Age-related changes in the transcriptional profile of mouse RPE/choroid

2003 ◽  
Vol 15 (3) ◽  
pp. 258-262 ◽  
Author(s):  
Hisashi Ida ◽  
Sharon A. Boylan ◽  
Andrea L. Weigel ◽  
Leonard M. Hjelmeland
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Cdna Probe ◽  
Gene Expression Profiles ◽  
Age Related Macular Degeneration ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Mouse Cdna ◽  
Age Related ◽  
Age Related Changes

To evaluate the age-related changes in gene expression occurring in the complex of retinal pigmented epithelium, Bruch’s membrane, and choroid (RPE/choroid), we examined the gene expression profiles of young adult (2 mo) and old (24 mo) male C57BL/6 mice. cDNA probe sets from individual animals were synthesized using total RNA isolated from the RPE/choroid of each animal. Probes were amplified using the Clontech SMART system, radioactively labeled, and hybridized to two different Clontech Atlas mouse cDNA arrays. From each age group, three independent triplicates were hybridized to the arrays. Statistical analyses were performed using the Significance Analysis of Microarrays program (SAM version 1.13; Stanford University). Selected array results were confirmed by semi-quantitative RT-PCR analysis. Of 2,340 genes represented on the arrays, ∼60% were expressed in young and/or old mouse RPE/choroid. A moderate fraction (12%) of all expressed genes exhibited a statistically significant change in expression with age. Of these 150 genes, all but two, HMG14 and carboxypeptidase E, were upregulated with age. Many of these upregulated genes can be grouped into several broad functional categories: immune response, proteases and protease inhibitors, stress response, and neovascularization. RT-PCR results from six of six genes examined confirmed the differential change in expression with age of these genes. Our study provides likely candidate genes to further study their role in the development of age-related macular degeneration and other aging diseases affecting the RPE/choroid.

scholarly journals Age-related differences in monocyte DNA methylation and immune function in healthy Kenyan adults and children

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Katherine R. Dobbs ◽  
Paula Embury ◽  
Emmily Koech ◽  
Sidney Ogolla ◽  
Stephen Munga ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Young Adults ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Innate Immune ◽  
Inflammatory Gene Expression ◽  
Cpg Sites ◽  
Age Related ◽  
Adults And Children

Abstract Background Age-related changes in adaptive and innate immune cells have been associated with a decline in effective immunity and chronic, low-grade inflammation. Epigenetic, transcriptional, and functional changes in monocytes occur with aging, though most studies to date have focused on differences between young adults and the elderly in populations with European ancestry; few data exist regarding changes that occur in circulating monocytes during the first few decades of life or in African populations. We analyzed DNA methylation profiles, cytokine production, and inflammatory gene expression profiles in monocytes from young adults and children from western Kenya. Results We identified several hypo- and hyper-methylated CpG sites in monocytes from Kenyan young adults vs. children that replicated findings in the current literature of differential DNA methylation in monocytes from elderly persons vs. young adults across diverse populations. Differentially methylated CpG sites were also noted in gene regions important to inflammation and innate immune responses. Monocytes from Kenyan young adults vs. children displayed increased production of IL-8, IL-10, and IL-12p70 in response to TLR4 and TLR2/1 stimulation as well as distinct inflammatory gene expression profiles. Conclusions These findings complement previous reports of age-related methylation changes in isolated monocytes and provide novel insights into the role of age-associated changes in innate immune functions.

scholarly journals Comparison of Stemness and Gene Expression between Gingiva and Dental Follicles in Children

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Chung-Min Kang ◽  
Seong-Oh Kim ◽  
Mijeong Jeon ◽  
Hyung-Jun Choi ◽  
Han-Sung Jung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Pcr Analysis ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Stem Cell Source ◽  
Regenerative Dentistry ◽  
Induced Pluripotent

