Down-regulated Probe Sets Research Articles

Erratum to: Transcriptome analysis reveals salt-stress-regulated biological processes and key pathways in roots of peanut (Arachis hypogaea L.)

The cultivated peanut is important oil crop and salt stress seriously influences its development and yield. Tolerant varieties produced using transgenic techniques can effectively increase peanut plantation area and enhance its yields. However, little is known about how gene expression is regulated by salt stress in peanut. In this study, we screened genes regulated by salt stress in peanut roots using microarray technique. In total, 4828 up-regulated and 3752 down-regulated probe sets were successfully identified in peanut roots subjected to 3 and 48 h of salt stress. Data analysis revealed that different response groups existed between the up and down-regulated probe sets. The main up-regulated biological processes involved in salt stress responses included transcription regulation, stress response, and metabolism and biosynthetic processes. The main down-regulated biological processes included transport processes, photosynthesis and development. The Kyoto encyclopedia of genes and genomes pathway analysis indicated that metabolic pathway, biosynthesis of unsaturated fatty acids and plant–pathogen interaction, were mainly up-regulated in peanut under salt stress. However, photosynthesis and phenylalanine metabolism were mainly down-regulated during salt stress. The function of some probe sets in salt stress regulation was not clarified (e.g., protein functioning in cell cycle regulation and xylem development). Many of the genes we identified lacked functional annotations and their roles in response to salt stress are yet to be elucidated. These results identified some candidate genes as potential markers and showed an overview of the transcription map, which may yield some useful insights into salt-mediated signal transduction pathways in peanut.

Abstract 4966: Establishment and characterization of a paired human non-small cell lung cancer cell lines from primary cancer and metastasis lymph node

Abstract Objective: Lung cancer has become one of the leading causes of cancer-related mortality in the world. Metastasis remains the overwhelming causes of death for lung cancer patients. 80-90% of lung cancer deaths are caused by metastasis. However, an ideal in vitro model to explore the regulation and the molecules that control the lymph node metastasis of lung cancer is required. Methods: A gene expression profiling for both of the cell lines were generated by microarray. Further investigations were carried out in the lung cancer cell lines and paired tissues by migration and invasion assays and immunohistochemistry. Results: Firstly, we established a paired human lung adenocarcinoma cell lines from primary tumor site (named DC cells) and metastatic paratracheal lymph nodes (named DL cells). The two cell lines have different biological characteristics which DC cells grow clump easily and DL cells grow more scattered. The doubling time of DC and DL cell lines were 32.6h and 45.2h, respectively. The detection of the human D1S80 and D17S30 VNTR locus verified the same individual for DC and DL cell lines. Secondary, Microarray analysis revealed 232 upregulated probe sets and 739 downregulated probe sets in DL cell line compared to DC cell line. Compared to DC cells, the transcriptional expression of 27 genes were dramatically increased (p < 0.05), including AIM2, IGFBP4, IL6 and IL6R in DL cells, while expression of 55 genes was clearly decreased (p < 0.05), including FGF13, FGF19, IL1A, IL1B, MCAM, MMP15, MMP7, and TGFB2 by real-time PCR analysis. Furthermore, we selected 32 genes and screened the expression of those genes in 10-paired human NSCLC primary lung cancer tissues and matched metastasis lymph nodes tissues by real-time PCR. The results showed: 8 out of 10 metastasis lymph node tissues got up-regulated of RGS7, VNN2, and 7 out of 10 metastasis lymph node tissues got up-regulated of PECAM1, VNN1, CLIC2 expression. On the other hand, 8 of 10 metastasis lymph node tissues got down-regulated expression of CEACAM6, and 7 out of 10 got down-regulated expression of PIAS3. At mean time, none of metastasis lymph node tissues got decreased expression of TNFRSF9, which only was seen in DL cells. The further study in other lung cancer cell lines indicated that CLIC2, PECAM1, RAB38, BCL9 and RGS7 genes influenc lung cancer cell migration ability in vitro. And these genes were also analyzed in more lung cancer tissues by immunohistochemistry staining. Conclusion: We established a paired human lung adenocarcinoma cell lines from primary tumor site and metastatic paratracheal lymph nodes. The CLIC2, PECAM1, RAB38, BCL9 and RGS7 genes involve in the lung cancer metastasis. Note: This abstract was not presented at the meeting. Citation Format: Jun Chen, Min Wang, Lingling Zu, Yongwen Li, Weiqiang Wang,Ying Li, Hongyu Liu. Establishment and characterization of a paired human non-small cell lung cancer cell lines from primary cancer and metastasis lymph node. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4966. doi:10.1158/1538-7445.AM2014-4966

A replication study for genome-wide gene expression levels in two layer lines elucidates differentially expressed genes of pathways involved in bone remodeling and immune responsiveness.

