Mirnas and Gene Expression Profiles in CD34+ Cells Are Dependent On the Source of Progenitor Cells Employed in Transplantation.

Abstract Abstract 3020 Introduction: Hematopoietic progenitors cells (HPCs) used in allogenic transplantation (allo-HSCT) may have different biological properties depending on their source of origin: mobilized peripheral blood (PB), bone marrow (BM) or umbilical cord (UC), which may be reflected in miRNAs or gene expression. The identification of different patterns of expression could have clinical implications. The aim of this study was to determine differences in miRNAs and gene expression patterns in the different sources of HPCs used in allo-HSCT. Materials and Method: CD34 + cells were isolated by immunomagnetic separation and sorting from 5 healthy donors per type of source: UC, BM and PB mobilized with G-CSF. A pool of samples from PB not mobilized was used as reference group. We analyzed the expression of 375 miRNAs using TaqMan MicroRNA Arrays Human v2.0 (Applied Biosystems), and gene expression using Whole Human Genome Oligo microarray kit 4×44K (Agilent). The expression levels of genes and miRNAs were obtained by the 2-ΔΔCTmethod. From expression data hierarchical clustering was performed using the Euclidean distance. To identify genes and miRNAs differentially expressed between the different sources of HPCs statistical Kruskal Wallis test was applied. All analysis were performed using the Multiexperiment Viewer 4.7.1. The function of the miRNAs and genes of interest was determined from the various databases available online (TAM database, Gene Ontology and TargetScan Human). Results: Forty-two miRNAs differentially expressed between the different sources were identified. As compared to BM or UC, in mobilized PB most miRNAs were overexpressed, including the miRNA family of miR515, which is characteristic of embryonic stem cells. On the other hand, 47 genes differentially expressed between the different sources were identified. Interestingly, a similar pattern of expression was observed between movilized PB and UC as compared to BM. Interestingly, 13 of these genes are targets of the miRNAs also identified in this study, which suggests that their expression might be regulated by these miRNAs. Conclusion: There are significant differences in miRNAs and gene expression levels between the different sources of HPCs Disclosures: No relevant conflicts of interest to declare.

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Molecular Determinants of Lymph Node Microenvironment Induced Host Immune Tolerance In CLL: Role for CAV1, PTPN6, and PKCβ In the Process

Blood ◽

10.1182/blood.v116.21.1367.1367 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1367-1367

Author(s):

Christine Gilling ◽

Amit Mittal ◽

Vincent Nganga ◽

Vicky Palmer ◽

Dennis D. Weisenburger ◽

...

Keyword(s):

Gene Expression ◽

Mouse Model ◽

Immune Tolerance ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cell Receptor ◽

Differentially Expressed ◽

Expression Levels ◽

Aggressive Disease

Abstract Abstract 1367 Previously, we have shown that gene expression profiles (GEP) of CLL cells from lymph nodes (LN), bone marrow (BM), and peripheral blood (PB) are significantly different from each other. Among the major pathways associated with differential gene expression, a “tolerogenic signature” involved in host immune tolerance is significant in regulating CLL progression. The genes associated with the tolerogenic signature are significantly differentially expressed in patient LN-CLL compared to BM-CLL and PB-CLL, suggesting that LN-CLL cells induce this immune tolerance. From 83 differentially expressed genes identified by GEP that are associated with immune dysregulation, we selected eleven genes (CAV1, PTPN6, PKCb, ZWINT, IL2Ra, CBLC, CDC42, ZNF175, ZNF264, IL10, and HLA-G) for validation studies to determine whether these genes are also dysregulated in the Emu-TCL1 mouse model of CLL. The results demonstrate a trend of upregulation of these genes as determined by qRT-PCR in the LN-tumor microenvironment. To further evaluate the kinetics of selected gene expression during tumor progression, we determined the expression levels of Cav1, Ptpn6, and Pkcb at 12, 24, and 36 weeks of CLL development in the Em-TCL1 mouse model. We found that the expression of all three genes increased as a function of age, indicating a correlation of gene expression with disease progression. In addition, as CLL progressed in these mice there was a marked decrease in CD4+ and CD8+ T cells. The murine data were further validated using CLL cells from the same patients with indolent versus aggressive disease indicating a similar trend in expression as CLL progressed (n=4). Furthermore, patient data analyzed by Kaplan Meier analyses of the expression levels of the selected genes indicated a significant association between down-regulation of PTPN6 (p=0.031) and up-regulation of ZWINT (p<0.001) with clinical outcome as determined by a shorter time to treatment (p<0.05). Functional analysis by knockdown of CAV1 and PKCb in primary patient CLL cells determined by MTT assay showed a decrease in proliferation following knockdown of these genes (p<0.005). Protein-interaction modeling revealed regulation of CAV1 and PTPN6 by one another. Additionally, the PTPN6 protein regulates B cell receptor (BCR) signaling and subsequently the BCR regulates PKCb. Therefore, these data from both mice and humans with CLL, argue that an aggressive disease phenotype is paralleled by expression of genes associated with immune suppression. In particular, evidence presented here suggests, dysregulation of CAV1, PTPN6, ZWINT, and PKCb expression promotes CLL progression. Disclosures: No relevant conflicts of interest to declare.

