Demethylation of Secreted Frizzled-Related Protein2( SFRP2) Promoter Upregulates Wnt/β-Catenin Activity in Endometriosis

Wnt/β-catenin signalling contributes to the metastasis and invasion in the etiology and pathogenesis of endometriosis (EMS), but why the WNT pathway is dysregulated in EMS remains unclear. This study aimed to explore the effects of demethylation of SFRP2 promoter on the Wnt/β-catenin activity in EMS. Aberrantly methylated-differentially expressed genes were identified from GEO database microarray data. 5 ectopic endometrium and 5 normal endometrium were get, subsequently, ectopic endometrium epithelial cells (EEECs) and normal endometrium epithelial cells (NEECs) were isolated in vitro. MSP, BSP, luciferase reporter assay, Lentivirus infection of high expression of SFRP2 gene vector, low expression of DNMT1 gene vector, and 5-Aza stimulation, RT-PCR and western blot were performed in the tissues or cells. It was found that compared with the normal endometrium and NEECs, the RNA and protein expression levels of SFRP2 were significantly increased while the SFRP2 promoter was demethylated in ectopic endometrium and EEECs. The 5-Aza treatment significantly upregulated SFRP2 mRNA and protein levels in EEECs. Furthermore, after the knockdown of DNMT1 expression, the demethylation of the SFRP2 promoter and upregulation of SFRP2 mRNA and protein in EEECs were observed. Meanwhile, the expression of lentivirus carrying SFRP2 cDNA up-regulates the activity of Wnt signaling and the protein expression of β-catenin in EEECs. In summary, the increased SFRP2 expression-induced Wnt/β-catenin signaling due to the demethylation of the SFRP2 promoter plays an important role in the pathogenesis of EMS, suggesting that SFRP2 might be a therapeutic target for EMS treatment.

SNHG3/miR-148a-3p Axis–mediated High Expression of DNMT1 Correlates with Poor Prognosis and Tumor Immune Infiltration of Hepatocellular Carcinoma

Background Epigenetic reprogramming plays an important role in the occurrence, development, and prognosis of hepatocellular carcinoma (HCC). DNA methylation is a key epigenetic regulatory mechanism, and DNA methyltransferase 1 (DNMT1) is the major enzyme responsible for maintenance methylation. Nevertheless, the role and mechanism of DNMT1 in HCC remains poorly defined. Methods In the current study, we conducted pan-cancer analysis for DNMT1’s expression and prognosis using The Cancer Genome Atlas (TCGA) data set. We conducted gene Set Enrichment Analysis (GSEA) between high-and-low DNMT1 expression groups to identify DNMT1-related functional significance. We also investigated the relationship between DNMT1 expression and tumor immune microenvironment, including immune cell infiltration and the expression of immune checkpoints. Through a combination series of computer analyses (including expression analyses, correlation analyses, and survival analyses), the noncoding RNAs (ncRNAs) that contribute to the overexpression of DNMT1 were ultimately identified. Results We found that DNMT1 was upregulated in 16 types of human carcinoma including HCC, and DNMT1 might be a biomarker predicting unfavorable prognosis in HCC patients. DNMT1 mRNA expression was statistically associated with age, histological grade, and the level of serum AFP. Moreover, DNMT1 level was significantly and positively linked to tumor immune cell infiltration, immune cell biomarkers, and immune checkpoint expression. Meanwhile, Gene Set Enrichment Analysis (GSEA) revealed that high-DNMT1 expression was associated with epithelial mesenchymal transition (EMT), E2F target, G2M checkpoint, and inflammatory response. Finally, through a combination series of computer analyses the SNHG3/hsa-miR-148a-3p/DNMT1 axis was confirmed as the potential regulatory pathway in HCC. Conclusion SNHG3/miR-148a-3p axis upregulation of DNMT1 may be related to poor outcome, tumor immune infiltration, and regulated malignant properties in HCC.

