scholarly journals Aberrant DNA Methylation-Mediated FOXF2 Dysregulation Is a Prognostic Risk Factor for Gastric Cancer

2021 ◽  
Vol 8 ◽  
Author(s):  
Cheng Zhang ◽  
Yong-Zhi Li ◽  
Dong-Qiu Dai
Keyword(s):  
Gastric Cancer ◽  
Dna Methylation ◽  
Western Blot ◽  
Methylation Level ◽  
Promoter Region ◽  
Target Genes ◽  
Risk Group ◽  
Rna Seq ◽  
Qrt Pcr ◽  
Aberrant Dna Methylation

Background: The prognosis of gastric cancer (GC) patients is poor. The effect of aberrant DNA methylation on FOXF2 expression and the prognostic role of FOXF2 methylation in GC have not yet been identified.Methods: The RNA-Seq and gene methylation HM450 profile data were used for analyzing FOXF2 expression in GC and its association with methylation level. Bisulfite sequencing PCR (BSP) was performed to measure the methylation level of the FOXF2 promoter region in GC cell lines and normal GES-1 cells. The cells were treated with the demethylation reagent 5-Aza-dC, and the mRNA and protein expression levels of FOXF2 were then measured by qRT-PCR and western blot assays. The risk score system from SurvivalMeth was calculated by integrating the methylation level of the cg locus and the corresponding Cox regression coefficient.Results: FOXF2 was significantly downregulated in GC cells and tissues. On the basis of RNA-Seq and Illumina methylation 450 data, FOXF2 expression was significantly negatively correlated with the FOXF2 methylation level (Pearson’s R = −0.42, p < 2.2e−16). The FOXF2 methylation level in the high FOXF2 expression group was lower than that in the low FOXF2 expression group. The BSP assay indicated that the methylation level of the FOXF2 promoter region in GC cell lines was higher than that in GES-1 cells. The qRT-PCR and western blot assay showed that FOXF2 mRNA and protein levels were increased in GC cells following treatment with 5-Aza-Dc. The methylation risk score model indicated that patients in the high risk group had poorer survival probability than those in the low risk group (HR = 1.84 (1.11–3.07) and p = 0.0068). FOXF2 also had a close transcriptional regulation network with four miRNAs and their corresponding target genes. Functional enrichment analysis of the target genes revealed that these genes were significantly related to several important signaling pathways.Conclusion: FOXF2 was downregulated due to aberrant DNA methylation in GC, and the degree of methylation in the promoter region of FOXF2 was related to the prognosis of patients. The FOXF2/miRNAs/target genes axis may play a vital biological regulation role in GC.

scholarly journals Association of Aberrant DNA Methylation Level in the CD4 and JAK-STAT-Pathway-Related Genes with Mastitis Indicator Traits in Chinese Holstein Dairy Cattle

Animals ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 65
Author(s):  
Tahir Usman ◽  
Nawab Ali ◽  
Yachun Wang ◽  
Ying Yu
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dairy Cattle ◽  
Methylation Level ◽  
Promoter Region ◽  
Promoter Regions ◽  
Cpg Sites ◽  
Cell Counts ◽  
Aberrant Dna Methylation ◽  
Stat Pathway

The present study was designed to evaluate the gene expression and DNA methylation level in the promoter region of the CD4 and the JAK-STAT-pathway-related genes. A total of 24 samples were deployed in the gene expression and 118 samples were used in the DNA methylation study. Student’s t-tests were used to analyze the gene expression and DNA methylation. The evaluation of DNA methylation in promoter regions of JAK2 and STAT5A revealed hypo-methylation levels of CpG sites and higher gene expression in cows diagnosed with mastitis as compared to the healthy control, and vice versa in those with CD4. DNA methylation was negatively correlated with gene expression in JAK2, STAT5A, and CD4 genes. Six, two, and four active transcription factors were identified on the CpG sites in the promoter regions of JAK2, STAT5A, and CD4 genes, respectively. Regarding correlation analysis, the DNA methylation levels of CD4 showed significantly higher positive correlations with somatic cell counts (p < 0.05). Findings of the current study inferred that aberrant DNA methylation in the CpG sites at the 1 kb promoter region in JAK2, STAT5A, and CD4 genes due to mastitis in cows can be used as potential epigenetic markers to estimate bovine mastitis susceptibility in dairy cattle.