The current replication study confirmed significant differences in gene expression profiles of the cerebrum among the two commercial layer lines Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB). Microarray analyses were performed for 30 LSL and another 30 LB laying hens kept in the small group housing system Eurovent German. A total of 14,103 microarray probe sets using customized Affymetrix ChiGene-1_0-st Arrays with 20,399 probe sets were differentially expressed among the two layer lines LSL and LB (FDR adjusted P-value <0.05). An at least 2-fold change in expression levels could be observed for 388 of these probe sets. In LSL, 214 of the 388 probe sets were down- and 174 were up-regulated and vice versa for the LB layer line. Among the 174 up-regulated probe sets in LSL, we identified 51 significantly enriched Gene ontology (GO) terms of the biological process category. A total of 63 enriched GO-terms could be identified for the 214 down-regulated probe sets of the layer line LSL. We identified nine genes significantly differentially expressed between the two layer lines in both microarray experiments. These genes play a crucial role in protection of neuronal cells from oxidative stress, bone mineral density and immune response among the two layer lines LSL and LB. Thus, the different regulation of these genes may significantly contribute to phenotypic trait differences among these layer lines. In conclusion, these novel findings provide a basis for further research to improve animal welfare in laying hens and these layer lines may be of general interest as an animal model.

Gene expression profiling and identification of resistance genes to low temperature in leaves of peanut (Arachis hypogaea L.)

The cultivated peanut (Arachis hypogaea L.) is important oil crop and cold stress seriously influences its development and yields. Tolerant varieties produced using transgenic techniques will effectively increase peanut plantation area and enhance yields. However, little is known about the network of gene expression regulation related to cold stress in peanut. Therefore, we screened genes regulated by cold stress in peanut. In total, 1478 up-regulated and 1510 down-regulated probe sets were successfully identified in leaves of peanut subjected to cold stress using the microarray technique. Data analysis indicated that the main biological processes involved in cold stress response included signal transduction, transcript regulations and accumulation of soluble metabolites. Regulation of cell division, translation, protein modification, transport and DNA/RNA-related processes were also involved in peanut cold acclimation. Our study also revealed that protein kinases, transcription factors, heat shock proteins, resistance proteins, stilbene synthase, and so on, may play important functions in cold stress regulation of peanut. The function of some probe sets in cold stress regulation had not been clarified (e.g. proteins functioning in glycerol ether metabolic process, microtubule-based movement or nutrient reservoir activity). Some genes we identified lack functional annotation and their roles in response to cold are yet to be elucidated. These results showed an overview of the transcription map of peanut under cold stress, which may yield some useful insights into cold-mediated signal transduction pathways in peanut and offer some candidate genes as potential markers of tolerance to cold stress.

3-M syndrome: a growth disorder associated with IGF2 silencing.

3-M syndrome is an autosomal recessive disorder characterised by pre- and post-natal growth restriction, facial dysmorphism, normal intelligence and radiological features (slender long bones and tall vertebral bodies). It is known to be caused by mutations in the genes encoding cullin 7, obscurin-like 1 and coiled-coil domain containing 8. The mechanisms through which mutations in these genes impair growth are unclear. The aim of this study was to identify novel pathways involved in the growth impairment in 3-M syndrome. RNA was extracted from fibroblast cell lines derived from four 3-M syndrome patients and three control subjects, hybridised to Affymetrix HU 133 plus 2.0 arrays with quantitative real-time PCR used to confirm changes found on microarray. IGF-II protein levels in conditioned cell culture media were measured by ELISA. Of the top 10 downregulated probesets, three represented IGF2 while H19 was identified as the 23rd most upregulated probeset. QRT-PCR confirmed upregulation of H19 (P<0.001) and downregulation of IGF2 (P<0.001). Levels of IGF-II secreted into conditioned cell culture medium were higher for control fibroblasts than those for 3-M fibroblasts (10.2±2.9 vs 0.6±0.9 ng/ml, P<0.01). 3-M syndrome is associated with a gene expression profile of reduced IGF2 expression and increased H19 expression similar to that found in Silver–Russell syndrome. Loss of autocrine IGF-II in the growth plate may be associated with the short stature seen in children with 3-M syndrome.

