scholarly journals Association between matrix Gla protein and ulcerative colitis according to DNA microarray data

2019 ◽  
Vol 8 (1) ◽  
pp. 66-75 ◽  
Author(s):  
Xu-Yang Dong ◽  
Mei-Xu Wu ◽  
Hui-Min Zhang ◽  
Hong Lyu ◽  
Jia-Ming Qian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ulcerative Colitis ◽  
Binding Site ◽  
Dna Microarray ◽  
Bioinformatics Analysis ◽  
Matrix Gla Protein ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Healthy Controls ◽  
Microarray Profiling

Abstract Background Matrix Gla protein (MGP) is a secreted protein contributed to the immunomodulatory functions of mesenchymal stromal cells. Microarray profiling found a significantly higher expression level of the extracellular matrix gene MGP in patients with ulcerative colitis (UC). However, little is known about the role of MGP in UC and its upstream signaling regulation. This study aimed to identify the expression of MGP in UC and its upstream regulator mechanism. Methods Colonic mucosa biopsies were obtained from patients with UC and healthy controls. DNA microarray profiling was used to explore underlying genes correlating with UC development. Mice were fed with water containing different concentrations of dextran sodium sulfate (DSS) to induce an experimental colitis model. Colonic tissues were collected and evaluated using immunohistochemistry, immunoblot, real-time polymerase chain reaction, and chromatin immunoprecipitation assay. Bioinformatics analysis was performed to identify candidate MGP gene-promoter sequence and transcription-initiation sites. Luciferase-reporter gene assay was conducted to examine the potential transcription factor of MGP gene expression. Results The expression of MGP was significantly increased in colonic tissues from UC patients and DSS-induced colitis models, and was positively correlated with disease severity. Bioinformatics analysis showed a conserved binding site for Egr-1 in the upstream region of human MGP gene. The significantly higher level of Egr-1 gene expression was found in UC patients than in healthy controls. The activity of luciferase was significantly enhanced in the Egr-1 expression plasmid co-transfected group than in the control group and was further inhibited when co-transfected with the Egr-1 binding-site mutated MGP promoter. Conclusions Up-regulated expression of MGP was found in UC patients and DSS-induced colitis. The expression of MGP can be regulated by Egr-1.

P0078INVESTIGATION THE VARIANTS AT THE BINDING SITE OF INFLAMMATORY TRANSCRIPTION FACTOR IN PATIENTS WITH ESRD

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
SUI-LUNG SU
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Disease Process ◽  
Binding Activity ◽  
Luciferase Reporter ◽  
Healthy Controls ◽  
Chip Assay ◽  
Potential Binding ◽  
Stage Renal Disease ◽  
End Stage

Abstract Background and Aims Inflammation is an important factor for enhancing the disease process from chronic kidney disease (CKD) to end-stage renal disease (ESRD). Nuclear factor-kappa B (NF-κB) is a transcription factor that regulates the expression of genes involved in inflammation. We investigated the potential association with the gene polymorphism of transcription factor binding site of NF-κB in ESRD patients. Method We used the Taiwan Biobank database, University of California, Santa Cruz, reference genome, chromatin immunoprecipitation sequencing database to find the SNPs at potential binding sites of NF-κB. In addition, we performed a case–control study and genotyped 847 patients with ESRD and 846 healthy controls at Tri-Service-General-Hospital from 2015 to 2016. Further we used ChIP-assay and Luciferase reporter assay to identify the binding activity at different genotype. Results Results of biometrics screening in the databases revealed 15 SNPs with the potential binding site of NF-κB. Genotype distributions of rs9395890 were significantly different in ESRD cases and healthy controls (P = 0.032). In the Dominant model, rs9395890 with T allele had a higher risk of ESRD (P = 0.032; odds ratio [OR] = 1.32, 95% confidence interval [CI] = 0.99–1.76). The ChIP assay reveals that around 1.49 times enrichment of NF-κB of the variant type TT when compared to that of the wild type GG in the rs9395890 (P<0.027; TT=3.20±0.16, GT=2.81±0.20, GG=1.71±0.18,) and the luciferase activity curve showed T allele was higher than G allele. Conclusion In conclusion, we demonstrate that rs9395890 may be associated with ESRD in the Taiwanese population.