The aim of this study was to compare the differential gene expression and stemness in the human gingiva and dental follicles (DFs) according to their biological characteristics. Gingiva (n=9) and DFs (n=9) were collected from 18 children. Comparative gene expression profiles were collected using cDNA microarray. The expression of development, chemotaxis, mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSs) related genes was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Histological analysis was performed using hematoxylin-eosin and immunohistochemical staining. Gingiva had greater expression of genes related to keratinization, ectodermal development, and chemotaxis whereas DFs exhibited higher expression levels of genes related to tooth and embryo development. qRT-PCR analysis showed that the expression levels of iPSc factors includingSOX2,KLF4, andC-MYCwere58.5±26.3,12.4±3.5, and12.2±1.9times higher in gingiva andVCAM1(CD146) andALCAM(CD166) were33.5±6.9and4.3±0.8times higher in DFs. Genes related to MSCs markers includingCD13,CD34,CD73,CD90, andCD105were expressed at higher levels in DFs. The results of qRT-PCR and IHC staining supported the microarray analysis results. Interestingly, this study demonstrated transcription factors of iPS cells were expressed at higher levels in the gingiva. Given the minimal surgical discomfort and simple accessibility, gingiva is a good candidate stem cell source in regenerative dentistry.

MLL-AF9 and MLL-ENL Induce Deregulation of Similar Downstream Candidate Genes: An Analysis across Experimental Protocols and Laboratories.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3372-3372
Author(s):  
Ashish R. Kumar ◽  
Robert K. Slany ◽  
Jay L. Hess ◽  
John H. Kersey
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Systems ◽  
Rt Pcr ◽  
Conditional Expression

Expression profiling has become an important tool for understanding gene deregulation in MLL-fusion leukemias. However, the results of gene profiling experiments are difficult to interpret when applied to leukemia cells because (i) leukemias arise in cells that differ greatly in their gene expression profiles, and (ii) leukemias most often require secondary genetic events in addition to the MLL fusion gene. Two principal model systems have been used to understand the direct effects of MLL-fusion genes. Knock-in models have the advantage of the fusion gene being under control of the physiologic promoter. On the other hand, conditional expression systems offer the ability to conduct short term experiments, permitting the analysis of direct effects on downstream genes. In the present combined-analysis, we used the Affymetrix U74Av2 oligonucleotide microarray to evaluate the effects of the MLL-fusion gene in vivo and in vitro respectively using two closely related MLL fusion genes - MLL-AF9 for knock-in and MLL-ENL for conditional expression. In the MLL-AF9 study, we compared gene expression profiles of bone marrow cells from MLL-AF9 knock-in mice (C57Bl/6, MLL-AF9+/−) to those of age-matched wild type mice (Kumar et. al. 2004, Blood). We used a t-test (p<0.05) to selected genes that showed significant changes in expression levels. In the MLL-ENL study, we transformed murine primary hematopoietic cells with a conditional MLL-ENL vector (MLL-ENL fused to the modified ligand-binding domain of the estrogen receptor) such that the fusion protein was active only in the presence of tamoxifen. We then studied the downstream effects of the fusion protein by comparing gene expression profiles of the cells in the presence and absence of tamoxifen. We used a pair-wise comparison analysis to select genes that showed a change in expression level of 1.5 fold or greater in at least two of three experiments (Zeisig et. al. 2004, Mol. Cell Biol.). Those genes that were up-regulated in both datasets were then compiled together. This list included Hoxa7, Hoxa9 and Meis1. The results for these 3 genes were confirmed by quantitative RT-PCR in both the MLL-AF9-knock-in and the MLL-ENL-conditional-expression systems. The remaining candidate genes in the common up-regulated gene set (not yet tested by quantitative RT-PCR) include protein kinases (Bmx, Mapk3, Prkcabp, Acvrl1, Cask), RAS-associated proteins (Rab7, Rab3b), signal transduction proteins (Notch1, Eat2, Shd, Fpr1), cell membrane proteins (Igsf4), chaperones (Hsp70.2), transcription factors (Isgf3g), proteins with unknown functions (Olfm1, Flot1), and hypothetical proteins. The results of the combined analysis demonstrate that these over-expressions are (i) a direct and sustained effect of the MLL-fusion protein, (ii) are independent of secondary events that might be involved in leukemogensis, and (iii) are independent of the two partner genes that participate in these fusions. The over-expression of a few genes in both the -in vitro and in vivo experimental systems makes these molecules very interesting for further studies, to understand the biology of MLL-fusion leukemias and for development of new therapeutic strategies.

