Microarray expression profile analysis of long non-coding RNAs in human aldosterone-producing adenoma
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE101894 | GEO | 2020/01/26
REPOSITORIES: GEO
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Project description:Recent advances in omics techniques have allowed detailed genetic characterization of aldosterone-producing adenoma (APA). The pathogenesis of APA is characterized by tumorigenesis-associated aldosterone synthesis. The pathophysiological intricacies of APAs have not yet been elucidated at the level of individual cells . Therefore, a single-cell level analysis is speculated to be valuable in studying the differentiation process of APA. We conducted single-nuclei RNA sequencing (snRNA-seq) of APAs and non-functional adenomas (NFA) obtained from three and two patients, respectively.The snRNA-seq revealed the intratumoral heterogeneity of APA and identified cell populations consisting of a shared cluster of NFA and APA. In addition, we extracted two cell differentiation pathways in APA and obtained a cell population specialized in aldosterone synthesis. Genes related to ribosomes and neurodegenerative diseases were upregulated in one of these pathways, whereas those related to the regulation of actin cytoskeleton were upregulated in the other pathway. Furthermore, the total RNA reads in the nucleus were higher in highly differentiated clusters, indicating a marked activation of transcription per cell. The snRNA-seq provides novel differentiation pathways within APA tumors, which will further support our understanding of its pathophysiology including endocrine function and tumorigenesis.
2023-12-01 | GSE242404 | GEO
Project description:The source of aldosterone in 30 to 40 % of patients with primary hyperaldosteronism (PA) is unilateral aldosterone-producing adenoma (APA). The mechanisms causing elevated aldosterone production in APA are unknown. Herein, we examined expression of G-protein coupled receptors (GPCR) in APA and demonstrate that compared to normal adrenals there is a general elevation of certain GPCR in many APA and/or ectopic expression of GPCR in others. RNA samples from normal adrenals (n = 5), APAs (n = 10), and cortisol-producing adenomas (CPAs) (n=13) were used on 15 genomic expression arrays, each of which included 223 GPCR transcripts presented in at least one out of 15 of the independent microarrays. The array results were confirmed using real-time RT-PCR (qPCR). Four GPCR transcripts exhibited a statistically significant increase that was greater than 3-fold compared to normal adrenals, suggesting a general increase in expression compared to normal adrenal glands. Four GPCR transcripts exhibited a greater than 15-fold increase of expression in one or more of the APA samples compared to normal adrenals. qPCR analysis confirmed array data and found the receptors with the highest fold increase in APA expression to be luteinizing hormone receptor (LH-R), serotonin receptor 4 (HTR4), gonadotropin-releasing hormone receptor (GnRHR), glutamate receptor metabotropic 3 (GRM3), endothelin receptor type B-like protein (GPR37), and ACTH receptor (MC2R). There are also sporadic increased expressions of these genes in the CPAs. Together, these findings suggest a potential role of altered GPCR expression in many cases of PA and provide candidate GPCR for further study. Keywords: disease state analysis
2007-07-19 | GSE8514 | GEO
Project description:To investigate the development of aldosterone-producing adenoma, we profiled cells of different areas of the adjacent adrenal cortex by snRNAseq to obtain possible trajectories towards adenoma formation.
2022-08-05 | GSE210381 | GEO
Project description:Analysis of aldosterone-producing adenoma (APA) samples from patients with primary hyperaldosteronism. These APAs have a somatic mutation in either KCNJ5, CACNA1D, or ATP1A1. Results provide insight into the different mechanisms each mutation may cause leading to elevated aldosterone production in APA. In this dataset, we include expression data from aldosterone-producing adenomas (APAs) with a somatic mutation in either KCNJ5, CACNA1D, or ATP1A1. These data are used to obtain differentially expressed genes between KCNJ5 mutant APAs and CACNA1D/ATP1A1 mutant APAs.