A Targeted Resequencing Study Allows the Detection of a Common Polymorphism At Mirna-223 Binding Site in 3′Untranslated Region Which Deregulates HSP90B1 Expression in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1776-1776
Author(s):  
Ana E Rodríguez ◽  
Dalia Qwaider ◽  
Rocío Benito ◽  
Irena Misiewicz-Krzeminska ◽  
María Hernández-Sánchez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Cell Line ◽  
B Lymphocytes ◽  
Prognostic Significance ◽  
Mrna Levels ◽  
Healthy Controls ◽  
Wild Type ◽  
Single Nucleotide ◽  
Custom Made

Abstract Abstract 1776 Array-based sequence capture (Roche NimbleGen) followed by next-generation sequencing (Roche GS FLX Titanium sequencing platform) was used to analyze genetic variations in 93 genes relevant in CLL and two chromosomal regions: 13q14.3 and 17p13.1. CD19+ cells from 4 patients with CLL and 4 patients with other hematological malignancies (used as controls) were studied. A custom-made data analysis pipeline was used to annotate detected variants, including known single-nucleotide polymorphisms (SNPs), amino acid consequences, genomic location and miRNA binding sites. The enrichment assay followed by NGS allowed the detection of over 1600 variations/sample (median 1721, range 1618–1823). All putative variants were first compared with published single nucleotide polymorphism (SNP) data (dbSNP build 130) and most of the variants detected were identified as known SNPs. Overall, 10% of variants detected in each sample were variations not previously described. Interestingly, a 4bp insertion/deletion polymorphism (rs2307842) in the 3′UTR of HSP90B1, target site for miR-223, was detected. There is an increasing evidence suggesting that SNPs in the 3′UTR targeted by miRNAs (known as miRSNPs) are associated with diseases by affecting gene expression. We hypothesized that this ‘GACT’ deletion disrupts the binding site for miR-223 thereby increasing the translation of HSP90B1 and we confirmed that miR-223 regulates HSP90B1 expression by 3′UTR reporter assays. The relative luciferase activity of the construct with wild-type 3′UTR (WT-3′UTR) was significantly repressed by 31% following miR-223 transfection (p<0.05). However, the presence of rs2307842 polymorphism in 3′UTR of HSP90B1 (VAR-3′UTR) abolished this suppression, suggesting that miR-223 directly binds to this site. We also validated HSP90B1 as a target gene of miR223 by transfecting MM1S and H929 cell lines with miR-223/NC mimics and then measuring HSP90B1 expression by semi-quantitative PCR and Western blot. Exogenous expression of miR-223 downregulated the expression levels of HSP90B1 in H929 cell line (WT-3′UTR) in both mRNA (p<0.05) and protein levels. By contrast, HSP90B1 expression was not modified in MM1S cell line (VAR-3′UTR). To evaluate the clinical impact of HSP90B1 3′UTR polymorphism, we expanded the study to 109 additional patients with CLL and 32 healthy controls. Sequencing of the HSP90B1 3′UTR region was performed by pyrosequencing (PyroMark Q24 system, Qiagen). The rs2307842 was detected in 27/109 (25%) patients and 8/32 (25%) healthy controls, as expected. Overall, we did not find any significant relationship between rs2307842 and clinical characteristics of CLL patients. To gain insight into its influence on gene expression, we measured HSP90B1 mRNA levels in paired samples (tumoral and normal) from CLL patients with rs2307842 (VAR-CLLs, n=6) and wild-type (WT-CLLs, n=12). PCR results showed that B lymphocytes (tumoral fraction) from VAR-CLLs have a higher expression of HSP90B1 than B lymphocytes from WT-CLLs (P=0.002) and also from the normal cells of the same patients (VAR-CLLs) (P=0.011). However, in WT-CLLs, no changes in mRNA expression were observed between tumor and normal fractions, being HSP90B1 mRNA levels similar to the normal fraction of VAR-CLLs. Thus, rs2307842 determined HSP90B1 overexpression only in the tumor fraction of the CLL patients with the polymorphism. Downregulation of miR-223 has prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients, and no target has been proposed or validated for miR-223 in CLL until date. Thus, this work provides novel information about how the downregulation of miR-223 can be determining the poor outcome of CLL patients, maybe through upregulation of HSP90B1 expression. Disclosures: No relevant conflicts of interest to declare.