Differential Gene Expression from Genome-Wide Microarray Analyses Distinguishes Lohmann Selected Leghorn and Lohmann Brown Layers

The Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB) layer lines have been selected for high egg production since more than 50 years and belong to the worldwide leading commercial layer lines. The objectives of the present study were to characterize the molecular processes that are different among these two layer lines using whole genome RNA expression profiles. The hens were kept in the newly developed small group housing system Eurovent German with two different group sizes. Differential expression was observed for 6,276 microarray probes (FDR adjusted P-value <0.05) among the two layer lines LSL and LB. A 2-fold or greater change in gene expression was identified on 151 probe sets. In LSL, 72 of the 151 probe sets were up- and 79 of them were down-regulated. Gene ontology (GO) enrichment analysis accounting for biological processes evinced 18 GO-terms for the 72 probe sets with higher expression in LSL, especially those taking part in immune system processes and membrane organization. A total of 32 enriched GO-terms were determined among the 79 down-regulated probe sets of LSL. Particularly, these terms included phosphorus metabolic processes and signaling pathways. In conclusion, the phenotypic differences among the two layer lines LSL and LB are clearly reflected in their gene expression profiles of the cerebrum. These novel findings provide clues for genes involved in economically important line characteristics of commercial laying hens.

Linked gene networks involved in nitrogen and carbon metabolism and levels of water-soluble carbohydrate accumulation in wheat stems

High levels of water-soluble carbohydrates (WSC) provide an important source of stored assimilate for grain filling in wheat. To better understand the interaction between carbohydrate metabolism and other metabolic processes associated with the WSC trait, a genome-wide expression analysis was performed using eight field-grown lines from the high and low phenotypic tails of a wheat population segregating for WSC and the Affymetrix wheat genome array. The 259 differentially expressed probe sets could be assigned to 26 functional category bins, as defined using MapMan software. There were major differences in the categories to which the differentially expressed probe sets were assigned; for example, probe sets upregulated in high relative to low WSC lines were assigned to category bins such as amino acid metabolism, protein degradation and transport and to be involved in starch synthesis-related processes (carbohydrate metabolism bin), whereas downregulated probe sets were assigned to cell wall-related bins, amino acid synthesis and stress and were involved in sucrose breakdown. Using the set of differentially expressed genes as input, chemical-protein network analyses demonstrated a linkage between starch and N metabolism via pyridoxal phosphate. Twelve C and N metabolism-related genes were selected for analysis of their expression response to varying N and water treatments in the field in the four high and four low WSC progeny lines; the two nitrogen/amino acid metabolism genes demonstrated a consistent negative association between their level of expression and level of WSC. Our results suggest that the assimilation of nitrogen into amino acids is an important factor that influences the levels of WSC in the stems of field-grown wheat.

Gene expression signatures of breast tissue before and after cross-sex hormone therapy in female-to-male transsexuals

To evaluate gene expression signatures of breast tissue in female-to-male (FtM) transsexuals under cross-sex hormone therapy (HT). Prospective cohort study. Academic research institution. Five hormone-naïve FtM transsexuals before and after HT. Breast tissue biopsy before and after 2 years of intramuscular testosterone undecanoate (1,000 mg every 12 wk) and oral lynestrenole (5 mg daily), and gene signature analysis by global gene expression array covering 28,869 genes. Differential regulation of specific genes and gene expression signatures. We identified 2,250 differentially expressed probe sets. One hundred twenty probe sets showed >2-fold change, of which 77 (64.2%) were up-regulated and 43 (35.8%) down-regulated. Genes involved in transcription were most overrepresented, with 43 out of 97 (44.3%) annotated probes, e.g., the transcription factor complex activator protein 1, including all three Jun genes (c-Jun, JunB, and JunD), two Fos genes (c-Fos and FosB), and activating transcription factor 3. In a Database for Annotation, Visualization, and Integrated Discovery analysis of the 2,007 down-regulated probe sets, proteins of the ribosome pathway and of two pathways involved in protein degradation, i.e., proteasome- and ubiquitin-mediated proteolysis, were significantly down-regulated. We identified eight breast cancer-associated gene expression signatures significantly overlapping with differentially regulated probe sets after cross-sex HT. Cross-sex HT in FtM transsexuals leads to the up-regulation and down-regulation of 243 and 2,007 distinct genes, respectively, and is associated with breast cancer-related gene expression signatures.