scholarly journals Regulation of RUNX3 expression by DNA methylation in prostate cancer

2020 ◽  
Author(s):  
Xin Yang ◽  
Shumei Wang ◽  
Alimu Reheman
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Dna Methylation ◽  
Western Blot ◽  
Cell Lines ◽  
Methylation Level ◽  
Aberrant Methylation ◽  
Normal Prostate ◽  
Qrt Pcr ◽  
Its Gene

Abstract Background: Runt-related transcription factor 3 (RUNX3) is a developmental regulator, and methylation of the RUNX3 is significantly associated with the occurrence and development of carcinogenesis. Previous studies have identified an association of increased methylation level of RUNX3 in prostate cancer (PCa); however, the role and molecular mechanism underlying aberrant methylation of the RUNX3 gene in prostate tumorigenesis remain elusive. In this study, we will investigate the role of RUNX3 promoter methylation and its gene expression in PCa cells. Methods: The methylation of the RUNX3 in the promoter region in PCa cells was detected by bisulfite-sequencing PCR (BSP). Following treatment of the PCa cells with DNA methylation transferase inhibitor 5-AZA-2'-deoxycytidine (AZA), the effect on methylation level and expression of RUNX3 were analyzed by qRT-PCR, Western blot, and BSP assays. Furthermore, we investigated the effect of the demethylated RUNX3 on proliferation, cell cycle and apoptosis of PCa cells using CCK-8 and flow cytometry assays. Using the DNA methylation transferase (DNMT3b) knockout or overexpression models, the relationship between DNMT3b and RUNX3 methylation was further assessed by qRT-PCR, Western blot and methylation-specific PCR (MSP). Results: The results indicated that the methylation level of RUNX3 in PCa cell lines was significantly higher than that of normal prostate epithelial (RWPE-1) cells. Furthermore, treatment with AZA not only promoted the demethylation of RUNX3 but also restored the mRNA and protein expression of RUNX3, and the reactivation of expression of the later exhibited its anti-tumor effects through regulation of the cycle progression in PCa cells. Moreover, DNMT3b could regulate the expression level of RUNX3 by altering the DNA methylation of the RUNX3 in PCa cells. Conclusion: RUNX3 is hypermethylated in a panel of PCa cell lines; Inhibits DNA methylation of RUNX3 could restored its gene expression, which in turn induced its anti-cancer effects. Thus, RUNX3 may serve as a novel putative molecular target gene for PCa therapy.

scholarly journals Long-term Changes In Aberrant DNA Methylation And Gastritis After Helicobacter Pylori Eradication Focused On Metachronous Gastric Cancer

2021 ◽  
Author(s):  
Ikko Tanaka ◽  
Shoko Ono ◽  
Yoshiyuki Watanabe ◽  
Hiroyuki Yamamoto ◽  
Ritsuko Oikawa ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Dna Methylation ◽  
Helicobacter Pylori ◽  
Methylation Level ◽  
Aberrant Methylation ◽  
Long Term Changes ◽  
Metachronous Gastric Cancer ◽  
Aberrant Dna Methylation ◽  
H Pylori