Interleukin 1β inhibits interleukin 6–mediated rat γ fibrinogen gene expression

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3466-3472 ◽  
Author(s):  
Zhixin Zhang ◽  
Gerald M. Fuller
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Intracellular Signaling ◽  
Rat Hepatocytes ◽  
Nuclear Factor Κb ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Acute Phase Reactants ◽  
Functional Analyses ◽  
Hepatic Genes

Interleukin (IL)-1β and IL-6 are the 2 major inducers of a group of hepatic genes during acute inflammation; however, each cytokine uses different intracellular signaling molecules. In most instances, the 2 cytokines interact positively to enhance hepatic gene expression, but in one class of acute-phase reactants, which includes fibrinogen, IL-1β exerts a transient inhibitory effect over the IL-6 stimulatory signal. This study explored the effects of IL-1β/nuclear factor κB (NF-κB) and IL-6/signal transducer and activator of transcription 3 (STAT3) combinatory signaling on the transcriptional regulation of the rat γ fibrinogen gene. Northern blot and functional analyses employing luciferase reporter constructs driven by the rat γ fibrinogen promoter demonstrated that IL-1β inhibited the IL-6-mediated transcription of this gene. Exposing primary rat hepatocytes to IL-1β had no effect on IL-6-mediated STAT3 activation; instead, IL-1β-activated NF-κB associated with 2 IL-6 responsive elements (STAT3 binding site) on the rat γ fibrinogen promoter and blocked STAT3 binding to these regions. The competitive binding of NF-κB and STAT3 on the overlapping binding site provides a mechanism for the inhibition by IL-1β of the IL-6-mediated transactivation of rat γ fibrinogen.

The role of NeuroD1 in the upregulation of SMYD3 by HBx.

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 183-183
Author(s):  
Junyao Xu ◽  
Qingqi Hong ◽  
Chuanchao He ◽  
Jie Wang
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Promoter Region ◽  
Gene Transfection ◽  
Histone Methyltransferase ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Cis Element ◽  
E Box

183 Background: SET and MYND Domain-Containing Protein 3 (SMYD3) is frequently overexpressed in hepatocellular carcinoma (HCC) exhibiting increased malignant phenotypes. It has also been known that the hepatitis B virus x protein (HBx) is strongly associated with HCC development and progression. Although overexpression of both proteins is related to HCC, the relationship between the two has not been well studied. Methods: Immunohistochemical staining was used to detect the expression of HBx and SMYD3 in HCC tumor tissues. HBx gene transfection, RNAi, and histone methyltransferase(H3-K4) activity assay were performed to reveal the transcrpitionally activation of HBx on functional SMYD3 gene expression. Chromatin immunoprecipitation (ChIP), Co-immunoprecipitation (Co-IP), Electrophoretic mobility shift assay (EMSA) were applied to investigate the underlying mechanism. Dual-luciferase reporter assay was used to search for the HBx responsive cis-element of SMYD3 gene. Results: Immunohistochemistry identified the positive correlation between HBx and SMYD3 expression in 42 HCC tissues. Up-regulation of HBx on SMYD3 expression was validated through experiments involving overexpression or knock-down of HBx in different HCC cell lines. And up-regulated SMYD3 is functionally active as histone methyltransferase. Next we found that HBx transcriptionally regulated SMYD3 gene expression by interacting with RNA polymerase IIand altering its binding site to a proximal promoter region(SD2) from a distant promoter region(SD6) of SMYD3. Truncated and mutant reporter assays revealed that the cis-element mapped in -178~-203bp in SMYD3 promotor is responsive for HBx-transactivation. And this 25bp cis-element contains a E-box 3 unit, which is a binding site for the transcriptional factor Neurogenic differentiation 1(NeuroD1). EMSA and Chip showed that HBx increased NeuroD1 binding to SMYD3 proximal promotor, however transcient expression of antisense NeuroD1 abolished HBx-induced SMYD3 expression. Conclusions: HBx transcriptionally up-regulates SMYD3 and that this process is mediated by NeuroD1 through binding to the E-box 3 site of SMYD3 promotor.