Dietary iron-deficient anemia induces a variety of metabolic changes and even apoptosis in rat liver: a DNA microarray study

Anemia can be induced by dietary iron deficiency, as well as by hemorrhagia. It may also be associated with changes in lipid metabolism. However, no global analysis detailing the consequences of iron deficiency in the liver has yet been conducted. Since the liver is a metabolically important organ and also a major iron-storing organ, we performed a comprehensive transcriptome analysis to determine the effects of iron deficiency on hepatic gene expression. Four-week-old rats were fed an iron-deficient diet, approximately 3 ppm iron, ad libitum for 16 days. These rats were compared with similar rats pair-fed a control diet with a normal iron level, 48 ppm iron. The 16-day iron-deficient diet apparently induced anemia. On day 17, the rats were killed under anesthesia, and their livers were dissected for DNA microarray analysis. We identified 600 upregulated and 500 downregulated probe sets that characterized the iron-deficient diet group. In the upregulated probe sets, genes involved in cholesterol, amino acid, and glucose metabolism were significantly enriched, while genes related to lipid metabolism were significantly enriched in the downregulated probe sets. We also found that genes for caspases 3 and 12, which mediate endoplasmic reticulum (ER)-specific apoptosis, were upregulated in the iron-deficient group. Combined, these results suggest that iron deficiency exerts various influences, not only on nutrient metabolism but also on apoptosis, as a consequence of ER stress in the liver.

Global gene expression analysis of in vitro root formation in Medicago truncatula

Medicago truncatula Gaertn. can generate roots in vitro through the formation of root stem cells from leaf explants cultured with auxin. To identify key genes involved in the early processes of root initiation, we compared gene expression in root-forming cultures (RFC) enriched for root stem cells with non-root-forming cultures (NRFC) and untreated leaves using the Affymetrix Medicago GeneChip. Comparing RFC (at 1 week, before root primordium formation) to normal leaf tissue, we identified 904 and 993 up- and downregulated probe sets. Comparing RFC and NRFC, we identified 92 and 182 up- and downregulated probe sets. By comparing all the samples we identified a set of 76 and 42 probe sets up- and downregulated that may be crucial to root stem cell formation and subsequent root initiation. Upregulated probe sets in RFC include Arabidopsis orthologs that are involved in root stem cell formation and root initiation. To validate the GeneChip results, quantitative real-time RT–PCR analysis was used to examine the expression of specific up- and downregulated genes, all of which positively correlated with the microarray data. We used bioinformatic tools developed to functionally annotate the Medicago genome array. This showed significant changes in metabolism, signalling and the expression of transcription factors including some with described roles in root organogenesis and other genes not previously linked to this process. This data facilitates the mapping of regulatory and metabolic networks in M. truncatula and provides candidates for further functional analysis of root initiation in vitro and in planta.

Differentially expressed genes identified by cross‐species microarray in the blind cavefish Astyanax

