scholarly journals Identification of a Novel Methylated Gene in Nasopharyngeal Carcinoma: TTC40

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Wajdi Ayadi ◽  
Nesrine Allaya ◽  
Hanèn Frikha ◽  
Emna Trigui ◽  
Abdelmajid Khabir ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Mrna Expression ◽  
Cpg Island ◽  
Repetitive Sequences ◽  
Aberrant Methylation ◽  
Sequencing Analysis ◽  
Reverse Transcription Pcr ◽  
Specific Pcr ◽  
Tetratricopeptide Repeat Domain ◽  
Arbitrarily Primed Pcr

To further explore the epigenetic changes in nasopharyngeal carcinoma (NPC), methylation-sensitive arbitrarily primed PCR was performed on NPC biopsies and nontumor nasopharyngeal samples. We have shown mainly two DNA fragments that appeared to be differentially methylated in NPCs versus nontumors. The first, defined as hypermethylated, corresponds to a CpG island at the 5′-end of the tetratricopeptide repeat domain 40 (TTC40) gene, whereas the second, defined as hypo-methylated, is located on repetitive sequences at chromosomes 16p11.1 and 13.1. Thereafter, the epigenetic alteration on the 5′-TTC40 gene was confirmed by methylation-specific PCR, showing a significant aberrant methylation in NPCs, compared to nontumors. In addition, the bisulfite sequencing analysis has shown a very high density of methylated cytosines in C15, C17, and X666 NPC xenografts. To assess whether TTC40 gene is silenced by aberrant methylation, we examined the gene expression by reverse transcription-PCR. Our analysis showed that the mRNA expression was significantly lower in tumors than in nontumors, which is associated with 5′-TTC40 gene hypermethylation. In conclusion, we found that the 5′-TTC40 gene is frequently methylated and is associated with the loss of mRNA expression in NPCs. Hypermethylation of 5′-TTC40 gene might play a role in NPC development; nevertheless, other studies are needed.

scholarly journals Inhibition of DNA methylation increases follistatin expression and secretion in the human adrenocortical cell line NCI-H295R

2006 ◽  
Vol 188 (2) ◽  
pp. 305-310 ◽  
Author(s):  
Pauliina Utriainen ◽  
Jianqi Liu ◽  
Tiina Kuulasmaa ◽  
Raimo Voutilainen
Keyword(s):  
Dna Methylation ◽  
Mrna Expression ◽  
Aberrant Methylation ◽  
Adrenocortical Cells ◽  
Adrenocortical Tumors ◽  
Specific Pcr ◽  
Methylation Inhibitor ◽  
Peptide Secretion ◽  
Dose Dependent Increase ◽  
Dose Dependent

Activin affects adrenocortical steroidogenesis and increases apoptosis, while follistatin (FS) acts as an activin antagonist by binding to activin, preventing attachment to its receptors. The regulation of FS expression in the adrenal cortex is poorly understood. Adrenocortical tumors often display aberrant methylation. In the present study, we investigated the effect of DNA methylation on FS mRNA expression and peptide secretion in adrenocortical cells. We treated human NCI-H295R adrenocortical cells with the methylation inhibitor 5-Aza-2′deoxycytidine (Azad; 0.1–100 μM for 1, 4 or 7 days) and measured FS mRNA expression by Northern blot and quantitative real time RT-PCR analyses as well as FS secretion by specific ELISA. Methylation-specific PCR showed decreased methylation in the FS promoter region after Azad treatment. A significant (P < 0.05) time- and dose-dependent increase in FS mRNA expression (up to 4.6-fold) and peptide secretion (up to 17.1-fold) was detected after Azad treatment. We conclude that FS gene expression and peptide secretion in NCI-H295R adrenocortical cells are regulated by DNA methylation. Thus, variable methylation in different adrenocortical tumors may influence activin bioactivity and its consequences in steroidogenesis and cell proliferation/apoptosis.

Aberrant methylation of ATG2B , ATG4D , ATG9A and ATG9B CpG island promoter is associated with decreased mRNA expression in sporadic breast carcinoma

Gene ◽  
2016 ◽  
Vol 590 (2) ◽  
pp. 285-292 ◽  
Author(s):  
Xuemei Zhang ◽  
Chuan Li ◽  
Da Wang ◽  
Qu Chen ◽  
Chang-Long Li ◽  
...  
Keyword(s):  
Breast Carcinoma ◽  
Mrna Expression ◽  
Cpg Island ◽  
Aberrant Methylation ◽  
Sporadic Breast Carcinoma

scholarly journals MicroRNA-135a Inhibits Nasopharyngeal Carcinoma Cell Proliferation Through Targeting Interleukin-17