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Molecular Phenotype of Malignant CD34+ Hematopoietic Stem and Progenitor Cells in Chronic Myelogenous Leukemia.

Blood ◽

10.1182/blood.v106.11.2863.2863 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2863-2863

Author(s):

Ralf Kronenwett ◽

Elena Diaz-Blanco ◽

Thorsten Graef ◽

Ulrich Steidl ◽

Slawomir Kliszewski ◽

...

Keyword(s):

Gene Expression ◽

Fatty Acid ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Pattern ◽

Leukemic Cell ◽

Differentially Expressed ◽

Specific Gene ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract In this study, we examined gene expression profiles of immunomagnetically enriched CD34+ cells from bone marrow (BM) of 9 patients with untreated CML in chronic phase and from 8 healthy volunteers using Affymetrix GeneChips. Additionally, in 3 patients CD34+ from peripheral blood (PB) were compared with those from BM. Differential expression of 12 candidate genes was corroborated by quantitative real-time RT-PCR. Following hybridization of labelled cRNA to Affymetrix GeneChips covering 8793 genes we used the statistical scripting language “R” for data analysis. For normalization a method of variance stabilization transformations was used. To identify significantly differentially expressed genes we used the Significance Analysis of Microarrays (SAM) algorithm. The intraindividual comparison of CD34+ cells from BM and PB in CML showed no differentially expressed genes which is different to normal CD34+ cells which had distinct gene expression patterns comparing circulating and sedentary CD34+ cells (Steidl et al., Blood, 2002). Comparing malignant BM CD34+ cells from CML with normal BM CD34+ cells 792 genes were significantly differentially expressed (fold change: >1.3; q-value: <0.03). 735 genes had a higher and 57 genes a lower expression in CML. Gene expression patterns reflected BCR-ABL-induced functional alterations such as increased cell-cycle and proteasome activity as well as decreased apoptosis. Downregulation of several genes involved in DNA repair and detoxification in CML might be the basis for DNA instability and progression to blast crisis. An interesting finding was an upregulation of fetal hemoglobin (Hb) components such as Hb gamma A and G in leukemic progenitor cells whereas no difference in adult Hb expression was observed suggesting an induction of fetal Hb synthesis in CML. Looking at genes involved in stem cell maintenance we found an upregulation of GATA2 and a reduced expression of proteins from the Wnt signalling pathway suggesting an increased self-renewal of CML hematopoietic stem cells compared to the normal counterpart. Moreover, several genes playing a role in ubiquitin-dependent protein catabolism and in fatty acid biosynthesis such as fatty acid synthase (FAS) were stronger expressed in CML. The functional role of FAS for leukemic cell growth was assessed in cell culture experiments. Incubation of the leukemic cell line K562 with the FAS inhibitor cerulenin (10 μg/ml) for 3 days resulted in death of 99% of cells suggesting that survival of leukemic cells depends upon endogenous fatty acid synthesis. In an attempt to find a specific gene expression pattern associated with response to imatinib therapy we divided the patients included in this study into two groups: maximal reduction of BCR-ABL transcript level <3-log vs. >3-log (major molecular remission) during therapy. Comparing pretherapeutic gene expression profiles of both groups we could not identify a pattern predictive for major molecular response. In conclusion, malignant CD34+ cells in CML have a specific gene expression pattern which seems not to be predictive for response to imatinib therapy.

