Sfnge array poster

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Sfnge array poster

  1. 1. Chronic Restraint Stress Modulates Expression of Genes Y. Wang1, Y. Zhang2, D. Yin2, J. Moorman2, Xiao Zeng1 1SuperArray Bioscience Corporation, Frederick, MD, 2Department of Internal Medicine, East Tennessee State University, Johnson City, TN. Abstract Materials and Methods Restraint stress mouse model: 6-8 week old male Balb/c mice from Charles River Laboratories were maintained in the Division of Laboratory Animal Resources at East Tennessee State University (ETSU). The mice were subjected to a chronic physical restraint protocol (3). Briefly, mice were placed in a 50-ml conical centrifuge tube filled with multiple punctures to allow ventilation. Mice were held horizontally in the tubes for a continuous 12 h followed by a 12-h rest, while food and water were provided ad libitum. Control mice were kept in their original cages and in the same diet schedule as the experimental group. Mice were physically restrained for two cycles. After physical restraint, the spleens were rapidly frozen in liquid nitrogen and stored at -80ºC for later isolation of total RNA. Genomic DNA interference was eliminated by DNase I treatment. Results Figure 3: Validation of Microarray Data Using Real-Time PCR RealFigure 1: Microarray Analysis of Apoptotic Genes in Restraint Stress Stress B A Control Stress Physical restraint stress alters apoptotic gene expression profiles in the spleen. Panel A displays representative images of the Oligo GEArray® Mouse Apoptosis Microarrays. The experiments were repeated three times. As an example, the up-regulation of Bnip3 is circled in red. Panel B lists the 23 genes whose expression levels are significantly changed in stressed versus unstressed mice. RNA using the TrueLabeling-AMP™ 2.0 Kit (Catalog No. GA-030). The resulting biotin-labeled cRNA probe was allowed to hybridize overnight to the Oligo GEArray® microarray in a standard hybridization oven at 60ºC. After blocking, washing and chemiluminescent detection steps, images were acquired by a Chemi-Doc imaging system (CCD camera). Raw signal intensity for individual genes on the microarray was extracted using web-based GEArray® Expression Analysis Suite software and normalized against signal intensities from housekeeping genes. Real-time RT-PCR: cDNA was synthesized from total RNA using the ReactionReady™ First Strand cDNA Synthesis Kit (Catalog No. C-01). Real-time PCR was performed using RT2 Real-Time™ SYBR Green Fluorescein PCR Master Mix (Catalog No. PA-011) on the Bio-Rad iCycler real-time PCR detection system. GAPDH and β-Actin were chosen as housekeeping genes for normalization. Threshold cycle numbers (Ct) were used for “∆∆Ct” analysis. Fold-changes between samples were then calculated as 2^(-∆∆Ct). The statistical significance was determined by one-way ANOVA and Bonferroni tests (p<0.05). 3.5 Fold 6.8 3.5 4.0 5.9 4.7 4.8 2.4 16.0 5.1 3.2 2.4 1.8 12.7 A Control Stress Physical restraint stress alters the p53signaling pathway in the spleen. Panel A displays representative images of the Oligo GEArray® Mouse p53 Signaling Pathway Microarrays. The experiments were repeated three times. As an example, the up-regulation of p21 is shown in red circles. Panel B lists the 24 genes whose expression levels are significantly increased in stressed versus unstressed mice, as well as Stat1 whose gene expression decreased. 3.0 2.5 2.0 1.5 1.0 0.5 0.0 FAS FADD p53 p21 Up-regulation of Fas, FADD, p53, and p21 genes in spleens from mice with physical restraint stress was confirmed by quantitative real-time PCR. The data are presented as the relative fold-changes normalized to GAPDH as the internal control. The data are representative of three experiments where the asterisk (*) denotes p < 0.01 when compared to control. 3.6 3.7 2.9 2.7 3.3 Conclusions 2.6 1.8 3.3 11.9 By focusing on apoptosis and p53 signaling pathways, we have found that 22.3% of the examined genes showed significant upregulated expression in splenocytes between stressed and unstressed mice. The up-regulation of FADD, FAS, p53 and p21 is further confirmed through real time PCR. B Position Description 3 Apaf1: Apoptotic peptidase activating factor 1 4 Apex1: Apurinic/apyrimidinic endonuclease 1 7 Aurkb: Aurora kinase B 8 Wdr39: WD repeat domain 39 10 Bak1: BCL2-antagonist/killer 1 11 Bap1: Brca1 associated protein 1 12 Bax: Bcl2-associated X protein 15 Bid: BH3 interacting domain death agonist 16 Birc5: Baculoviral IAP repeat-containing 5 18 Brca1: Breast cancer 1 19 Brca2: Breast cancer 2 20 Btg2: B-cell translocation gene 2, anti-proliferative 25 Ccng2: Cyclin G2 26 Ccnh: Cyclin H Cdc25a: Cell division cycle 25 homolog A (S. 27 cerevisiae) 28 Cdc25c: Cell division cycle 25 homolog C (S. cerevisiae) 29 Cdc2a: Cell division cycle 2 homolog A (S. pombe) Cdk7: Cyclin-dependent kinase 7 (homolog of Xenopus 31 MO15 cdk-activating kinase) 32 Cdkn1a: Cyclin-dependent kinase inhibitor 1A (P21) 34 Chek1: Checkpoint kinase 1 homolog (S. pombe) 35 Chek2: CHK2 checkpoint homolog (S. pombe) 39 Cyr61: Cysteine rich protein 61 41 Daxx: Fas death domain-associated protein Gadd45a: Growth arrest and DNA-damage-inducible 45 53 alpha 95 Stat1: Signal transducer and activator of transcription 1 Control Stress 4.9 Figure 2: Microarray Analysis of p53-Mediated Signaling in Restraint Stress p53- Oligo GEArray® System: Two cataloged Oligo GEArray® microarrays from SuperArray were used in this study. The Oligo GEArray® Mouse Apoptosis Microarray (Catalog No. OMM-012) contains 112 genes involved in apoptosis. The Oligo GEArray® Mouse p53 Signaling Pathway Microarray (Catalog No. OMM-027) is designed to profile the expression of 113 key genes involved in the p53 signaling pathway. Microarray processing: Biotinylated cRNA target was synthesized from total Position Description 2 Akt1: Thymoma viral proto-oncogene 1 5 Api5: Apoptosis inhibitor 5 14 Bax: Bcl2-associated X protein Bnip3l: BCL2/adenovirus E1B interacting protein 325 like 28 Birc1e: Baculoviral IAP repeat-containing 1e 31 Birc2: Baculoviral IAP repeat-containing 2 Bnip2: BCL2/adenovirus E1B interacting protein 1, 35 NIP2 Bnip3: BCL2/adenovirus E1B interacting protein 1, 36 NIP3 37 Casp3: Caspase 3 38 Bok: Bcl-2-related ovarian killer protein 48 Casp2: Caspase 2 52 Casp8: Caspase 8 53 Dsip1: TSC22 domain family 3 Cradd: CASP2 and RIPK1 domain containing adaptor 58 with death domain 65 Fadd: Fas (TNFRSF6)-associated via death domain 71 Ltbr: Lymphotoxin B receptor Nfkb1: Nuclear factor of kappa light chain gene 74 enhancer in B-cells 1, p105 75 Zc3hc1: Zinc finger, C3HC type 1 87 Rnf7: Ring finger protein 7 Tnfrsf21: Tumor necrosis factor receptor superfamily, 95 member 21 98 Fas: Fas (TNF receptor superfamily member) 109 Trp53: Transformation related protein 53 Trp53inp1: Transformation related protein 53 111 inducible nuclear protein 1 F o ld C h a n g e Psychological and physical stress can alter the immune system in both human and animals (1). It has been reported that mice subjected to chronic 12-hour daily physical restraint for two days showed dramatic apoptosis in splenocytes (2). To identify genes that contribute to the splenocyte apoptosis, we compared gene expression in the spleens of restraint stressed mice with that in the spleens of unstressed mice using oligo microarrays consisting of 225 genes. We report here that mice subjected to chronic 12-hour daily physical restraint for two days exhibited significantly altered expression of 48 out of 225 genes. These genes included pro-apoptotic genes. We selected four genes of interest, and confirmed the microarray results by real time PCR. Although these findings are not specific for restraint stress-induced apoptosis in splenocytes, they identify a potentially important component of pro-apoptotic activity in restraint stress and suggest a possible target for anti-apoptotic therapy in stress-induced apoptosis in splenocytes. Our mouse model and identified biomarkers would be very useful for further study of the intercommunication of the immune and the nervous systems. Fold 3.1 4.8 3.8 3.7 2.2 2.6 3.8 2.1 3.1 3.8 4.7 2.0 2.5 3.4 Our data provide additional evidence that apoptotic machinery underlines lymphopenia during stress. It remains to be further investigated whether the apoptotic genes provide a marker for individuals with chronic stress. Pathway-focused microarray analysis is a simple and easy approach to screen for genes of interest. 3.7 3.4 3.6 References 2.6 5.3 4.8 3.0 3.5 2.7 1. Padgett,D.A. and Glaser,R. (2003). How stress influences the immune response. Trends Immunol. 24, 444-448. 3.0 2. Yin,D., Tuthill,D., Mufson,R.A., and Shi,Y. (2000). Chronic restraint stress promotes lymphocyte apoptosis by modulating CD95 expression. J. Exp. Med. 191, 1423-1428. 0.5 3. Sheridan,J.F., Dobbs,C., Jung,J., Chu,X., Konstantinos,A., Padgett,D., and Glaser,R. (1998). Stress-induced neuroendocrine modulation of viral pathogenesis and immunity. Ann. N. Y. Acad. Sci. 840, 803-808.

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