Abstract BackgroundA persistently high methylation level in gastric mucosa after Helicobacter pylori (H. pylori) eradication is presumed to be a risk for metachronous gastric cancer (MGC); however, long-term changes in aberrant DNA methylation and histological gastritis have been unclear. Our aim was to examine changes in DNA methylation and histological gastritis according to the occurrence of MGC.MethodsSubjects were classified into 3 groups: 25 patients in whom metachronous gastric cancers occurred after the initial endoscopic resection (ER) for early gastric cancer and H. pylori eradication (MGC group), 17 patients in whom MGC did not occur for more than 5 years after initial ER and H. pylori eradication (non-MGC group) and 29 patients without a history of gastric cancer who succeeded in eradication more than 5 years ago (HP group). Aberrance of DNA methylation in 3 genes (miR-124a-3, EMX1, NKX6-1) and histological score of atrophy and intestinal metaplasia (IM) were evaluated using biopsy samples before and more than a mean of 5 years after H. pylori eradication.ResultsThe methylation level of miR-124a-3 in the HP group and non-MGC group and that of EMX1 in the HP group significantly decreased in the long term after eradication. In the MGC group, H. pylori eradication did not improve aberrant methylation, and the Z-score significantly increased. There were significant positive correlations between methylation levels in miR-124a-3 and EMX1 and histological findings after eradication.ConclusionsA persistently high methylation level after H. pylori eradication reflected precancerous mucosal conditions and led to long-term MGC.

scholarly journals DNA-Methylation-Mediated lncRNA HOXB-AS4 Promotes Gastric Cancer Progression Through Regulating miR-130a-5p/PKP4

2022 ◽  
Author(s):  
Xiaoyan Wang ◽  
Rong He ◽  
Yan Wang ◽  
Yunyun Liu ◽  
Yuxin Wang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Dna Methylation ◽  
In Situ Hybridization ◽  
Western Blot ◽  
Fluorescence In Situ Hybridization ◽  
Cancer Cells ◽  
Chromatin Immunoprecipitation ◽  
Qrt Pcr ◽  
Specific Pcr

Abstract Background：Gastric cancer (GC) is one of the most common cancer in the world, possessing the second leading cause of cancer-related mortality. Long noncoding RNAs (lncRNAs) have been shown to play important roles in tumorigenesis. However, the effect of lncRNA HOXB-AS4 in GC progression and the underlying mechanisms remain unknown. Methods：Firstly, the expression of lncRNA HOXB-AS4 in gastric cancer tissues and cancer cells was investigated according to GEPIA database and Real time fluorescence quantitative PCR(qRT-PCR). Then, MTT, clone formation, Transwell and Western blot were used to study the effects of overexpression or down-regulation of HOXB-AS4 on the proliferation, invasion and epithelial mesenchymal transformation of cancer cells. We further studied the molecular mechanism of HOXB-AS4 by fluorescence in situ hybridization, bioinformatics analysis, luciferase reporting, methylation specific PCR (MSP) and chromatin immunoprecipitation (chip).Results:In the study, the GEPIA database and quantitative Real-Time PCR (qRT-PCR) assay showed that HOXB-AS4 was upregulated in GC tissues and cells. Then, MTT, clone formation, transwell, and western blot assays suggested that overexpression of HOXB-AS4 increased cell proliferation, migration, and invasion, and regulated epithelial-mesenchymal transition (EMT) markers expression, while knockdown of HOXB-AS4 showed the opposite effect. Fluorescence in situ hybridization (FISH) assay found that HOXB-AS4 localized in the cytoplasm of the GSE-1 and AGS cells. Further mechanism experiments, including bioinformatics, luciferase reporter, qRT-PCR, and western blot assays showed that HOXB-AS4 sponged to miR-130a-5p to regulate the PKP4 expression. Knockdown of miR-130a-5p obliterated the effect of HOXB-AS4, which was further abolished by knockdown of PKP4 in vitro and in vivo. Methylation-specific PCR (MSP) and chromatin immunoprecipitation (CHIP) assay showed that overexpression of HOXB-AS4 in GC was mediated by SP1-dependent DNA methylation. Abnormal upregulation of lncRNA HOXB-AS4 contributed to GC progression, which was mediated by DNA methylation. The study clarified that DNA-methylation-mediated HOXB-AS4 played its role through miR-130a-5p/PKP4 axis.Conclusions: Our study provides new insights for the understanding of epigenetic regulation on lncRNA expression in GC, and indicates that HOXB-AS4 could be a biomarker of GC prognosis. Moreover, targeting HOXB-AS4 /miR-130a-5p/PKP4 axis might be a promising strategy to treat GC.