Changes in gene expression were examined by microarray analysis during development of the eyed surface dwelling (surface fish) and blind cave-dwelling (cavefish) forms of the teleost Astyanax mexicanus De Filippi, 1853. The cross-species microarray used surface and cavefish RNA hybridized to a DNA chip prepared from a closely related species, the zebrafish Danio rerio Hamilton, 1822. We identified a total of 67 differentially expressed probe sets at three days post-fertilization: six upregulated and 61 downregulated in cavefish relative to surface fish. Many of these genes function either in eye development and/or maintenance, or in programmed cell death. The upregulated probe set showing the highest mean fold change was similar to the human ubiquitin specific protease 53 gene. The downregulated probe sets showing some of the highest fold changes corresponded to genes with roles in eye development, including those encoding gamma crystallins, the guanine nucleotide binding proteins Gnat1 and Gant2, a BarH-like homeodomain transcription factor, and rhodopsin. Downregulation of gamma-crystallin and rhodopsin was confirmed by in situ hybridization and immunostaining with specific antibodies. Additional downregulated genes encode molecules that inhibit or activate programmed cell death. The results suggest that cross-species microarray can be used for identifying differentially expressed genes in cavefish, that many of these genes might be involved in eye degeneration via apoptotic processes, and that more genes are downregulated than upregulated in cavefish, consistent with the predominance of morphological losses over gains during regressive evolution.

D-Serine exposure resulted in gene expression changes implicated in neurodegenerative disorders and neuronal dysfunction in male Fischer 344 rats

D-Serine, an endogenous amino acid, is involved in many physiological processes through its interaction with the glycine binding site of the N-methyl-D-aspartate (NMDA) receptor. It has important roles in development, learning, and cell death signaling. Recent evidence suggests that decreased function of the NMDA receptor is related to the etiology of schizophrenia, and the use of D-serine as add-on therapy is beneficial in alleviating the symptoms of treatment-refractory schizophrenia. The NMDA receptor also plays a major role in neuronal cell death and neurodegeneration mediated by excitatory amino acid toxicity in ischemia, epilepsy, and trauma. Due to its co-activator function, D-serine can markedly potentiate NMDA-mediated excitotoxicity. To investigate potential adverse effects of D-serine treatment, we investigated gene expression changes in the forebrain of male F-344 rats treated with a single intraperitoneal injection of D-serine (5, 20, 50, 200, or 500 mg/kg) at 96 h post-treatment. Gene expression profiling using Affymetrix Rat Genome 230 2.0 arrays revealed that D-serine treatment resulted in up- and down-regulation of 134 and 52 genes, respectively, based on the common genes identified using three statistical methods, i.e. t test (p < 0.01 over two consecutive doses), ANOVA (with adjusted Bonferonni correction for multiple testing) and significance analysis of microarray (SAM). Self organized map (SOM) clustering analysis of the differentially expressed genes showed two clusters, one with all 134 up-regulated probe sets and the other with all 52 down-regulated probe sets. The dose-response pattern of the down-regulated cluster showed nearly a perfect mirror image of that of the up-regulated one. Gene ontology analysis revealed that pathways implicated in neuronal functions and/or neurodegenerative disorders are over-represented among the differentially expressed genes. Specifically, genes involved in vesicle-mediated transport, endocytosis, ubiquitin conjugation pathway, regulation of actin filament polymerization/depolymerization, focal adhesion, Wnt signaling, and insulin signaling were up-regulated, while genes involved in RNA metabolism/splicing/processing and Notch signaling were down-regulated. Consistent with this finding, pathway analysis using GenMAPP showed a significant number of differentially expressed genes in these pathways. In addition, the GenMAPP result also showed activation of the signaling pathways of several proinflammatory cytokines (including IL-2, IL-3, IL-5, IL-6 and TNF-alpha), which might suggest the onset of neuroinflammation. Biological association network analysis showed that several nuclear factors implicated in transcription regulation (including Taf1, Max, Myc, and Hnf4a) are highly connected to a large number of up-regulated genes. While the transcript levels of these transcription factors were not changed, their connections to Ddx3x, a gene involved in mRNA processing and translation initiation, raise the possibility that they may be up-regulated at the post-transcriptional level. The observation that Ubqln1 and Ube2d, two differentially expressed genes involved in ubiquitin-mediated proteolysis and implicated in neurodegenerative disorders, are highly connected in this network suggests a role of ubiquitination proteasome pathway in response to D-serine exposure. This finding is consistent with the result of gene ontology analysis and suggests that D-serine treatment might result in damage to cellular proteins and subsequent up-regulation of ubiquitination proteasome pathway to clear these damaged proteins. In summary, D-serine exposure resulted in perturbation of a number of pathways implicated in neuronal functions and neurodegenerative disorders. However, activation of cellular response to counter the toxic effects of D-serine might be hindered due to the down-regulation of such important cellular machinery like RNA metabolism, splicing and processing. Consequently, cell damage might be further exacerbated. Taken together, these findings highlight the potential impacts of D-serine exposure on neuronal functions.