Vapor cannabis exposure generationally affects male reproductive functions in mice

This study was performed to examine whether vapor exposure to cannabis plant matter negatively impacts male reproductive functions and testis development in mice. Adult CD-1 male mice (F0) were exposed to air (control) or 200 mg of vaporized cannabis plant matter 3x/day over a 10 day period. Subsequently, F0 males were bred with drug naive CD-1 females to generate F1 males, and F1 offspring were used to generate F2 males. Cannabis vapor exposure decreased sperm count and/or motility in F0 and F1 males and disrupted the progression of germ cell development, as morphometric analyses exhibited an abnormal distribution of the stages of spermatogenesis in F0 males. Although plasma levels of testosterone were not affected by cannabis exposure in any ages or generations of males, dysregulated steroidogenic enzymes, Cyp11a1 and Cyp19a1, were observed in F0 testis. In the neonatal testis from F1 males, while apoptosis was not altered, DNA damage and DNMT1, but not DNMT3A and DNMT3B, were increased in germ cells following cannabis exposure. In contrast, the alterations of DNA damage and DNMT1 expression were not observed in F2 neonatal males. These results suggest that cannabis vapor exposure generationally affects male reproductive functions, probably due to disruption of spermatogenesis in the developing testis.

Bioinformatics analysis of DNMT1 expression and its role in head and neck squamous cell carcinoma prognosis

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of malignancy in the world. DNA cytosine-5-methyltransferase 1 (DNMT1) play key roles in carcinogenesis and regulation of the immune micro-environment, but the gene expression and the role of DNMT1 in HNSCC is unknown. In this study, we utilized online tools and databases for pan-cancer and HNSCC analysis of DNMT1 expression and its association with clinical cancer characteristics. We also identified genes that positively and negatively correlated with DNMT1 expression and identified eight hub genes based on protein–protein interaction (PPI) network analysis. Enrichment analyses were performed to explore the biological functions related with of DNMT1. The Tumor Immune Estimation Resource (TIMER) database was performed to explore the relationship between DNMT1 expression and immune-cell infiltration. We demonstrated that DNMT1 gene expression was upregulated in HNSCC and associated with poor prognosis. Based on analysis of the eight hub genes, we determined that DNMT1 may be involved in cell cycle, proliferation and metabolic related pathways. We also found that significant difference of B cells infiltration based on TP 53 mutation. These findings suggest that DNMT1 related epigenetic alterations have close relationship with HNSCC progression, and DNMT1 could be a novel diagnostic biomarker and a promising therapeutic target for HNSCC.

TERT and DNMT1 expression predict sensitivity to decitabine in gliomas

Background Decitabine (DAC) is an FDA-approved DNA methyltransferase (DNMT) inhibitor that is used in the treatment of patients with myelodysplastic syndromes. Previously, we showed that DAC marks antitumor activity against gliomas with isocitrate dehydrogenase 1 (IDH1) mutations. Based on promising preclinical results, a clinical trial has been launched to determine the effect of DAC in IDH-mutant gliomas. The next step is to comprehensively assess the efficacy and potential determinants of response to DAC in malignant gliomas. Methods The expression and activity of telomerase reverse transcriptase (TERT) and DNMT1 were manipulated in patient-derived IDH1-mutant and -wildtype glioma lines, followed by assessment of cell proliferation with DAC treatment alone or in combination with telomerase inhibitors. RNA sequencing, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and correlation analysis were performed. Results IDH1-mutant glioma tumorspheres with hemizygous codeletion of chromosome arms 1p/19q were particularly sensitive to DAC and showed significant inhibition of DNA replication genes. Our transcriptome analysis revealed that DAC induced expression of cyclin-dependent kinase inhibitor 1A/p21 (CDKN1A), along with downregulation of TERT. These molecular changes were also observed following doxorubicin treatment, supporting the importance of DAC-induced DNA damage in contributing to this effect. We demonstrated that knockdown of p21 led to TERT upregulation. Strikingly, TERT overexpression increased DNMT1 levels and DAC sensitivity via a telomerase-independent mechanism. Furthermore, RNA inhibition (RNAi) targeting of DNMT1 abrogated DAC response in TERT-proficient glioma cells. Conclusions DAC downregulates TERT through p21 induction. Our data point to TERT and DNMT1 levels as potential determinants of response to DAC treatment.