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Gene Expression Profiles of CD34+ Cells in Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v112.11.5078.5078 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5078-5078

Author(s):

Monika Belickova ◽

Alzbeta Vasikova ◽

Eva Budinska ◽

Jaroslav Cermak

Keyword(s):

Gene Expression ◽

Differentially Expressed Genes ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Profiles ◽

Statistical Testing ◽

Differentially Expressed ◽

Control Group ◽

Regulation Of Transcription ◽

Cd34 Cells

Abstract Myelodysplatic syndrome (MDS) represents a heterogeneous group of clonal disorders with ineffective hematopoiesis that is characterized by dysplasia and peripheral cytopenia of one or more cell lineages. We studied gene expression profiles in CD34+ cells of 42 MDS patients and 6 healthy controls using Illumina cDNA microarray technology. Nine patients had RA, 7 patients had RCMD, 17 patients had RAEB and 9 had RAEB-T. CD34+ cells were isolated from bone marrow samples using MACS magnetic columns. The quality of total extracted RNA was confirmed with the Agilent Bioanalyzer 2100. 200ng of total RNA was amplified using Illumina RNA amplification kit. cRNA targets were hybridized on the Sentrix HumanRef-8 BeadChips (> 24 000 probes), which were scanned on the Illumina BeadStation 500. The data were pre-processed and normalized by lumi R package designed to preprocess the Illumina microarray data. Normalized data were filtered by detection p-value <0.01, resulting in total number of 10 091 genes. This gene set was tested for differential expression between clinical groups and control group. For this purpose, statistical testing by ANOVA with correction for multiple testing problem by Bayesian thresholding was performed. Additionally, analysis by random-forests (RAFT) was performed. Significant genes from both analyses were merged resulting in 332 differentially expressed genes detected. Out of these, 79 genes showed ≥2.5 fold changes in gene expression between controls and all MDS groups (22 up-regulated and 57 down-regulated). Our findings were confirmed by real-time quantitative PCR for several genes (TaqMan Gene Expression Assays). We used DAVID database to annotate 79 selected genes: 8 of 22 up-regulated genes in MDS patients were recognized to play a role in regulation of transcription (LEO1, E2F6 and several zing finger proteins). A half of these over-expressed genes could not be annotated due to still unknown biological function. Within the set of the down-regulated genes in MDS patients those biological processes were predominantly detected: cell differentiation (KLF4, FOSL2, STK17B, BCL3, SNF1LK, ID2 etc.), response to stress (CXCL12, SMAD7, CYGB, etc.) and cell proliferation (MXD1, OSM, FTH1, KLF10 etc.). In the set of 31 genes with 5 fold decreased expression, we identified 8 genes involved in B-cell development. (VPREB1, VPREB3, CD79A, EBI2, LEF1, CXCL12, CTGF, GALNAC4S-6ST). RAFT analysis was performed also in the set of 332 statistically differentially expressed genes in order to evaluate accuracy of grouping the patients according their diagnosis. We detected strong heterogeneity in gene expression patterns within the MDS patients, especially in the RAEB group reflecting clinical diversity of MDS. Clustering analysis (Spearman correlation) showed that most of the RAEB-2 patients (7 out of 9) were clustered together with REAB-T whereas RAEB-1 clustered with RCMD or RA. These results underline the need of distinguishing RAEB-1 and RAEB-2 diagnosis according to WHO classification system, since their expression profiles are significantly different.

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A Distinct Expression of Various Gene Subsets in CD34+ Cells Obtained From Patients with Early and Advanced Myelodysplastic Syndrome.

