Microarray Analysis Of Global Gene Expression Research Articles

Global gene deregulations in FASN silenced retinoblastoma cancer cells: molecular and clinico-pathological correlations.

Activation of fatty acid synthase (FASN) enzyme in the de novo lipogenic pathway has been reported in various cancers including retinoblastoma (RB), a pediatric ocular cancer. The present study investigates lipogenesis-dependent survival of RB cancer cells and the associated molecular pathways in FASN silenced RB cells. The siRNA-mediated FASN gene knockdown in RB cancer cells (Y79, WERI RB1) repressed FASN mRNA and protein expressions, and decreased cancer cell viability. Global gene expression microarray analysis was performed in optimized FASN siRNA transfected and untransfected RB cells. Deregulation of various downstream cell signaling pathways such as EGFR (n = 55 genes), TGF-beta (n = 45 genes), cell cycle (n = 41 genes), MAPK (n = 39 genes), lipid metabolism (n = 23 genes), apoptosis (n = 21 genes), GPCR signaling (n = 21 genes), and oxidative phosporylation (n = 18 genes) were observed. The qRT-PCR validation in FASN knockdown RB cells revealed up-regulation of ANXA1, DAPK2, and down-regulation of SKP2, SREBP1c, RXRA, ACACB, FASN, HMGCR, USP2a genes that favored the anti-cancer effect of lipogenic inhibition in RB. The expression of these genes in primary RB tumor tissues were correlated with FASN expression, based on their clinico-pathological features. The differential phosphorylation status of the various PI3K/AKT pathway proteins (by western analysis) indicated that the FASN gene silencing indeed mediated apoptosis in RB cells through the PI3K/AKT pathway. Scratch assay clearly revealed that FASN silencing reduced the invading property of RB cancer cells. Dependence of RB cancer cells on lipid metabolism for survival and progression is implicated. Thus targeting FASN is a promising strategy in RB therapy.

Recent progress in host immunity to avian coccidiosis: IL-17 family cytokines as sentinels of the intestinal mucosa

The molecular and cellular mechanisms leading to immune protection against coccidiosis are complex and include multiple aspects of innate and adaptive immunities. Innate immunity is mediated by various subpopulations of immune cells that recognize pathogen associated molecular patterns (PAMPs) through their pattern recognition receptors (PRRs) leading to the secretion of soluble factors with diverse functions. Adaptive immunity, which is important in conferring protection against subsequent reinfections, involves subtypes of T and B lymphocytes that mediate antigen-specific immune responses. Recently, global gene expression microarray analysis has been used in an attempt to dissect this complex network of immune cells and molecules during avian coccidiosis. These new studies emphasized the uniqueness of the innate immune response to Eimeria infection, and directly led to the discovery of previously uncharacterized host genes and proteins whose expression levels were modulated following parasite infection. Among these is the IL-17 family of cytokines. This review highlights recent progress in IL-17 research in the context of host immunity to avian coccidiosis.

LAIR2-expressing extravillous trophoblasts associate with maternal spiral arterioles undergoing physiologic conversion

Leukocyte-associated immunoglobulin-like receptor 2 (LAIR2) was identified on a global gene expression microarray analysis of surplus chorionic villus sampling (CVS) tissues as down-regulated in the first trimester of preeclampsia pregnancies. LAIR2 is the soluble receptor counterpart to LAIR1, an inhibitory receptor found on multiple immune cell subsets. In situ and immunohistochemical studies have previously shown that placental expression of LAIR2 expression is highly restricted, confined to the more distal portions of extravillous trophoblast (EVT) cell columns. This study examines LAIR2 expression in deeper layers of trophoblasts in the placental implantation site, maternal decidua and maternal spiral arterioles. Immunohistochemical staining detected LAIR2 expression on a subset of EVT within the implantation site. This trophoblast included the invasive EVT infiltrating the maternal decidual vessels and the EVT forming the endovascular trophoblastic plugs. More specifically, LAIR2-positive EVT showed a striking predilection for maternal decidual arterioles and the immediately surrounding decidua. Moreover, the appearance of EVT expressing LAIR2 in these areas was contemporaneous with the process of spiral arteriole remodeling. Based on these findings, we suggest that LAIR2-expressing EVT may play an important role in the remodeling of maternal spiral arterioles.