2013-07-14 | GSE48303 | GEO
Project description:The source of aldosterone in 30 to 40 % of patients with primary hyperaldosteronism (PA) is unilateral aldosterone-producing adenoma (APA). The mechanisms causing elevated aldosterone production in APA are unknown. Herein, we examined expression of G-protein coupled receptors (GPCR) in APA and demonstrate that compared to normal adrenals there is a general elevation of certain GPCR in many APA and/or ectopic expression of GPCR in others. RNA samples from normal adrenals (n = 5), APAs (n = 10), and cortisol-producing adenomas (CPAs) (n=13) were used on 15 genomic expression arrays, each of which included 223 GPCR transcripts presented in at least one out of 15 of the independent microarrays. The array results were confirmed using real-time RT-PCR (qPCR). Four GPCR transcripts exhibited a statistically significant increase that was greater than 3-fold compared to normal adrenals, suggesting a general increase in expression compared to normal adrenal glands. Four GPCR transcripts exhibited a greater than 15-fold increase of expression in one or more of the APA samples compared to normal adrenals. qPCR analysis confirmed array data and found the receptors with the highest fold increase in APA expression to be luteinizing hormone receptor (LH-R), serotonin receptor 4 (HTR4), gonadotropin-releasing hormone receptor (GnRHR), glutamate receptor metabotropic 3 (GRM3), endothelin receptor type B-like protein (GPR37), and ACTH receptor (MC2R). There are also sporadic increased expressions of these genes in the CPAs. Together, these findings suggest a potential role of altered GPCR expression in many cases of PA and provide candidate GPCR for further study. Keywords: disease state analysis Normal human adult adrenals were obtained through the Cooperative Human Tissue Network (Philadelphia, PA) and Clontech (Palo Alto, CA). All the normal adrenal samples came from patients that underwent adrenalectomy along with a renalectomy due to renal carcinoma. The adrenal samples acquired from autopsies were obtained no more than six hours after death, and it was confirmed that the causes of death were unrelated to adrenal function. Real-time quantitative RT-PCR was used to check the legitimacy of the normal control adrenal samples. Examination of tissue sections from each adrenal gland also suggested normal histology. The APA samples were obtained from the Departments of Medicine at UT Southwestern and Division of Endocrinology at the University of Padua. All APA samples were from Conn’s Syndrome patients with significantly elevated circulating aldosterone that was lowered to the normal range after surgical removal of the tumor. Thirty-two adenomas were collected from patients with primary hyperaldosteronism. Twenty-eight of these adenomas had levels of CYP11B2 that were two standard deviations (SD) greater than seen in normal adrenal glands and these samples were used for analyses. It is currently not clear if the adenomas with low CYP11B2 expression are the source of aldosterone in these patients. Other studies suggest that subcapsular micronodules have varying expression of steroidogenic enzymes-some with aldosterone synthase and some without (Shigematsu et al., 2006). Future studies are required to determine if some tumor-bearing adrenals from primary hyperaldosteronsim patients also have small CYP11B2 positive micronodules as the source of aldosterone production. The cortisol-producing adenoma (CPA) tissues were obtained from Division of Endocrinology at the University of Padua from patients with Cushing’s syndrome. Each of the patients was cured following surgical removal of the adenoma. The use of these tissues was approved by the Institutional Review Boards of the University of Texas Southwestern Medical Center (Dallas, TX), the Medical College of Georgia (Augusta, GA), and University of Padua (Italy). In addition, this study was approved by the ethical committee at the University of Padua, and informed consent was obtained from every patient. Microarray Analysis Total RNA isolated from normal adult adrenal glands (n = 5) and APA (n = 10) were used on 15 genomic expression arrays. In brief, RNA was hybridized to an Affymetrix human HG-U133+2 oligonucleotide microarray set containing 54,675 probe sets representing approximately 40,500 independent human genes. The arrays were scanned at high resolution using an Affymetrix GeneChip Scanner 3000 located at Medical College of Georgia Microarray Core Facility (Augusta, GA). Results were analyzed using GeneSpring GX 7.3.1 software (Silicon Genetics, Redwood City, CA) to identify differences in expression of GPCR between normal adult adrenal and APA. RNA Extraction The tissue was pulverized in liquid nitrogen to a powder. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA). The purity and integrity of the RNA were assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and its quantity was determined by NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA Synthesis 2 µg of total RNA was reverse transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) following manufacturer recommendations and incubated at 25 ºC for 10 min, and 37 ºC for 2 h. The synthesized cDNA was subjected to 1:10 dilution and stored at -20ºC. Real-Time Quantitative PCR Primers and probes for the amplification of the selected GPCR sequences were performed using the TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) based on published sequences for genes encoding the respective human GPCR. The gene symbols and AB assay numbers are listed in Table 1. The primer and probe set for human CYP11B2 was designed using Primer Express 3.0 (Applied Biosystems, Foster City, CA) and purchased from IDT (Integrated DNA Technologies, Inc, IA) as published previously (Saner-Amigh et al., 2006). In brief: Forward: 5’-GGCAGAGGCAGAGATGCTG-3’, Reverse: 5’- CTTGAGTTAGTGTCTCCACCAGGA-3’, Probe: 5’-CTGCACCACGTGCTGAAGCACT-3’. PCR reactions were performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) with a total volume of 20 μl per reaction following the reaction parameters recommended by the manufacturer, which includes denaturation at 95ºC for 20 s followed by amplification for 40 cycles (95ºC at 3 s, 60ºC at 30 s, fluorescence measurement). For each GPCR the 20 μl total volume consisted of 10 µl TaqMan Fast Universal PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 900 nM of each primer, 400 nM of the probe and 5 μl of each first-strand cDNA sample. CYP11B2 reaction mix consisted of 10µl TaqMan PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 100 nM of primer/probe mix and 5 μl of each first-strand cDNA sample. 18s rRNA was detected and quantified using the TaqMan Ribosomal RNA Control Reagents (Vic Probe) (Applied Biosystems, Foster City, CA). Each reaction included 10 µl of TaqMan PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 100 nM probe and 50 nM primers. Negative controls contained water instead of first-strand cDNA. Quantitative normalization of cDNA in each tissue-derived sample was performed using expression of 18s rRNA as an internal control. The generated Ct value of each gene was normalized by its respected Ct value of 18s rRNA (∆Ct). Then each gene was further normalized using the average ∆Ct value of the normal adult adrenal (∆∆Ct). The final fold-expression changes were calculated using the equation 2-∆∆Ct (Livak & Schmittgen, 2001). Statistical Analysis Data were analyzed and compared to control values (mean of normal adrenal samples) using the Mann-Whitney Rank Sum test with the SigmaStat 3.0 software package (SPSS, Chicago, IL). Results were considered significantly different when p value was 0.05.