2018 ◽  
Vol 46 (6) ◽  
pp. 2232-2238 ◽  
Author(s):  
Li-Xin Wang ◽  
Zhao-Peng Kang ◽  
Zhi-Chao Yang ◽  
Rui-Xia Ma ◽  
Yan Tan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nasopharyngeal Carcinoma ◽  
Mrna Expression ◽  
Normal Subjects ◽  
Significant Inhibition ◽  
Enzyme Linked Immunosorbent Assay ◽  
Epithelial Cell Line ◽  
Interleukin 17 ◽  
Reverse Transcription Pcr ◽  
Tnf Α

Background/Aims: The objective of this study was to investigate the potential role of IL-17 in the development of nasopharyngeal carcinoma (NPC) and to screen microRNAs (miRNAs) that potentially target IL-17 in NPC cells. Methods: Blood was collected from NPC patients and normal subjects, and plasma IL-17 concentration was quantified by enzyme-linked immunosorbent assay. An immortalized normal human nasopharyngeal epithelial cell line, NP69, was treated with or without human IL-17 (15 ng/mL) for various times, and expression of IL-1ß, IL-6, IL-12, and TNF-α mRNA was assessed by real-time reverse transcription PCR. The candidate miRNAs that potentially target IL-17 were predicted by a bioinformatics strategy. The selected miR-135a mimic was transfected into primary NPC cells, and cell proliferation was assessed by MTT assay. Results: The concentration of plasma IL-17 was significantly higher in the NPC patients (92.5 ± 7.3 pg/mL) than in the control subjects (56.8 ± 2.9 pg/mL). In response to IL-17 treatment, the mRNA expression of IL-1ß and IL-6 was significantly upregulated and reached a peak at 12 h, followed by a slight decrease at 24 h, while the mRNA expression of IL-12 and TNF-α was significantly upregulated at 12 h and remained high even at 48 h after exposure to IL-17. Moreover, miR-135a specifically targets IL-17 and was dramatically downregulated in NPC cells compared with NP69 cells. Transfection of exogenous miR-135a mimic resulted in significant suppression of IL-17 secretion and subsequent inhibition of NPC cell proliferation. Conclusions: Blood IL-17 was significantly higher in NPC patients compared with normal subjects. Expression of miR-135a in the cancer cells isolated from nasopharyngeal tumors was significantly lower than that in NP69 cells, and suppression of IL-17 by miR-135a mimic resulted in significant inhibition of NPC cell proliferation. These findings suggested that downregulation of miR-135a may contribute to the development of NPC via the mechanism of IL-17 stimulation of proinflammatory cytokine expression.

scholarly journals B cell-specific GPR55 deficiency promotes atherosclerosis

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
R Guillamat-Prats ◽  
D Hering ◽  
M Rami ◽  
C Haerdtner ◽  
L Bindila ◽  
...  
Keyword(s):  
Body Weight ◽  
B Cells ◽  
B Cell ◽  
Mrna Expression ◽  
Cell Function ◽  
Metabolic Effects ◽  
Atherosclerotic Plaques ◽  
Funding Source ◽  
Sequencing Analysis ◽  
B Cell Function

Abstract Background Atherosclerosis is accompanied by an imbalance between resolving and pro-inflammatory lipid mediators. Targeting lipid signaling pathways might offer a new anti-inflammatory therapy for improving the clinical outcome in cardiovascular disease patients. We considered lysophosphatidylinositol (LPI) and its receptor G protein-coupled receptor (GPR)55 as a potential modulator of atherosclerosis. Its role in regulating atherosclerosis and B cell function is unknown. Hypothesis We assessed the hypothesis that GPR55 signaling causally affects atherosclerosis and whether it has a specific role in regulating B cell function in this disease. Methods Atherosclerotic plaques were compared between apolipoprotein E deficient (ApoE−/−) and ApoE−/−Gpr55−/− mice after 4 to 16 weeks Western Diet (WD; 0.15% cholesterol; n=12–15 per group). To specifically test the role of B cell GPR55 in atherosclerosis, we generated mixed chimeras by lethally irradiating low density lipoprotein receptor deficient (Ldlr−/−) mice and reconstituting with a mixture of μMT and wildtype (control) or μMT and Gpr55−/− bone marrow cells. Circulating B cells were sorted and bulk RNA sequencing analysis was performed. We performed lipid and immunostainings of murine aortic root plaques, qPCR and ELISA of tissue lysates, as well as multiplex analysis of plasma immunoglobulins. Leukocyte plasma and tissue counts were determined by flow cytometry. Results GPR55 expression in mouse and human atherosclerotic plaques was detected by immunostaining. Furthermore, we confirmed murine Gpr55 mRNA expression on sorted circulating B220+B cells via qPCR, which was higher compared to CD3+ T cells, while CD11+ myeloid cells as well as NK cells had only low Gpr55 mRNA levels. ApoE−/−Gpr55−/− mice had significantly larger plaques after 4&16 weeks WD compared to ApoE−/− controls, with more pronounced body weight increases and higher cholesterol levels at the 16 weeks WD time point. In addition, global Gpr55 deficiency resulted in enhanced aortic pro-inflammatory cytokine mRNA expression (IL-1β, IL-6, TNFα) and a massively upregulated IgG1 plasma levels and increased percentages of splenic germinal center and plasma cells. B-cell RNA-seq analysis showed 460 differential expressed regulated genes in the ApoE−/−Gpr55−/− compared to ApoE−/−. The main pathways affected were calcium ion transport, immunoglobulin production, negative regulation of phosphorylation, and cellular component morphogenesis, suggesting a dsysregulation of B cell function. B cell specific Gpr55 deficiency blunted the metabolic effects on body weight and cholesterol, but still translated in larger atherosclerotic plaques and elevated plasma IgG levels compared to the respective controls. Conclusion Both global and B cell-restricted Gpr55 deficiency promotes atherosclerosis and is associated with a more pro-inflammatory phenotype. Our findings suggest a novel role for GPR55 in regulating B cell development and function. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)