Genome-Wide Differential Gene Expression Profiles in Broiler Chickens with Gangrenous Dermatitis

Gangrenous dermatitis (GD) is a disease of poultry characterized by necrosis of the skin and severe cellulitis of the subcutaneous tissues caused by infection with Clostridium septicum (CS) and/or Clostridium perfringens (CP) type A. While GD causes significant morbidity, mortality, and economic loss to the poultry industry, the fundamental mechanisms underlying this host-pathogen interaction are relatively unknown. This study used comparative global gene expression microarray analysis of GD-affected and clinically healthy chickens from a recent GD outbreak to glean insights into the molecular and cellular changes associated with this disease process. Histopathologic and immunohistochemical analyses confirmed extensive muscle damage and prominent leukocyte infiltration in the skin of GD-affected birds but not in healthy controls. The levels of mRNAs in the skin and underlying muscle corresponding to 952 microarray elements were altered in GD-afflicted birds compared with healthy controls, with 468 being increased and 484 decreased. From these, a subset of 386 genes was identified and used for biologic function and pathway analyses. The biologic functions that were most significantly associated with the differentially expressed genes were "inflammatory response" and "cellular growth and proliferation" classified under the categories of "disease and disorders" and "molecular and cellular functions," respectively. The biologic pathway that was most significantly associated with the differentially expressed genes was the nuclear factor-erythroid 2-related factor 2 (NRF2)-mediated oxidative stress pathway. Finally, in vitro infection of chicken macrophages with CS or CP modified the levels of mRNAs encoding interferon (IFN)-alpha, IFN-gamma, interleukin (IL)-1beta, IL-6, IL-12p40, tumor necrosis factor superfamily 15 (downregulated), IL-8, and IL-10 (upregulated), thus confirming the suppressive effect of GD on the chicken immune system.

Variations in Discovery‐Based Preeclampsia Candidate Genes

Preeclampsia is a common and potentially lethal pregnancy disorder with lifelong increased risk of cardiovascular disease in survivors. Our prior global gene expression microarray analysis led to a novel set of 36 candidates in first trimester placentas of women who subsequently developed preeclampsia. In this report, we present preliminary studies demonstrating biomarkers of genotype and methylation variations in a subset of these candidate genes in maternal leukocyte and fetoplacental DNA of 28 case and 27 control dyads. We tested 84 single nucleotide polymorphisms (SNPs) using MassArray iPLEX and 50 CpG sites using EpiTYPER assays. Promising prediction modeling was identified with 25 SNPs selected using Fisher's exact tests (p ≤ 0.05) and 20 CpG sites selected on fold change. Genotype Distribution Analysis identified SNP variations that differed between nine paired cases versus paired controls. The findings validate the examined candidate genes and support feasibility of methods for further biomarker development. The integrative approach that was implemented begins to translate the 36 candidates toward clinical utility as a screening modality for preeclampsia.

Identification of Gene Networks and Pathways Associated with Guillain-Barré Syndrome

BackgroundThe underlying change of gene network expression of Guillain-Barré syndrome (GBS) remains elusive. We sought to identify GBS-associated gene networks and signaling pathways by analyzing the transcriptional profile of leukocytes in the patients with GBS.Methods and FindingsQuantitative global gene expression microarray analysis of peripheral blood leukocytes was performed on 7 patients with GBS and 7 healthy controls. Gene expression profiles were compared between patients and controls after standardization. The set of genes that significantly correlated with GBS was further analyzed by Ingenuity Pathways Analyses.256 genes and 18 gene networks were significantly associated with GBS (fold change ≥2, P<0.05). FOS, PTGS2, HMGB2 and MMP9 are the top four of 246 significantly up-regulated genes. The most significant disease and altered biological function genes associated with GBS were those involved in inflammatory response, infectious disease, and respiratory disease. Cell death, cellular development and cellular movement were the top significant molecular and cellular functions involved in GBS. Hematological system development and function, immune cell trafficking and organismal survival were the most significant GBS-associated function in physiological development and system category. Several hub genes, such as MMP9, PTGS2 and CREB1 were identified in the associated gene networks. Canonical pathway analysis showed that GnRH, corticotrophin-releasing hormone and ERK/MAPK signaling were the most significant pathways in the up-regulated gene set in GBS.ConclusionsThis study reveals the gene networks and canonical pathways associated with GBS. These data provide not only networks between the genes for understanding the pathogenic properties of GBS but also map significant pathways for the future development of novel therapeutic strategies.

