle-encapsulated miRNAs from endothelial cells. [Journal Article]
- Physiol Genomics 2014 Nov 15; 46(22):833-40.
MicroRNAs (miRNAs) encapsulated within microparticles (MPs) are likely to have a role in cell-to-cell signaling in a variety of diseases, including atherosclerosis. However, little is known about the mechanisms by which different cell types release and transfer miRNAs. Here, we examined TNF-α-induced release of MP-encapsulated miR-126, miR-21, and miR-155 from human aortic endothelial cells (ECs) and their transfer to recipient cells. ECs were treated with TNF-α (100 ng/ml) in the presence or absence of inhibitors that target different MP production pathways. MPs released in response to TNF-α were characterized by: 1) 70-80% decrease in miRNA/MP levels for miR-126 and -21 but a significant increase in pre-miR-155 and miR-155 (P < 0.05), 2) 50% reduction in uptake by recipient cells (P < 0.05), and 3) diminished ability to transfer miRNA to recipient cells. Cotreatment of donor ECs with TNF-α and caspase inhibitor (Q-VD-OPH, 10 μM) produced MPs that had: 1) 1.5- to 2-fold increase in miRNA/MP loading, 2) enhanced uptake by recipient cells (2-fold), and 3) increased ability to transfer miR-155. Cotreatment of ECs with TNF-α and Rho-associated kinase (ROCK) inhibitor (10 μM) produced MPs with features similar to those produced by TNF-α treatment alone. Our data indicate that TNF-α induced the production of distinct MP populations: ROCK-dependent, miRNA-rich MPs that effectively transferred their cargo and were antiapoptotic, and caspase-dependent, miRNA-poor MPs that were proapoptotic. These data provide insight into the relationship between MP production and extracellular release of miRNA, as well as the potential of encapsulated miRNA for cell-to-cell communication.
- Dietary supplementation with polyunsaturated fatty acid during pregnancy modulates DNA methylation at IGF2/H19 imprinted genes and growth of infants. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Oct 7.
Epigenetic regulation of imprinted genes is regarded as a highly plausible explanation for linking dietary exposures in early life with the onset of diseases during childhood and adulthood. We sought to test whether prenatal dietary supplementation with docosahexaenoic acid (DHA) during pregnancy may modulate epigenetic states at birth. This study was based on a randomized intervention trial conducted in Mexican pregnant women supplemented daily with 400 mg of DHA or a placebo from gestation week 18-22 to parturition. We applied quantitative profiling of DNA methylation states at IGF2 promoter 3 (IGF2 P3), IGF2 differentially methylated region (DMR), and H19 DMR in cord blood mononuclear cells of the DHA-supplemented group (n=131) and the control group (n=130). In stratified analyses, DNA methylation levels in IGF2 P3 were significantly higher in the DHA group than the control group in preterm infants (p=0.04). We also observed a positive association between DNA methylation levels and maternal body mass index (BMI); IGF2 DMR methylation was higher in the DHA group than the control group in infants of overweight mothers (p=0.03). In addition, at H19 DMR, methylation levels were significantly lower in the DHA group than the control group in infants of normal weight mothers (p=0.01). Finally, methylation levels at IGF2/H19 imprinted regions were associated with maternal BMI. These findings suggest that epigenetic mechanisms may be modulated by DHA, with potential impacts on child growth and development.
- Identification of genes whose expression is altered by obesity throughout the arterial tree. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Sep 30.
We used next-generation RNA sequencing (RNA-Seq) technology to identify genes whose expression is consistently affected by obesity across multiple arteries. Specifically, transcriptional profiles of the iliac artery as well as the feed artery, first, second, and third branch order arterioles in the soleus, gastrocnemius and diaphragm muscles from obese Otsuka Long-Evans Tokushima Fatty (OLETF) and lean Long-Evans Tokushima Otsuka (LETO) rats were examined. Within the gastrocnemius and soleus muscles, the number of genes differentially expressed with obesity tended to increase with increasing branch order arteriole number (i.e., decreasing size of the artery). This trend was opposite in the diaphragm. We found a total of 15 genes that were consistently upregulated with obesity (MIS18A, CTRB1, FAM151B, FOLR2, PXMP4, OAS1B, KLRA17, SLC25A44, SNX10, SLFN3, MEF2BNB, IRF7, RAD23A, LGALS3BP) and 5 genes that were consistently downregulated with obesity (C2, GOLGA7, RIN3, PCP4, CYP2E1). A small fraction (~9%) of the genes affected by obesity was modulated across all arteries examined. In conclusion, the present study identifies a select number of genes (i.e., 20 genes) whose expression is consistently altered throughout the arterial network in response to obesity and provides further insight into the heterogeneous vascular effects of obesity. Although there is no known direct function of the majority of 20 genes related to vascular health, the obesity-associated upregulation of SREBF2, LGALS3BP, IRF7, and FOLR2 across all arteries is evidence of a systemic pro-atherogenic phenotype switch with obesity. These data may serve as an important resource for identifying novel therapeutic targets against obesity-related vascular complications.