Gene Expression Signature and the Prediction of Ulcerative Colitis–Associated Colorectal Cancer by DNA Microarray

2007 ◽  
Vol 13 (2) ◽  
pp. 415-420 ◽  
Author(s):  
Toshiaki Watanabe ◽  
Takashi Kobunai ◽  
Etsuko Toda ◽  
Takamitsu Kanazawa ◽  
Yoshihiro Kazama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
Ulcerative Colitis ◽  
Dna Microarray ◽  
Gene Expression Signature ◽  
Expression Signature

scholarly journals Corrigendum to: Association between matrix Gla protein and ulcerative colitis according to DNA microarray data

2020 ◽  
Vol 8 (3) ◽  
pp. 252-252
Author(s):  
Xu-Yang Dong ◽  
Mei-Xu Wu ◽  
Hui-Min Zhang ◽  
Hong Lyu ◽  
Jia-Ming Qian ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Dna Microarray ◽  
Microarray Data ◽  
Matrix Gla Protein ◽  
Dna Microarray Data

Interleukin 1β inhibits interleukin 6–mediated rat γ fibrinogen gene expression

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3466-3472 ◽  
Author(s):  
Zhixin Zhang ◽  
Gerald M. Fuller
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Intracellular Signaling ◽  
Rat Hepatocytes ◽  
Nuclear Factor Κb ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Acute Phase Reactants ◽  
Functional Analyses ◽  
Hepatic Genes

Abstract Interleukin (IL)-1β and IL-6 are the 2 major inducers of a group of hepatic genes during acute inflammation; however, each cytokine uses different intracellular signaling molecules. In most instances, the 2 cytokines interact positively to enhance hepatic gene expression, but in one class of acute-phase reactants, which includes fibrinogen, IL-1β exerts a transient inhibitory effect over the IL-6 stimulatory signal. This study explored the effects of IL-1β/nuclear factor κB (NF-κB) and IL-6/signal transducer and activator of transcription 3 (STAT3) combinatory signaling on the transcriptional regulation of the rat γ fibrinogen gene. Northern blot and functional analyses employing luciferase reporter constructs driven by the rat γ fibrinogen promoter demonstrated that IL-1β inhibited the IL-6-mediated transcription of this gene. Exposing primary rat hepatocytes to IL-1β had no effect on IL-6-mediated STAT3 activation; instead, IL-1β-activated NF-κB associated with 2 IL-6 responsive elements (STAT3 binding site) on the rat γ fibrinogen promoter and blocked STAT3 binding to these regions. The competitive binding of NF-κB and STAT3 on the overlapping binding site provides a mechanism for the inhibition by IL-1β of the IL-6-mediated transactivation of rat γ fibrinogen.

Post-Transcriptional Regulation of Cardiac Sarcomere Protein Titin Through 3’ Untranslated Regions

2013 ◽  
Vol 9 ◽  
Author(s):  
Tara A Shrout
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Striated Muscle ◽  
Binding Capacity ◽  
Structural Features ◽  
Neonatal Rat ◽  
Regulatory Control ◽  
Untranslated Regions ◽  
Luciferase Reporter ◽  
Regulatory Effects

Titin is the largest known protein in the human body, and forms the backbone of all striated muscle sarcomeres. The elastic nature of titin is an important component of muscle compliance and functionality. A significant amount of energy is expended to synthesize titin, thus we postulate that titin gene expression is under strict regulatory control in order to conserve cellular resources. In general, gene expression is mediated in part by post-transcriptional control elements located within the 5’ and 3’ untranslated regions (UTRs) of mature mRNA. The 3’UTR in particular contains structural features that affect binding capacity to other RNA components, such as MicroRNA, which control mRNA localization, translation, and degradation. The degree and significance of the regulatory effects mediated by two determined variants of titin’s 3’ UTR were evaluated in Neonatal Rat Ventricular Myocyte and Human Embryonic Kidney cell lines. Recombinant plasmids to transfect these cells lines were engineered by insertion of the variant titin 3’UTR 431- and 1047-base pairs sequences into luciferase reporter vectors. Expression due to an unaltered reporter vector served as the control. Quantitative changes in luciferase activity due to the recombinants proportionally represented the effect titin’s respective 3’UTR conferred on downstream post-transcriptional expression relative to the control. The effect due to titin’s shorter 3’UTR sequence was inconclusive; however, results illustrated that titin’s longer 3’UTR sequence caused a 35 percent decrease in protein expression. Secondary structural analysis of the two sequences revealed differential folding patterns that affect the stability and degree of MicroRNA-binding within titin’s variant 3’UTR sequences.

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