Bridging global gene expression candidates in first trimester placentas with susceptibility loci from linkage studies of preeclampsia

Preeclampsia is as a leading cause of maternal and perinatal morbidity and mortality. Prevention, early identification, and individualized treatments may become feasible if reliable early biomarkers can be developed. Towards a systems biology framework, this review synthesizes prior linkage studies and genome scans of preeclampsia with candidates identified in a global gene expression microarray analysis of chorionic villus sampling (CVS) specimens from women who subsequently developed preeclampsia. Nearly 40% of these CVS candidate genes occurred in previously identified susceptibility loci for preeclampsia. Integration of genetic epidemiologic and functional gene expression data could help to elucidate preeclampsia as a complex disease resulting from multiple maternal and fetal/placental factors that each contributes a greater or lesser effect. These loci and related candidate genes are set to substantially improve insights into the first trimester pathogenesis of this pregnancy disorder.

LAIR2 localizes specifically to sites of extravillous trophoblast invasion

Purpose A global gene expression microarray analysis of surplus chorionic villus sampling (CVS) tissues identified leukocyte-associated immunoglobulin-like receptor 2 (LAIR2) as down-regulated in the first trimester of pregnancies destined for preeclampsia. Neither the localization nor the function of LAIR2 has been examined in the placenta. Localization studies were conducted in placental tissues to determine the precise sites of LAIR2 mRNA production and protein binding. Results Quantitative real time polymerase chain reaction (qRT-PCR) indicated LAIR2 expression in CVS, but none in breast, lymph node, kidney, skin, uterus, or third trimester placentas. In situ hybridization (ISH) revealed a highly restricted LAIR2 localization. LAIR2 mRNA was found only in the more distal portions of trophoblast anchoring cell columns, adjacent to the invading extravillous trophoblast (EVT). Immunohistochemistry (IHC) detected intracellular LAIR2 staining in these same cells. Extracellular staining of this soluble receptor was found in the acellular material between invasive EVT cells distal to the anchoring cell columns. Conclusions ISH and IHC staining for LAIR2 detected specific, highly localized expression at the leading edge of EVT anchoring cell columns in first trimester placentas. This staining likely identifies the site of production for this soluble receptor. Following secretion, the receptor appears to bind extracellular material among the invasive EVT. The precise restriction of this protein only to the sites of EVT invasion strongly suggests that it functions to regulate this invasion. The decreased LAIR2 expression noted in first trimester placentas that ultimately developed preeclampsia further suggests that alterations in LAIR2 may play an etiologic role in preeclampsia.

DNA Methyltransferase 1 Is Essential for and Uniquely Regulates Hematopoietic Stem and Progenitor Cells.

Abstract 392DNA methylation is essential for development and plays crucial roles in a variety of biological processes. The DNA methyltransferase Dnmt1 serves to maintain parental cell methylation patterns on daughter DNA strands in mitotic cells, however, the precise role of Dnmt1 in regulation of quiescent adult stem cells is not known. To examine the role of Dnmt1 in adult hematopoietic stem cells (HSCs), we crossed Dnmt1fl/fl mice with Mx1-Cre transgenic mice, and by injection of poly(I)-poly(C) we selectively deleted Dnmt1 in the hematopoietic system (Dnmt1Δ/Δ). In Dnmt1Δ/Δ mice, peripheral blood counts and mature multilineage composition of the bone marrow was found to be normal. Interestingly, specific defects were observed in Dnmt1Δ/Δ HSC self-renewal as assessed by long-term and secondary competitive transplantation, in retention of Dnmt1Δ/Δ HSCs within the bone marrow niche, and in the ability of Dnmt1Δ/Δ HSCs to give rise to multilineage hematopoiesis. Loss of Dnmt1 also had unique impact on myeloid progenitor cells (including common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythrocyte progenitors), regulating their cycling and transcriptional lineage fidelity. To determine the molecular mechanisms underlying these defects, we performed global gene expression microarray analysis and bisulfite sequencing of select loci (IAP, Car1, and Gata1) in purified populations of control and Dnmt1Δ/Δ long-term HSCs, short-term HSCs/multipotent progenitor cells, and myeloid restricted progenitor cells. Through this approach, we demonstrate that loss of Dnmt1 has cell type-specific molecular consequences. For example, demethylation of the Car1 and Gata1 loci in Dnmt1Δ/Δ long-term HSCs is not sufficient to activate gene transcription, whereas demethylation of these genes in Dnmt1Δ/Δ short-term HSCs is associated with activation of transcription. In Dnmt1Δ/Δ myeloid restricted progenitor cells, we observed increases in DNA methylation at specific gene loci such as Car1, indicating that methylation can be established by other methyltransferases in the absence of Dnmt1. Our global gene expression microarray analysis clearly demonstrates that Dnmt1 regulates expression of distinct gene families in these closely related, primitive hematopoietic populations. We were unable to attribute specific functional defects in Dnmt1Δ/Δ hematopoietic stem and progenitor cells to alterations in expression of previously characterized genes, supporting the existence of novel, uncharacterized regulators of HSC and progenitor cell function to be explored from candidates in our data set. We conclude that maintenance methylation induced by Dnmt1 appears to be especially important for HSC and progenitor cell state transitions, such as the stepwise differentiation of long-term HSCs to multipotent progenitors, multipotent progenitors to myeloid restricted progenitors, stem cell mobilization, and regulating cell cycle entry. These findings establish a unique and critical role for Dnmt1 in the primitive hematopoietic compartment. Furthermore, our evidence suggests that epigenetic regulation, at least with respect to DNA methylation, of adult stem cells is distinct from embryonic stem cells and other somatic cell types. Disclosures:No relevant conflicts of interest to declare.