- Using normalization to resolve RNA-Seq biases caused by amplification from minimal input. [Journal Article]
- Physiol Genomics 2014 Nov 1; 46(21):808-20.
RNA-Seq has become a widely used method to study transcriptomes, and it is now possible to perform RNA-Seq on almost any sample. Nevertheless, samples obtained from small cell populations are particularly challenging, as biases associated with low amounts of input RNA can have strong and detrimental effects on downstream analyses. Here we compare different methods to normalize RNA-Seq data obtained from minimal input material. Using RNA from isolated medaka pituitary cells, we have amplified material from six samples before sequencing. Both synthetic and real data are used to evaluate different normalization methods to obtain a robust and reliable pipeline for analysis of RNA-Seq data from samples with very limited input material. The analysis outlined here shows that quantile normalization outperforms other more commonly used normalization procedures when using amplified RNA as input and will benefit researchers employing low amounts of RNA in similar experiments.
- Maternal PRKAA1 and EDNRA genotypes are associated with birth weight, and PRKAA1 with uterine artery diameter and metabolic homeostasis at high altitude. [Journal Article]
- Physiol Genomics 2014 Sep 15; 46(18):687-97.
Low birth weight and intrauterine growth restriction (IUGR) increase the risk of mortality and morbidity during the perinatal period as well as in adulthood. Environmental and genetic factors contribute to IUGR, but the influence of maternal genetic variation on birth weight is largely unknown. We implemented a gene-by-environment study wherein we utilized the growth restrictive effects of high altitude. Multigenerational high-altitude residents (Andeans) are protected from altitude-associated IUGR compared with recent migrants (Europeans). Using a combined cohort of low- and high-altitude European and Andean women, we tested 63 single nucleotide polymorphisms (SNPs) from 16 natural selection-nominated candidate gene regions for associations with infant birth weight. We identified significant SNP associations with birth weight near coding regions for two genes involved in oxygen sensing and vascular control, PRKAA1 and EDNRA, respectively. Next, we identified a significant association for the PRKAA1 SNP with an intermediate phenotype, uterine artery diameter, which has been shown to be related to Andean protection from altitude-associated reductions in fetal growth. To explore potential functional relationships for the effect of maternal SNP genotype on birth weight, we evaluated the relationship between maternal PRKAA1 SNP genotype and gene expression patterns in general and, in particular, of key pathways involved in metabolic homeostasis that have been proposed to play a role in the pathophysiology of IUGR. Our observations suggest that maternal genetic variation within genes that regulate oxygen sensing, metabolic homeostasis, and vascular control influence fetal growth and birth weight outcomes and hence Andean adaptation to high altitude.
- Gonadal transcriptomic analysis of yellow catfish (Pelteobagrus fulvidraco): identification of sex-related genes and genetic markers. [Journal Article]
- Physiol Genomics 2014 Nov 1; 46(21):798-807.
Yellow catfish (Pelteobagrus fulvidraco) has been recognized as a vital freshwater aquaculture species in East and Southeast Asia. In addition to its commercial interest, it is also attracted much attention because of its value in studying sex-determination mechanisms. A comprehensive gonadal transcriptome analysis is believed to provide a resource for genome annotation, candidate gene identification, and molecular marker development. Herein, we performed a de novo assembly of yellow catfish gonad transcriptome by high-throughput Illumina sequencing. A total of 82,123 contigs were obtained, ranging from 351 to 21,268 bp, and N50 of 2,329 bp. Unigenes of 21,869 in total were identified. Of these, 229 and 1,188 genes were found to be specifically expressed in XY gonad tissue for 1 yr and 2 yr old yellow catfish, respectively; correspondingly, 51 and 40 genes were identified in XX gonad tissue at those two stages. Gene ontology and KEGG analysis were conducted and classified all contigs into different categories. A large number of unigenes involved in sex determination were identified, as well as microsatellites and SNP variants. The expression patterns of sex-related genes were then validated by quantitative real-time PCR (qRT-PCR) suggesting the high reliability of RNA-Seq results. In this study, the transcriptome of yellow catfish gonad was first sequenced, assembled, and characterized; it provides a valuable genomic resource for better understanding of yellow catfish sex determination as well as development of molecular markers, thereby assisting in the production of monosex yellow catfish for aquaculture.