Oxidative stress mediates the apoptosis and epigenetic modification of the Bcl-2 promoter via DNMT1 in a cigarette smoke-induced emphysema model

Background: Emphysema is a crucial pathological characteristic of chronic obstructive pulmonary disease (COPD). Oxidative stress, apoptosis and epigenetic mechanisms contribute to the pathogenesis of emphysema. However, an attempt to accurately identify whether these mechanisms interact with each other and how they are triggered has never been conducted.Method: The total reactive oxygen species (ROS) level, pulmonary apoptosis and B-cell lymphoma/ leukemia-2 (Bcl-2) expression, an apoptosis regulator, were detected in samples from COPD patients. Bisulfite sequencing PCR (BSP) was conducted to observe the alterations in the methylation of the Bcl-2 promoter in specimens. The dysregulation of DNA methyltransferase enzyme 1 (DNMT1), a vital DNA methyltransferase enzyme, in the lungs of patients was confirmed through western blotting. To find out interactions between oxidative stress and DNA methylation in emphysema, mouse models were built with antioxidant treatment and DNMT1 silencing, and were examined with the pulmonary apoptosis, Bcl-2 and DNMT1 expression, and epigenetic alterations of Bcl-2.Results: Higher reactive oxygen species (ROS) levels and pulmonary apoptosis were observed in COPD patients than in healthy controls. Downregulated Bcl-2 expression with increased promoter methylation and DNMT1 protein expression was found in COPD patients. Antioxidant treatment reduced the level of ROS and dysregulation of DNMT1 expression and emphysematous progression in the smoking models. Following DNMT1 blockage, smoking models showed improved lung function, pulmonary apoptosis, emphysematous progression, and increased Bcl-2 protein expression with less promoter methylation than emphysema mice.Conclusion: Cigarette-induced oxidative stress mediates pulmonary apoptosis and hypermethylation of the Bcl-2 promoter in emphysema models through DNMT1.

Oxidative stress mediates the apoptosis and epigenetic modification of the Bcl-2 promoter via DNMT1 in a cigarette smoke-induced emphysema model

Background: Emphysema is a crucial pathological characteristic of chronic obstructive pulmonary disease (COPD). Oxidative stress, apoptosis and epigenetic mechanisms contribute to the pathogenesis of emphysema. However, an attempt to accurately identify whether these mechanisms interact with each other and how they are triggered has never been conducted. Method: The total reactive oxygen species (ROS) level, pulmonary apoptosis and B-cell lymphoma/ leukemia-2 (Bcl-2) expression, an apoptosis regulator, were detected in samples from COPD patients. Bisulfite sequencing PCR (BSP) was conducted to observe the alterations in the methylation of the Bcl-2 promoter in specimens. The dysregulation of DNA methyltransferase enzyme 1 (DNMT1), a vital DNA methyltransferase enzyme, in the lungs of patients was confirmed through western blotting. To find out interactions between oxidative stress and DNA methylation in emphysema, mouse models were built with antioxidant treatment and DNMT1 silencing, and were examined with the pulmonary apoptosis, Bcl-2 and DNMT1 expression, and epigenetic alterations of Bcl-2. Results: Higher reactive oxygen species (ROS) levels and pulmonary apoptosis were observed in COPD patients than in healthy controls. Downregulated Bcl-2 expression with increased promoter methylation and DNMT1 protein expression was found in COPD patients. Antioxidant treatment reduced the level of ROS and dysregulation of DNMT1 expression and emphysematous progression in the smoking models. Following DNMT1 blockage, smoking models showed improved lung function, pulmonary apoptosis, emphysematous progression, and increased Bcl-2 protein expression with less promoter methylation than emphysema mice.Conclusion: Cigarette-induced oxidative stress mediates pulmonary apoptosis and hypermethylation of the Bcl-2 promoter in emphysema models through DNMT1.