Gene Ontology-driven transcriptional analysis of CD34+cell-initiated megakaryocytic cultures identifies new transcriptional regulators of megakaryopoiesis

Differentiation of hematopoietic stem and progenitor cells is an intricate process controlled in large part at the level of transcription. While some key megakaryocytic transcription factors have been identified, the complete network of megakaryocytic transcriptional control is poorly understood. Using global gene expression microarray analysis, Gene Ontology-based functional annotations, and a novel interlineage comparison with parallel, isogenic granulocytic cultures as a negative control, we closely examined the mRNA level of transcriptional regulators in megakaryocytes derived from human mobilized peripheral blood CD34(+) hematopoietic cells. This approach identified 199 differentially expressed transcription factors or transcriptional regulators. We identified and detailed the transcriptional kinetics of most known megakaryocytic transcription factors including GATA1, FLI1, and MAFG. Furthermore, many genes with transcription factor activity or transcription factor binding activity were identified in megakaryocytes that had not previously been associated with that lineage, including BTEB1, NR4A2, FOXO1A, MEF2C, HDAC5, VDR, and several genes associated with the tumor suppressor p53 (HIPK2, FHL2, and TADA3L). Protein expression and nuclear localization were confirmed in megakaryocytic cells for four of the novel candidate megakaryocytic transcription factors: FHL2, MXD1, E2F3, and RFX5. In light of the hypothesis that transcription factors expressed in a particular differentiation program are important contributors to such a program, these data substantially expand our understanding of transcriptional regulation in megakaryocytic differentiation of stem and progenitor cells.

Analysis of the Human Fetal Liver Hematopoietic Microenvironment

In the adult, hematopoietic stem cells (HSCs) are resident in the bone marrow (BM) compartment and are in direct association with the BM stromal microenvironment. However, human adult HSCs are largely quiescent and undergo limited self-renewal. This is in contrast to the higher frequency of cycling HSCs undergoing self-renewal during fetal development when hematopoiesis is transiently localized to the fetal liver (FL), suggesting that FL provides a more conducive microenvironment to support HSCs. Here, we provide phenotypic and molecular characterization of primary human FL stromal cells capable of supporting human repopulating progenitors. Qualitative and quantitative analysis revealed several properties unique to FL stromal cells compared to adult BM-derived stroma that included a greater than 10-fold enhanced proliferative capacity of FL stromal vs adult BM, and a 2-fold increase in the number of N-cadherin- and osteopontin-expressing cells. Supportive of extrinsic influences likely to modulate HSC expansion, global gene expression microarray analysis revealed that FL stroma has higher expression of regulators of the Wnt signaling pathway compared to adult BM stroma, which demonstrated an increased expression of the Notch signaling pathway. Our results suggest that human FL stromal cells provide a unique microenvironment to HSCs compared to adult BM stroma by controlling Wnt signaling of HSCs during human fetal hematopoietic development, while Notch signaling is tightly regulated by the HSC microenvironment in the adult. We propose that the human HSC niche is ontogenically controlled during human development to provide appropriate expansion of fetal HSCs and subsequent maintenance of adult HSCs.