Galectin-3 is modulated in pancreatic cancer cells under hypoxia and nutrient deprivation

2020 ◽  
Vol 401 (10) ◽  
pp. 1153-1165 ◽  
Author(s):  
Antônio F. da Silva Filho ◽  
Lucas B. Tavares ◽  
Maira G. R. Pitta ◽  
Eduardo I. C. Beltrão ◽  
Moacyr J. B. M. Rêgo
Keyword(s):  
Pancreatic Cancer ◽  
Cell Death ◽  
Mrna Expression ◽  
Cancer Cells ◽  
Ductal Adenocarcinoma ◽  
Reverse Transcription Pcr ◽  
Pancreatic Cancer Cells ◽  
Through Flow ◽  
Galectin 3

AbstractPancreatic ductal adenocarcinoma is one of the most aggressive tumors with a microenvironment marked by hypoxia and starvation. Galectin-3 has been evaluated in solid tumors and seems to present both pro/anti-tumor effects. So, this study aims to characterize the expression of Galectin-3 from pancreatic tumor cells and analyze its influence for cell survive and motility in mimetic microenvironment. For this, cell cycle and cell death were accessed through flow cytometry. Characterization of inside and outside Galectin-3 was performed through Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), immunofluorescence, Western blot, and ELISA. Consequences of Galectin-3 extracellular inhibition were investigated using cell death and scratch assays. PANC-1 showed increased Galectin-3 mRNA expression when cultivated in hypoxia for 24 and 48 h. After 24 h in simultaneously hypoxic/deprived incubation, PANC-1 shows increased Galectin-3 protein and secreted levels. For Mia PaCa-2, cultivation in deprivation was determinant for the increasing in Galectin-3 mRNA expression. When cultivated in simultaneously hypoxic/deprived condition, Mia PaCa-2 also presented increasing for the Galectin-3 secreted levels. Treatment of PANC-1 cells with lactose increased the death rate when cells were incubated simultaneously hypoxic/deprived condition. Therefore, it is possible to conclude that the microenvironmental conditions modulate the Galectin-3 expression on the transcriptional and translational levels for pancreatic cancer cells.

scholarly journals Molecular Characterization of Ciliate Diversity in Stream Biofilms

2008 ◽  
Vol 74 (6) ◽  
pp. 1740-1747 ◽  
Author(s):  
Andrew Dopheide ◽  
Gavin Lear ◽  
Rebecca Stott ◽  
Gillian Lewis
Keyword(s):  
18S Rrna ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Sequence Diversity ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Specific Pcr ◽  
Stream Biofilms

ABSTRACT Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

scholarly journals Genome-Wide Analysis of Glucocorticoid-Responsive Transcripts in the Hypothalamic Paraventricular Region of Male Rats

Endocrinology ◽  
2018 ◽  
Vol 160 (1) ◽  
pp. 38-54 ◽  
Author(s):  
Keiichi Itoi ◽  
Ikuko Motoike ◽  
Ying Liu ◽  
Sam Clokie ◽  
Yasumasa Iwasaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Receptor Gene ◽  
Male Rats ◽  
Regulatory Mechanisms ◽  
High Dose ◽  
Sequencing Analysis ◽  
Reverse Transcription Pcr ◽  
Genome Wide ◽  
A Genome