2007-07-18 | E-GEOD-8514 | biostudies-arrayexpress
Project description:Analysis of aldosterone-producing adenoma (APA) samples from patients with primary hyperaldosteronism. These APAs have a somatic mutation in either KCNJ5, CACNA1D, or ATP1A1. Results provide insight into the different mechanisms each mutation may cause leading to elevated aldosterone production in APA. In this dataset, we include expression data from aldosterone-producing adenomas (APAs) with a somatic mutation in either KCNJ5, CACNA1D, or ATP1A1. These data are used to obtain differentially expressed genes between KCNJ5 mutant APAs and CACNA1D/ATP1A1 mutant APAs. A total of 13 samples were analyzed (8 KCNJ5 mutant APAs and 5 CACNA1D/ATP1A1 mutant APAs). 43 genes had a false discovery rate (FDR) <0.5% and were >2-fold different between KCNJ5 mutant APAs and 5 CACNA1D/ATP1A1 mutant APAs. The two sets of genotypes were separated on unsupervised hierarchical clustering of 1475 genes correlating >0.6 with CYP11B2.
2013-07-14 | E-GEOD-48303 | biostudies-arrayexpress
Project description:Aldosterone-producing adenomas (APA) are heterogeneous. The objectives of this study were to compare the transcriptional profiles in APA and normal adjacent adrenal gland (AAG). This was done through the Illumina beadchip analysis of RNA from eight paired APA-AAG. Other tissues (phaeochromocytoma, cushing, and hyperplastic adrenals) were included as controls.
2020-12-01 | GSE156931 | GEO
Project description:Learn about the transcriptome profiling of zona glomerulosa (ZG), zona fasciculata (ZF) and aldosterone-producing adenomas (APA) in human adrenals
2015-09-15 | GSE64957 | GEO
Project description:Learn about the transcriptome profiling of zona glomerulosa (ZG), zona fasciculata (ZF) and aldosterone-producing adenomas (APA) in human adrenals 21 pairs of zona fasciculata (ZF) and zona glomerulosa (ZG), and 14 paired aldosterone-producing adenomas (APAs) from 14 Conn’s syndrome patients and 7 phaeochromocytoma patients were assayed on the Affymetrix Human Genome U133 Plus 2.0 Array. Laser capture microdissection was used to acquire samples of ZF, ZG and APA as previously described (Azizan EA, et al. J Clin Endocrinol Metab. 2012;97:E819-E829). For differentiation of ZG from ZF, sections were stained with cresyl violet using the LCM Staining Kit (AM1935, Ambion, USA). Data processing and analysis was performed using AffymetrixGeneChip Command Console Software and PartekGenomicSuite 6.5 (Partek Inc., St. Louis, MO). Gene expressions were portrayed as the summarized log-signal of the Robust Multichip Average (RMA) with quantilenormalisation and median polish for probe set summarisation. Validation by qPCR was performed on genes >10 fold up-regulated in zona glomerulosa (compared to zona fasciculata) and >10 fold up-regulated in aldosterone-producing adenomas (compared to zona glomerulosa).
2015-09-15 | E-GEOD-64957 | biostudies-arrayexpress
Project description:Sepsis is a life-threatening disease that often causes multiple organ dysfunctions. Long noncoding RNAs (lncRNAs) are involved in the pathogenesis and development of sepsis. However, there is little known about the function of lncRNAs in sepsis-induced myocardial dysfunction.To test the hypothesis that dysregulating lncRNAs may be involved in sepsis-induced myocardial dysfunction, we identified differentially expressed (DE) lncRNAs and mRNAs in cardiac tissue by high-throughput sequencing. We constructed coding and non-coding co-expression (CNC) and lncRNA-micro RNA (miRNA)-mRNA competing endogenous networks. The findings will provide useful information for exploring therapeutic candidate targets and new molecular biomarkers of septic myocardial injury.
2023-05-17 | GSE153086 | GEO