scholarly journals Regulation of RUNX3 expression by DNA methylation in prostate cancer

2020 ◽  
Author(s):  
Xin Yang ◽  
Shumei Wang ◽  
Alimu Reheman
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Dna Methylation ◽  
Western Blot ◽  
Cell Lines ◽  
Methylation Level ◽  
Aberrant Methylation ◽  
Normal Prostate ◽  
Qrt Pcr ◽  
Its Gene

Abstract Background: Runt-related transcription factor 3 (RUNX3) is a developmental regulator, and methylation of the RUNX3 is significantly associated with the occurrence and development of carcinogenesis. Previous studies have identified an association of increased methylation level of RUNX3 in prostate cancer (PCa); however, the role and molecular mechanism underlying aberrant methylation of the RUNX3 gene in prostate tumorigenesis remain elusive. In this study, we will investigate the role of RUNX3 promoter methylation and its gene expression in PCa cells. Methods: The methylation of the RUNX3 in the promoter region in PCa cells was detected by bisulfite-sequencing PCR (BSP). Following treatment of the PCa cells with DNA methylation transferase inhibitor 5-AZA-2'-deoxycytidine (AZA), the effect on methylation level and expression of RUNX3 were analyzed by qRT-PCR, Western blot, and BSP assays. Furthermore, we investigated the effect of the demethylated RUNX3 on proliferation, cell cycle and apoptosis of PCa cells using CCK-8 and flow cytometry assays. Using the DNA methylation transferase (DNMT3b) knockout or overexpression models, the relationship between DNMT3b and RUNX3 methylation was further assessed by qRT-PCR, Western blot and methylation-specific PCR (MSP). Results: The results indicated that the methylation level of RUNX3 in PCa cell lines was significantly higher than that of normal prostate epithelial (RWPE-1) cells. Furthermore, treatment with AZA not only promoted the demethylation of RUNX3 but also restored the mRNA and protein expression of RUNX3, and the reactivation of expression of the later exhibited its anti-tumor effects through regulation of the cycle progression in PCa cells. Moreover, DNMT3b could regulate the expression level of RUNX3 by altering the DNA methylation of the RUNX3 in PCa cells. Conclusion: RUNX3 is hypermethylated in a panel of PCa cell lines; Inhibits DNA methylation of RUNX3 could restored its gene expression, which in turn induced its anti-cancer effects. Thus, RUNX3 may serve as a novel putative molecular target gene for PCa therapy.

scholarly journals EPEN-21. IMPAIRED NEURONAL-GLIAL FATE SPECIFICATION IN PEDIATRIC EPENDYMOMA REVEALED BY SINGLE-CELL RNA-SEQ

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii311-iii312
Author(s):  
Bernhard Englinger ◽  
Johannes Gojo ◽  
Li Jiang ◽  
Jens M Hübner ◽  
McKenzie L Shaw ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Regulatory Networks ◽  
Target Genes ◽  
Target Identification ◽  
Rna Seq ◽  
Cell Models ◽  
Pediatric Ependymoma ◽  
Glial Fate ◽  
Anatomic Locations

Abstract Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets including IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

scholarly journals EGFR DNA Methylation Correlates With EGFR Expression, Immune Cell Infiltration, and Overall Survival in Lung Adenocarcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Zhanyu Xu ◽  
Fanglu Qin ◽  
Liqiang Yuan ◽  
Jiangbo Wei ◽  
Yu Sun ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Protein Expression ◽  
Lung Adenocarcinoma ◽  
Methylation Level ◽  
Promoter Region ◽  
Body Region ◽  
Gene Body ◽  
Immune Infiltration ◽  
Cpg Sites ◽  
Gene Body Region