Blood ◽

10.1182/blood.v114.22.3795.3795 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3795-3795

Author(s):

Monika Belickova ◽

Jaroslav Cermak ◽

Alzbeta Vasikova ◽

Eva Budinska

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Differentially Expressed Genes ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Differentially Expressed ◽

Abnormal Development ◽

Statistical Hypothesis ◽

P Value ◽

Cd34 Cells

Abstract Abstract 3795 Poster Board III-731 Gene expression profiles of CD34+ cells were compared between a cohort of 51 patients with MDS or AML from MDS and 7 healthy controls. The patients were classified according to the WHO criteria as follows: 5q- syndrome (n=7), RA (n=3), RARS (n=2), RCMD (n=10), RAEB-1 (n=7), RAEB-2 (n=15), and AML with MLD (multilineage dysplasia) (n=7). HumanRef-8 v2 Expression Bead Chips (Illumina) were used to generate expression profiles of the samples for >22,000 transcripts. The raw data were normalized data with the R software, lumi package. Normalized data were filtered by detection p-value <0.01, resulting in total number of 9811genes. To identify differentially expressed genes we performed two parallel statistical hypothesis testings: Analysis of Variance (ANOVA) together with Tukey test and empirical bayesian thresholding correction for multiple testing problem; and Significance Analysis of Microarrays (SAM). The results were confirmed by real-time quantitative PCR for six genes (TaqMan Gene Expression Assays). Hierarchical clustering of significantly differentially expressed genes clearly separated patients and controls, 5q-syndrome and RAEB-1 as a separate entities confirming usefulness of WHO classification subgroups. The most up-regulated genes in all patients included HBG2, HBG1, CYBRD1, HSPA1B, ANGPT1, and MYC. We assume that expression changes in globin genes, both fetal and adult globins (HBG2, HBG1 and HBA1, HBB) may play role not only in dysregulation of erythropoiesis but also in the disease progression or leukemic transformation of MDS. Among the most down-regulated genes, 13 genes related to B-lymphopoiesis (e.g. POU2AF1, VPREB1, VPREB3, CD79A, EBF1, LEF1, BCL3, IRF8 & IRF4) were detected, suggesting the abnormal development of B-cell progenitors in all MDS patients. Some of these genes (e.g. VPREB3, LEF1) showed decreasing trend in expression level from early to advanced MDS with the lowest expression in AML with MLD. Patients with advanced MDS had significantly decreased expression of genes involved in in the mitotic cell cycle, DNA replication, and chromosome segregation compared to early MDS where these gene subsets were up-regulated. The DAVID database also identified de-regulation in the cell cycle pathway through its 7 genes (CDC25C, CDC7, CDC20, ORC1L, CCNB2, BUB1, & CCNA2). On the other hand, advanced MDS patients showed significant up-regulation of proto-oncogenes (BMI1, MERTK) and genes related to angiogenesis (ANGPT1), anti-apoptosis (VNN1). The results confirm on molecular basis that increased cell proliferation and resistance to apoptosis together with a loss of cell cycle control, damaged DNA repair and altered immune response may play an important role in the expansion of malignant clone in MDS patients. The study was supported by Grant NR-9235 obtained from the Ministry of Health, Czech Republic. Disclosures: No relevant conflicts of interest to declare.

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The G-CSF Produces Long-Term Changes in Gene and Mirnas Expression Profiles in CD34+ From Healthy Donors

Blood ◽

10.1182/blood.v120.21.588.588 ◽

2012 ◽

Vol 120 (21) ◽

pp. 588-588

Author(s):

Alicia Báez ◽

Concepción Prats-Martín ◽

Isabel Álvarez-Laderas ◽

Estefania Garcia ◽

Luis Ignacio Sanchez-Abarca ◽

...