Functional Somatic and Germline Variants in the NF-κB Pathway in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 560-560 ◽  
Author(s):  
Ma. Reina Improgo ◽  
Adam Kiezun ◽  
Yaoyu Wang ◽  
Lillian Werner ◽  
Petar Stojanov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Transcriptional Activity ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Wild Type ◽  
Rt Pcr ◽  
Germline Variants

Abstract Abstract 560 Nuclear factor kappa B (NF-κB) encompasses a family of transcription factors involved in oncogenic processes including cellular proliferation and apoptotic inhibition. Constitutive activation of NF-κB has been observed in hematologic malignancies and is thought to confer resistance to chemotherapeutic agents. Here, we examine the role of the NF-κB pathway in chronic lymphocytic leukemia (CLL). Whole-exome sequencing was performed using tumor and matched germline DNA from 167 CLL patients. We identified 51 patients (30%) carrying 53 non-silent somatic variants in genes of the canonical NF-κB pathway, which consists of 272 genes as defined by the Ingenuity Pathway Analysis tool. Of the 99 patients whose germline sequences have been analyzed to date, 27 patients (27%) carry 34 non-silent germline variants in NF-κB pathway genes. A total of 67 patients (40%) have at least one non-silent somatic or germline variant. Variants in the NFKB1 gene, itself, were also observed: a somatic variant, H66R, found in two patients, and two germline variants, Y89F and R849W, each found in one patient. To evaluate the functional consequences of the NFKB1 variants, we performed site-directed mutagenesis to generate full-length NFKB1 cDNAs encoding these variants. We subsequently measured transcriptional activity of wild-type and mutant NFKB1 via luciferase assays in HEK293T cells using reporter cassettes containing the NFKB1 response element. Transcriptional activity of the three NFKB1 variants was found to be at least 2-fold higher than that of wild-type NFKB1 (p<0.0001). We further hypothesized that this increased transcriptional activity would lead to increased expression of NFKB1 downstream target genes. Analysis of gene expression profiles from Affymetrix HG-U133 Plus 2.0 Arrays of 65 CLL patient samples showed that the NFKB1 downstream targets CCL3, CCL4, and CD69 are upregulated in NFKB1 variants. To validate these results, we performed quantitative RT-PCR in patients with (n=3) or without (n=9) NFKB1 variants and confirmed upregulation of CCL3 (p=0.0286), CCL4 (p=0.0384), and CD69 (p=0.0263). Direct transfection of HEK293T cells with NFKB1 variants also resulted in a 3.3-fold upregulation of CCL3 (p=0.05). To test the hypothesis that deregulation of the NF-κB pathway is a key mechanism in CLL, we compared gene expression profiles of NF-κB pathway genes between CLL patient samples (n=146) and normal B cells (n=16) and found overall upregulation of the NF-κB pathway in CLL (Kolmogorov-Smirnov test, p=2.2e-16). K-means clustering and principal component analysis (PCA) further revealed that CLL patients can be divided into two subgroups exhibiting differential magnitude of NF-κB pathway upregulation. Studies in progress aim to identify the clinical significance of these subgroups. Finally, we assessed the effect of inhibiting the NF-κB pathway using the cell permeant NF-κB inhibitor, SN50. We performed Annexin V/PI staining 24 hours post-treatment in CLL cells with (n=9) or without (n=3) NF-κB pathway variants. SN50 increased cell death 1.8-fold in all cells tested (p<0.0001). Quantitative RT-PCR also showed a 59% decrease in expression of CCL3 one hour post-treatment, confirming inhibition of the NF-κB pathway. In conclusion, our findings demonstrate that a high proportion of CLL patients harbor somatic and germline variants in NF-κB pathway genes, some of which appear to be functional. Furthermore, the NF-κB pathway is upregulated in CLL and pharmacological inhibition of the pathway leads to increased cancer cell death. Functional characterization of NF-κB pathway variants offers mechanistic insight into the disease, providing novel targets for therapy. Disclosures: No relevant conflicts of interest to declare.