Mathematical Modeling of Corticosteroid Pharmacogenomics in Rat Muscle following Acute and Chronic Methylprednisolone Dosing

The pharmacogenomic effects of a corticosteroid (CS) were assessed in rat skeletal muscle using microarrays. Adrenalectomized (ADX) rats were treated with methylprednisolone (MPL) by either 50 mg/kg intravenous injection or 7-day 0.3 mg/kg/h infusion through subcutaneously implanted pumps. RNAs extracted from individual rat muscles were hybridized to Affymetrix Rat Genome Genechips. Data mining yielded 653 and 2316 CS-responsive probe sets following MPL bolus and infusion treatments. Of these, 196 genes were controlled by MPL under both dosing conditions. Cluster analysis revealed that 124 probe sets exhibited three typical expression dynamic profiles following acute dosing. Cluster A consisted of up-regulated probe sets which were grouped into five subclusters each exhibiting unique temporal patterns during the infusion. Cluster B comprised down-regulated probe sets which were divided into two subclusters with distinct dynamics during the infusion. Cluster C probe sets exhibited delayed down-regulation under both bolus and infusion conditions. Among those, 104 probe sets were further grouped into subclusters based on their profiles following chronic MPL dosing. Several mathematical models were proposed and adequately captured the temporal patterns for each subcluster. Multiple types of dosing regimens are needed to resolve common determinants of gene regulation as chronic exposure results in unexpected differences in gene expression compared to acute dosing. Pharmacokinetic/pharmacodynamic (PK/PD) modeling provides a quantitative tool for elucidating the complexities of CS pharmacogenomics in skeletal muscle.

Estrogen, not intrinsic aging, is the major regulator of delayed human wound healing in the elderly.

Analysis of gene expression in male elderly and young human wounds suggests that estrogen has a more profound influence on aging than previously thought.

Species-specific differences in coumarin-induced hepatotoxicity as an example toxicogenomics-based approach to assessing risk of toxicity to humans

One expected result from toxicogenomics technology is to overcome the barrier because of species-specific differences in prediction of clinical toxicity using animals. The present study serves as a model case to test if the well-known species-specific difference in the toxicity of coumarin could be elucidated using comprehensive gene expression data from rat in-vivo, rat in-vitro, and human in-vitro systems. Coumarin 150 mg/kg produced obvious pathological changes in the liver of rats after repeated administration for 7 days or more. Moreover, 24 h after a single dose, we observed minor and transient morphological changes, suggesting that some early events leading to hepatic injury occur soon after coumarin is administered to rats. Comprehensive gene expression changes were analyzed using an Affymetrix GeneChip approach, and differentially expressed probe sets were statistically extracted. The changes in expression of the selected probe sets were further examined in primary cultured rat hepatocytes exposed to coumarin, and differentially expressed probe sets common to the in-vivo and in-vitro datasets were selected for further study. These contained many genes related to glutathione metabolism and the oxidative stress response. To incorporate human data, human hepatocyte cultured cells were exposed to coumarin and changes in expression of the bridging gene set were examined. In total, we identified 14 up-regulated and 11 down-regulated probe sets representing rat-human bridging genes. The overall responsiveness of these genes to coumarin was much higher in rats than humans, consistent with the reported species difference in coumarin toxicity. Next, we examined changes in expression of the rat-human bridging genes in cultured rat and human hepatocytes treated with another hepatotoxicant, diclofenac sodium, for which hepatotoxicity does not differ between the species. Both rat and human hepatocytes responded to the marker genes to the same extent when the same concentrations of diclofenac sodium were exposed. We conclude that toxicogenomics-based approaches show promise for overcoming species-specific differences that create a bottleneck in analysis of the toxicity of potential therapeutic treatments.

Global transcriptome analysis of murine embryonic stem cell-derived cardiomyocytes

Microarray analysis reveals that the specific pattern of gene expression in cardiomyocytes derived from embryonic stem cells reflects the biological, physiological and functional processes occurring in mature cardiomyocytes.