- S-adenosylmethionine mediates inhibition of inflammatory response and changes in DNA methylation in human macrophages. [Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.]
- Physiol Genomics 2014 Sep 1; 46(17):617-23.
S-adenosylmethionine (SAM), the unique methyl donor in DNA methylation, has been shown to lower lipopolysaccharide (LPS)-induced expression of the proinflammatory cytokine TNF-α and increase the expression of the anti-inflammatory cytokine IL-10 in macrophages. The aim of this study was to assess whether epigenetic mechanisms mediate the anti-inflammatory effects of SAM. Human monocytic THP1 cells were differentiated into macrophages and treated with 0, 500, or 1,000 μmol/l SAM for 24 h, followed by stimulation with LPS. TNFα and IL-10 expression levels were measured by real-time PCR, cellular concentrations of SAM and S-adenosylhomocysteine (SAH), a metabolite of SAM, were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and DNA methylation was measured with LC-MS/MS and microarrays. Relative to control (0 μmol/l SAM), treatment with 500 μmol/l SAM caused a significant decrease in TNF-α expression (-45%, P < 0.05) and increase in IL-10 expression (+77%, P < 0.05). Treatment with 1,000 μmol/l SAM yielded no significant additional benefits. Relative to control, 500 μmol/l SAM increased cellular SAM concentrations twofold without changes in SAH, and 1,000 μmol/l SAM increased cellular SAM sixfold and SAH fourfold. Global DNA methylation increased 7% with 500 μmol/l SAM compared with control. Following treatment with 500 μmol/l SAM, DNA methylation microarray analysis identified 765 differentially methylated regions associated with 918 genes. Pathway analysis of these genes identified a biological network associated with cardiovascular disease, including a subset of genes that were differentially hypomethylated and whose expression levels were altered by SAM. Our data indicate that SAM modulates the expression of inflammatory genes in association with changes in specific gene promoter DNA methylation.
- Dynamic Gene Expression Patterns in Animal Models of Early and Late Heart Failure Reveal Biphasic-Bidirectional Transcriptional Activation of Signaling Pathways. [JOURNAL ARTICLE]
- Physiol Genomics 2014 Aug 26.
Background: Altered cardiac gene expression in heart failure (HF) has mostly been identified by single-point analysis of end-stage disease. This may miss earlier changes in gene expression that are transient and/or directionally opposite to those observed later. Results: Myocardial datasets from the largest microarray data repository (Gene Expression Omnibus) yielded six HF studies with time-course data. Differentially expressed transcripts between non-failing controls, early-HF (<3 days after cardiac insult) and late-HF (usually >2 weeks) were determined, and analysis of KEGG pathways and predicted regulatory control elements performed. We found that gene expression followed varying patterns: Downregulation of metabolic pathways occurred early and was sustained into late stage-HF. In contrast, most signaling pathways undergo a complex biphasic pattern: Calcium signaling, p53, apoptosis and MAP-kinase pathways displayed a bi-directional response, declining early but rising late. These profiles were compatible with specific miRNA and transcription regulators: Estrogen-related receptor-α (Esrra) and myocyte-enhancer factor-2 (Mef2) binding sites were overrepresented in the promoter regions of downregulated transcripts. Concurrently, there were overrepresented binding sites for E2f and ETS family members (E-Twenty Six, including Gabp, Elf1, and Ets2), serum response (Srf) and interferon regulated factor (Irf) in biphasic-bidirectional and late-upregulated transcripts. Binding sites for miRNAs downregulated by HF were more common in up-regulated transcripts (e.g. miRNA-22,-133a/b, and -150 in early-HF and miRNA-1,-9,-499 in late-HF). Conclusions: During the development of HF, metabolic gene classes show early and sustained downregulation in HF, while signaling pathways undergo a complex biphasic pattern with early down- and more pronounced late upregulation.