Keyword(s):

Gene Expression ◽

Conflicts Of Interest ◽

Reference Group ◽

Expression Profiles ◽

Differentially Expressed ◽

Long Term Effects ◽

Time Points ◽

Healthy Donors ◽

Mirnas Expression

Abstract Abstract 588 Introduction: The G-CSF (granulocyte colony-stimulating factor) is the cytokine most commonly used for the mobilization of hematopoietic progenitor cells (HPCs) from healthy donors used in allogenic transplantation (allo-HSCT). Although the administration of G-CSF is considered safe, the knowledge about its long-term effects, especially in HPCs, is limited. The aim of this study was to analyze whether G-CSF induces changes in gene and miRNAs expression profiles in HPCs from healthy donors, and determine whether or not these changes persist at the long term. Materials and Method: CD34 + cells were isolated by immunomagnetic separation and sorting from 5 healthy donors before mobilization with G-CSF and at afterwards at 5, 30 and 365 days after mobilization. A pool of samples from PB not mobilized was used as reference group. We analyzed the expression of 375 miRNAs using TaqMan MicroRNA Arrays Human v2.0 (Applied Biosystems), and the gene expression profile using Whole Human Genome Oligo microarray kit 4×44K (Agilent). The expression levels of genes and miRNAs were obtained by the 2-ΔΔCTmethod. From expression data hierarchical clustering was performed using the Euclidean distance. To identify genes and miRNAs differentially expressed due to the effect of G-CSF at different time-points a cut-off value of level expression 2,5 above or below the control values was used and non-parametric Mann-Whitney test was applied. All analysis were performed using the Multi-experiment Viewer 4.7.1. The function of the miRNAs and genes of interest was determined from the various databases available online (TAM database, Gene Ontology, TargetScan Human). Results: Seven miRNAs were differentially expressed in HPCs at 5, 30 and 365 days after mobilization with G-CSF, as compared to HPCs obtained from not mobilized PB. In addition, 67 genes were also differentially expressed after administration of G-CSF, whose expression profiles remained abnormal 1 year after mobilization. These genes are involved in biological processes such as hematopoietic cell proliferation, ribosomal protein synthesis, cell metabolism and transmembrane transportation. Interestingly, 8 of these genes are target of the miRNAs also identified in the current study, which suggests that their expression might be regulated by these miRNAs. Conclusion: The G-CSF modifies gene expression profiles and miRNAs in HPCs from healthy donors. Remarkably, these changes were observed from early time-points and persisted at least 1 year after exposure to the drug. This effect of G-CSF on HPCs have not been previously reported and might be clinically relevant. Disclosures: No relevant conflicts of interest to declare.

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Comprehensive Comparison of Amnion Stromal Cells and Chorion Stromal Cells by RNA-Seq

International Journal of Molecular Sciences ◽

10.3390/ijms22041901 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1901

Author(s):

Brielle Jones ◽

Chaoyang Li ◽

Min Sung Park ◽

Anne Lerch ◽

Vimal Jacob ◽

...

Keyword(s):

Gene Expression ◽

Stromal Cells ◽

Expression Profiles ◽

Expression Patterns ◽

Principal Component ◽

Differentially Expressed ◽

Inflammatory Factor ◽

Next Generation Sequencing Technology ◽

Angiogenic Potential ◽

Anti Inflammatory

Mesenchymal stromal cells derived from the fetal placenta, composed of an amnion membrane, chorion membrane, and umbilical cord, have emerged as promising sources for regenerative medicine. Here, we used next-generation sequencing technology to comprehensively compare amniotic stromal cells (ASCs) with chorionic stromal cells (CSCs) at the molecular and signaling levels. Principal component analysis showed a clear dichotomy of gene expression profiles between ASCs and CSCs. Unsupervised hierarchical clustering confirmed that the biological repeats of ASCs and CSCs were able to respectively group together. Supervised analysis identified differentially expressed genes, such as LMO3, HOXA11, and HOXA13, and differentially expressed isoforms, such as CXCL6 and HGF. Gene Ontology (GO) analysis showed that the GO terms of the extracellular matrix, angiogenesis, and cell adhesion were significantly enriched in CSCs. We further explored the factors associated with inflammation and angiogenesis using a multiplex assay. In comparison with ASCs, CSCs secreted higher levels of angiogenic factors, including angiogenin, VEGFA, HGF, and bFGF. The results of a tube formation assay proved that CSCs exhibited a strong angiogenic function. However, ASCs secreted two-fold more of an anti-inflammatory factor, TSG-6, than CSCs. In conclusion, our study demonstrated the differential gene expression patterns between ASCs and CSCs. CSCs have superior angiogenic potential, whereas ASCs exhibit increased anti-inflammatory properties.