BackgroundThe epidermal growth factor receptor (EGFR) is a primary target of molecular targeted therapy for lung adenocarcinoma (LUAD). The mechanisms that lead to epigenetic abnormalities of EGFR in LUAD are still unclear. The purpose of our study was to evaluate the abnormal methylation of EGFR CpG sites as potential biomarkers for LUAD.MethodsTo assess the differentially methylation CpG sites of EGFR in LUAD, we used an integrative study of Illumina HumanMethylation450K and RNA-seq data from The Cancer Genome Atlas (TCGA). We evaluated and compared EGFR multiple-omics data to explore the role of CpG sites located in EGFR promoter regions and gene body regions and the association with transcripts, protein expression levels, mutations, and somatic copy number variation. We calculated the correlation coefficients between CpG sites of EGFR and immune infiltration fraction (by MCPcounter and ESTIMATE) and immune-related pathways in LUAD. Finally, we validated the differential methylation of clinically and prognostically relevant CpG sites using quantitative methylation-specific PCR (qMSP).ResultsWe found that the methylation level of many EGFR CpGs in the promoter region was negatively correlated with the transcription level, protein expression, and SCNV, while the methylation at the gene body region was positively correlated with these features. The methylation level of EGFR CpGs in the promoter region was positively correlated with the level of immune infiltration and IFN-γ signature, while the opposite was found for methylation of the gene body region. The qMSP results showed that cg02316066 had a high methylation level, while cg02166842 had a low methylation level in LUAD. There was a high degree of co-methylation between cg02316066 and cg03046247.ConclusionOur data indicate that EGFR is an epigenetic regulator in LUAD acting through DNA methylation. Our research provides a theoretical basis for the further detection of EGFR DNA methylation as a predictive biomarker for LUAD survival and immunotherapy.

scholarly journals Small RNA sequencing evaluation of renal microRNA biomarkers in dogs with X-linked hereditary nephropathy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Candice P. Chu ◽  
Shiguang Liu ◽  
Wenping Song ◽  
Ethan Y. Xu ◽  
Mary B. Nabity
Keyword(s):  
Small Rna ◽  
Target Genes ◽  
Differentially Expressed ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Ckd Progression ◽  
Critical Pathways ◽  
Qrt Pcr ◽  
Hereditary Nephropathy ◽  
Differentially Expressed Mirnas

AbstractDogs with X-linked hereditary nephropathy (XLHN) are an animal model for Alport syndrome in humans and progressive chronic kidney disease (CKD). Using mRNA sequencing (mRNA-seq), we have characterized the gene expression profile affecting the progression of XLHN; however, the microRNA (miRNA, miR) expression remains unknown. With small RNA-seq and quantitative RT-PCR (qRT-PCR), we used 3 small RNA-seq analysis tools (QIAGEN OmicSoft Studio, miRDeep2, and CPSS 2.0) to profile differentially expressed renal miRNAs, top-ranked miRNA target genes, and enriched biological processes and pathways in CKD progression. Twenty-three kidney biopsies were collected from 5 dogs with XLHN and 4 age-matched, unaffected littermates at 3 clinical time points (T1: onset of proteinuria, T2: onset of azotemia, and T3: advanced azotemia). We identified up to 23 differentially expressed miRNAs at each clinical time point. Five miRNAs (miR-21, miR-146b, miR-802, miR-142, miR-147) were consistently upregulated in affected dogs. We identified miR-186 and miR-26b as effective reference miRNAs for qRT-PCR. This study applied small RNA-seq to identify differentially expressed miRNAs that might regulate critical pathways contributing to CKD progression in dogs with XLHN.

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