The critical role of surgical pathology in personalized medicine: The impact of biopsy cavities in breast cancer samples on recurrence risk when assessed by quantitative RT-PCR

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e22016-e22016
Author(s):  
F. L. Baehner ◽  
J. Anderson ◽  
C. Millward ◽  
C. Sangli ◽  
C. Quale ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Expression Profiles ◽  
Critical Role ◽  
Recurrence Score ◽  
Pcr Analysis ◽  
Breast Cancers ◽  
Rt Pcr ◽  
Poorly Differentiated ◽  
The Impact

e22016 Background: Tumor gene expression analysis using the Recurrence Score (RS) assay is frequently used in ER+ breast cancer. Manual microdissection is performed in cases where biopsy cavities (BxC) are present in the submitted specimen. The objective of this was to characterize by quantitative RT-PCR the impact of BxC on 21 gene expression profiles and the RS. Methods: 48 (15 well, 18 moderate, and 15 poorly differentiated) breast cancers were evaluated for gene expression differences between whole sections (WS; containing BxC) and enriched tumor (ET; BxC excluded). Standardized quantitative RT-PCR analysis for the 21 Oncotype DX genes was performed; reference normalized gene expression measurements ranged from 0 to 15, where each 1-unit reflects an approximate 2-fold change in RNA. Analyses of individual genes and the RS were performed on the entire sample set and stratified by tumor grade. Correlation analyses used Pearson's R, concordance analysis used Lin's sample concordance and paired t- tests to characterize differences. Results: There were statistically significant differences in reference normalized gene expression between ET and WS in 6 genes: BAG1 (ET-WS: 0.13 units, p=0.0025), CD68 (ET-WS: -0.64 units, p<0.0001), ER (ET-WS: 0.29 units, p=0.0012), GSTM1 (ET-WS: 0.18 units p=0.0025), STK15 (ET-WS: -0.18 units, p=0.0041) and STMY3 (ET-WS: 0.62 units, p<0.0001). Expression of the macrophage marker CD68 was higher and expression of ER was lower in WS containing BxC. The correlation (0.95) and concordance (0.92) were generally high between WS and ET for RS overall however among moderately differentially tumors, there was a statistically significant mean increase in RS for WS of 3.3 units (p = 0.0012) while among poorly differentiated tumors there was a trend toward a statistically significant decrease in RS for WS of 2.2 units (p=0.0569). Conclusions: Histologic identification of invasive carcinoma and exclusion of BxC is essential for precise RS assessment. Inclusion of BxC in breast cancer specimens is associated with significant changes in the expression of individual genes and impacts the RS. Removal of BxC from breast cancer specimens assessed for gene expression levels is warranted. [Table: see text]

scholarly journals RNA-seq Analysis Reveals Gene Expression Profiling of Female Fertile and Sterile Ovules of Pinus Tabulaeformis Carr. during Free Nuclear Mitosis of the Female Gametophyte

2018 ◽  
Vol 19 (8) ◽  
pp. 2246 ◽  
Author(s):  
Yang Yao ◽  
Rui Han ◽  
Zaixin Gong ◽  
Caixia Zheng ◽  
Yuanyuan Zhao
Keyword(s):  
Gene Expression ◽  
Female Gametophyte ◽  
Expression Profiles ◽  
Signal Transduction Pathway ◽  
Gene Expression Profiles ◽  
Pinus Tabulaeformis ◽  
Cdna Libraries ◽  
Pcr Analysis ◽  
Transcriptional Networks ◽  
Ovule Development

The development of the female gametophyte (FG) is one of the key processes of life cycle alteration between the haploid gametophyte and the diploid sporophytes in plants and it is required for successful seed development after fertilization. It is well demonstrated that free nuclear mitosis (FNM) of FG is crucial for the development of the ovule. However, studies of the molecular mechanism of ovule and FG development focused mainly on angiosperms, such as Arabidopsis thaliana and further investigation of gymnosperms remains to be completed. Here, Illumina sequencing of six transcriptomic libraries obtained from developing and abortive ovules at different stages during free nuclear mitosis of magagametophyte (FNMM) was used to acquire transcriptome data and gene expression profiles of Pinus tabulaeformis. Six cDNA libraries generated a total of 71.0 million high-quality clean reads that aligned with 63,449 unigenes and the comparison between developing and abortive ovules identified 7174 differentially expressed genes (DEGs). From the functional annotation results, DEGs involved in the cell cycle and phytohormone regulation were highlighted to reveal their biological importance in ovule development. Furthermore, validation of DEGs from the phytohormone signal transduction pathway was performed using quantitative real-time PCR analysis, revealing the dynamics of transcriptional networks and potential key components in the regulation of FG development in P. tabulaeformis were identified. These findings provide new insights into the regulatory mechanisms of ovule development in woody gymnosperms.

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