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Regional variations in ABC transporter expression along the mouse intestinal tract

Physiological Genomics ◽

10.1152/physiolgenomics.00150.2003 ◽

2004 ◽

Vol 17 (1) ◽

pp. 11-20 ◽

Cited By ~ 40

Author(s):

David M. Mutch ◽

Pascale Anderle ◽

Muriel Fiaux ◽

Robert Mansourian ◽

Karine Vidal ◽

...

Keyword(s):

Gene Expression ◽

Abc Transporter ◽

Expression Profile ◽

Abc Transporters ◽

Intestinal Tract ◽

Expression Profiles ◽

Expression Patterns ◽

Assessment Model ◽

Differentially Expressed ◽

Distal Intestine

The ATP-binding cassette (ABC) family of proteins comprise a group of membrane transporters involved in the transport of a wide variety of compounds, such as xenobiotics, vitamins, lipids, amino acids, and carbohydrates. Determining their regional expression patterns along the intestinal tract will further characterize their transport functions in the gut. The mRNA expression levels of murine ABC transporters in the duodenum, jejunum, ileum, and colon were examined using the Affymetrix MuU74v2 GeneChip set. Eight ABC transporters (Abcb2, Abcb3, Abcb9, Abcc3, Abcc6, Abcd1, Abcg5, and Abcg8) displayed significant differential gene expression along the intestinal tract, as determined by two statistical models (a global error assessment model and a classic ANOVA, both with a P < 0.01). Concordance with semiquantitative real-time PCR was high. Analyzing the promoters of the differentially expressed ABC transporters did not identify common transcriptional motifs between family members or with other genes; however, the expression profile for Abcb9 was highly correlated with fibulin-1, and both genes share a common complex promoter model involving the NFκB, zinc binding protein factor (ZBPF), GC-box factors SP1/GC (SP1F), and early growth response factor (EGRF) transcription binding motifs. The cellular location of another of the differentially expressed ABC transporters, Abcc3, was examined by immunohistochemistry. Staining revealed that the protein is consistently expressed in the basolateral compartment of enterocytes along the anterior-posterior axis of the intestine. Furthermore, the intensity of the staining pattern is concordant with the expression profile. This agrees with previous findings in which the mRNA, protein, and transport function of Abcc3 were increased in the rat distal intestine. These data reveal regional differences in gene expression profiles along the intestinal tract and demonstrate that a complete understanding of intestinal ABC transporter function can only be achieved by examining the physiologically distinct regions of the gut.

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Is gene expression among women with rheumatoid arthritis dysregulated during a postpartum flare?

Arthritis Research & Therapy ◽

10.1186/s13075-021-02418-w ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Matthew Wright ◽

Mette K. Smed ◽

J. Lee Nelson ◽

Jørn Olsen ◽

Merete L. Hetland ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Gene Expression ◽

Disease Activity ◽

Activity Index ◽

Expression Profiles ◽

Expression Patterns ◽

Time Frame ◽

Differentially Expressed ◽

Healthy Women ◽

Immune Related Genes

Abstract Background To evaluate our hypotheses that, when rheumatoid arthritis (RA) flares postpartum, gene expression patterns are altered compared to (a) healthy women, (b) RA women whose disease activity is low or in remission postpartum, and (c) pre-pregnancy expression profiles. Methods Twelve women with RA and five healthy women were included in this pilot study. RA disease activity and postpartum flare were assessed using the Clinical Disease Activity Index (CDAI). Total RNA from frozen whole blood was used for RNA sequencing. Differential gene expression within the same women (within-group) over time, i.e., postpartum vs. third trimester (T3) or pre-pregnancy (T0), were examined, using a significance threshold of q < 0.05 and fold-change ≥ 2. Results Nine of the women with RA experienced a flare postpartum (RAFlare), while three had low disease activity or were in remission (RANoFlare) during that time frame. Numerous immune-related genes were differentially expressed postpartum (vs. T3) during a flare. Fold-changes in expression from T3 to postpartum were mostly comparable between the RAFlare and healthy groups. At 3 months postpartum, compared to healthy women, several genes were significantly differentially expressed only among the RAFlare women, and not among the RANoFlare women. Some of these genes were among those whose “normal” expression was significantly modulated postpartum, and the postpartum expression patterns were significantly altered during the RA flare. There were also some genes that were significantly differentially expressed in RAFlare compared to both healthy and RANoFlare women, even though their expression was not significantly modulated postpartum. Furthermore, while postpartum expression profiles were similar to those at pre-pregnancy among healthy women, significant differences were found between those time points among the RAFlare women. Conclusions The large majority of gene expression changes between T3 and 3 months postpartum among RA women who flared postpartum reflected normal postpartum changes also seen among healthy women. Nonetheless, during a postpartum flare, a set of immune-related genes showed dysregulated expression compared to healthy women and women with RA whose disease activity was low or in remission during the same time frame, while other genes demonstrated significant differences in expression compared to RA pre-pregnancy levels.