Abstract Glucocorticoids (GCs) are essential for stress adaptation, acting centrally and in the periphery. Corticotropin-releasing factor (CRF), a major regulator of adrenal GC synthesis, is produced in the paraventricular nucleus of the hypothalamus (PVH), which contains multiple neuroendocrine and preautonomic neurons. GCs may be involved in diverse regulatory mechanisms in the PVH, but the target genes of GCs are largely unexplored except for the CRF gene (Crh), a well-known target for GC negative feedback. Using a genome-wide RNA-sequencing analysis, we identified transcripts that changed in response to either high-dose corticosterone (Cort) exposure for 12 days (12-day high Cort), corticoid deprivation for 7 days (7-day ADX), or acute Cort administration. Among others, canonical GC target genes were upregulated prominently by 12-day high Cort. Crh was upregulated or downregulated most prominently by either 7-day ADX or 12-day high Cort, emphasizing the recognized feedback effects of GC on the hypothalamic-pituitary-adrenal (HPA) axis. Concomitant changes in vasopressin and apelin receptor gene expression are likely to contribute to HPA repression. In keeping with the pleotropic cellular actions of GCs, 7-day ADX downregulated numerous genes of a broad functional spectrum. The transcriptome response signature differed markedly between acute Cort injection and 12-day high Cort. Remarkably, six immediate early genes were upregulated 1 hour after Cort injection, which was confirmed by quantitative reverse transcription PCR and semiquantitative in situ hybridization. This study may provide a useful database for studying the regulatory mechanisms of GC-dependent gene expression and repression in the PVH.

scholarly journals A 5-Methylcytosine Site of Growth Differentiation Factor 9 (GDF9) Gene Affects Its Tissue-Specific Expression in Sheep

Animals ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 200
Author(s):  
Zhangyuan Pan ◽  
Xiangyu Wang ◽  
Ran Di ◽  
Qiuyue Liu ◽  
Wenping Hu ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Methylation Level ◽  
Cpg Island ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Directed Mutation ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9 ◽  
Basal Transcriptional Activity

Growth differentiation factor 9 (GDF9) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine GDF9 in different tissues by real-time PCR. GDF9 exhibits significantly higher levels of expression (p < 0.01) in the ovary, relative to other tissues, indicating that its expression is tissue specific. To explore the regulatory mechanism of this tissue-specific expression, the methylation level of one CpG island (−1453 to −1854) of GDF9 promoter in ovary and heart was determined. In this region (−1987 to −1750), only the mC-4 site was present in the Sp4 binding site showed differential methylation between the heart and ovary; with increased (p < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with GDF9 mRNA expression (R = −0.75, p = 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of GDF9. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (p < 0.05) basal transcriptional activity of GDF9 promoter in oocytes. These results imply that methylation of GDF9 promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the GDF9 promoter region in sheep, thereby regulating GDF9 expression and resulting in a tissue-specific expression.

scholarly journals Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting cuct

2013 ◽  
Vol 305 (7) ◽  
pp. F1006-F1013 ◽  
Author(s):  
Zhiyuan Yu ◽  
Qun Kong ◽  
Bruce C. Kone
Keyword(s):  
Promoter Methylation ◽  
Dna Methyltransferase ◽  
Cpg Island ◽  
Collecting Duct ◽  
Epigenetic Mechanism ◽  
Sequencing Analysis ◽  
Control Of Gene Expression ◽  
Bisulfite Treatment ◽  
High Level ◽  
Interfering Rna

Aldosterone increases tubular Na+ absorption largely by increasing α-epithelial Na+ channel (αENaC) transcription in collecting duct principal cells. How aldosterone reprograms basal αENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethylation in the epigenetic control of basal and aldosterone-stimulated αENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines in a CpG island near the transcription start site of the αENaC promoter. 5-Aza-CdR treatment or small interfering RNA-mediated knockdown of DNMT3b or methyl-CpG-binding domain protein (MBD)-4 derepressed basal αENaC transcription, indicating that promoter methylation suppresses basal αENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet)2 at the αENaC promoter, consistent with active demethylation. 5-Aza-CdR mimicked aldosterone by enhancing Sp1 binding to the αENaC promoter. We conclude that DNMT3b- and MBD4-dependent methylation of the αENaC promoter limits basal αENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates the dispersal of DNMT3b and recruitment of Tet2 to demethylate the αENaC promoter to induce αENaC transcription. These results disclose a novel epigenetic mechanism for the control of basal and aldosterone-induced αENaC transcription that adds to previously described epigenetic controls exerted by histone modifications.

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