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171 THE EFFECT OF TRICHOSTATIN A ON THE DEVELOPMENT AND THE GLOBAL GENE EXPRESSION PATTERNS IN MOUSE EMBRYOS DEVELOPING IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab171 ◽

2008 ◽

Vol 20 (1) ◽

pp. 165

Author(s):

X. S. Cui ◽

X. Y. Li ◽

T. Kim ◽

N.-H. Kim

Keyword(s):

Gene Expression ◽

Trichostatin A ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Profiles ◽

Mouse Embryos ◽

Differentially Expressed ◽

Gene Expression Patterns ◽

Cell Stage

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P < 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

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The Expression of Human Placental Genes Related to Carotenoid/retinoid Metabolism and Pathways (P02-005-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz029.p02-005-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Xiaoming Gong ◽

Lewis Rubin

Keyword(s):

Gene Expression ◽

Microarray Analysis ◽

Fetal Development ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Set Enrichment Analysis ◽

Differentially Expressed ◽

Retinoid Metabolism ◽

Expression Of Genes ◽

Β Carotene

Abstract Objectives Carotenoid/retinoids status and metabolism are essential for normal placental and fetal development. Both deficiencies and excess of retinoids and some carotenoids are associated with adverse pregnancy outcomes, such as preeclampsia and preterm birth. A group of important genes involved in regulating carotenoid/retinoid metabolism and maternal to fetal transfer in human placenta. The objective of this study is to analyze (a) the expression of genes critical for regulating carotenoid/retinoid metabolism and maternal-fetal transport in human trophoblasts and (b) placental transcriptional profiles of these pathways in response to carotenoid exposure. Methods Human cytotrophoblasts (CTBs) were isolated from term placentas. CTB RNA was used to analyze the expression of genes involved in carotenoid/retinoid metabolism and pathways by qRT-PCT. First trimester-like trophoblasts (HTR-8/SVneo) were treated with either β-carotene or lycopene. RNAs were isolated and gene expression were analyzed by DNA microarrays. Results Human CTBs express retinoid metabolism and pathways-related genes, including Stra6, Lrat, Rdh5, Rdh10, Aldh1a1, Aldh1a2, Aldh1a3, Aldh8a1, Cyp26a1, and Cyp26b1, but not carotenoid metabolism genes, BCO1 and BCO2. Microarray analysis of placental gene expression profile revealed a total of 872 and 756 differentially expressed genes, respectively, compared to the control. Gene set enrichment analysis and functional annotation clustering was performed to characterize the genes differentially expressed in either β-carotene or lycopene-treated HTR-8/SVneo cells. Many known retinoid metabolism related genes and genes involved in regulation of retinoid signaling were found, and the expression profiles of these genes were markedly different in response to β-carotene treatments. Finally, the qRT-PCR and microarray analysis results showed similar gene expression patterns of carotenoid/retinoid metabolism and pathways. Conclusions These findings suggest that placental expression of genes involved in retinoid metabolism and transport in trophoblasts is critical for regulating retinoid homeostasis during placental and fetal development. Carotenoid exposure in early placental development, significantly modify the placenta gene expression related to retinoid pathways and maternal to fetal transfer. Funding Sources NIH HD421174.

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