Increased Promoter Methylation Research Articles

Abstract 108: Inflammasome Activation Via BRCC3-mediated NLRP3 Deubiquitylation Promotes Atherosclerosis In Tet2 Clonal Hematopoiesis

Clonal hematopoiesis (CH) has emerged as an independent risk factor for atherosclerotic cardiovascular disease (CVD) with activation of macrophage inflammasomes as a potential underlying mechanism. The NLRP3 inflammasome has a key role in promoting atherosclerosis in mouse models of Tet2 CH, while inhibition of the inflammasome product IL-1β appeared to particularly benefit patients with TET2 CH in CANTOS. TET2 is an epigenetic modifier that decreases promoter methylation. However, the mechanisms underlying macrophage NLRP3 inflammasome activation in TET2 deficiency and potential links with epigenetic modifications are poorly understood. We used cholesterol-loaded TET2 deficient murine and embryonic stem cell derived isogenic human macrophages to evaluate mechanisms of NLRP3 inflammasome activation in vitro and hypercholesterolemic Ldlr -/- mice modeling TET2 CH to assess the role of NLRP3 inflammasome activation in atherosclerosis. Tet2 deficiency acted synergistically with cholesterol loading in cell culture and with hypercholesterolemia in vivo to increase JNK1 phosphorylation and NLRP3 inflammasome activation. The mechanism of JNK activation in TET2 deficiency was increased promoter methylation and decreased expression of the JNK-inactivating dual specificity phosphatase, Dusp10 . Active Tet1-deadCas9 targeted editing of Dusp10 promoter methylation abolished cholesterol-induced inflammasome activation in Tet2 -deficient macrophages. Increased JNK1 signaling led to NLRP3 deubiquitylation and activation by the deubiquitinase BRCC3. Accelerated atherosclerosis and NETosis in Tet2 CH mice were reversed by holomycin, a BRCC3 deubiquitinase inhibitor, and also by hematopoietic deficiency of Abro1 , an essential scaffolding protein in the BRCC3-containing cytosolic complex. Hypercholesterolemia and TET2 deficiency converge on a common pathway of NLRP3 inflammasome activation mediated by JNK1 activation and BRCC3-mediated NLRP3 deubiquitylation with potential therapeutic implications for the prevention of CVD in TET2 CH.

Pan-cancer analysis reveals potential immunological and prognostic roles of METTL7A in human cancers

Methyltransferase-like protein 7A (METTL7A) is an m6A RNA methyltransferase that has been linked to cancer prognosis and drug resistance. However, a comprehensive analysis of METTL7A is lacking. The expression of METTL7A, prognostic performance, correlation with microsatellite instability (MSI), tumor mutational burden (TMB), and immune infiltration was investigated in The Cancer Genome Atlas (TCGA). Immunohistochemistry staining was applied to detect METTL7A in 6 tumors. METTL7A was significantly decreased in 19 cancers in TCGA including LUAD. Alterations of METTL7A include amplification and mutation, and epigenetic alterations revealed increased promoter methylation may result in down-regulation of METTL7A in LUAD. We also found that METTL7A was linked to both TMB and MSI in LUAD. METTL7A was increasingly correlated with invasive immune cells, while being negatively associated with Macrophages M0, Mast cells activated, activated memory CD4 T cells, CD8 T cells, and follicular helper T cells in several tumors. Additionally, METTL7A showed similar correlation with immune therapy-related genes across cancers. Our biological validation found that the protein levels of METTL7A were down-regulated in breast cancer (BRCA), endometrioid cancer (UCEC), colon cancer (COAD), prostate cancer (PRAD), and kidney clear cell carcinoma (KIRC), as detected by immunohistochemistry staining. Overall, our work indicates that METTL7A may serve as promising diagnostic and prognostic indicator of LUAD, and our work sheds light on the potential immunological and prognostic roles of METTL7A in human cancers.

BRCC3-Mediated NLRP3 Deubiquitylation Promotes Inflammasome Activation and Atherosclerosis in Tet2 Clonal Hematopoiesis.

Clonal hematopoiesis (CH) has emerged as an independent risk factor for atherosclerotic cardiovascular disease, with activation of macrophage inflammasomes as a potential underlying mechanism. The NLRP3 (NLR family pyrin domain containing 3) inflammasome has a key role in promoting atherosclerosis in mouse models of Tet2 CH, whereas inhibition of the inflammasome product interleukin-1β appeared to particularly benefit patients with TET2 CH in CANTOS (Cardiovascular Risk Reduction Study [Reduction in Recurrent Major CV Disease Events]). TET2 is an epigenetic modifier that decreases promoter methylation. However, the mechanisms underlying macrophage NLRP3 inflammasome activation in TET2 (Tet methylcytosine dioxygenase 2) deficiency and potential links with epigenetic modifications are poorly understood. We used cholesterol-loaded TET2-deficient murine and embryonic stem cell-derived isogenic human macrophages to evaluate mechanisms of NLRP3 inflammasome activation in vitro and hypercholesterolemic Ldlr-/- mice modeling TET2 CH to assess the role of NLRP3 inflammasome activation in atherosclerosis. Tet2 deficiency in murine macrophages acted synergistically with cholesterol loading in cell culture and with hypercholesterolemia in vivo to increase JNK1 (c-Jun N-terminal kinase 1) phosphorylation and NLRP3 inflammasome activation. The mechanism of JNK (c-Jun N-terminal kinase) activation in TET2 deficiency was increased promoter methylation and decreased expression of the JNK-inactivating dual-specificity phosphatase Dusp10. Active Tet1-deadCas9-targeted editing of Dusp10 promoter methylation abolished cholesterol-induced inflammasome activation in Tet2-deficient macrophages. Increased JNK1 signaling led to NLRP3 deubiquitylation and activation by the deubiquitinase BRCC3 (BRCA1/BRCA2-containing complex subunit 3). Accelerated atherosclerosis and neutrophil extracellular trap formation (NETosis) in Tet2 CH mice were reversed by holomycin, a BRCC3 deubiquitinase inhibitor, and also by hematopoietic deficiency of Abro1, an essential scaffolding protein in the BRCC3-containing cytosolic complex. Human TET2-/- macrophages displayed increased JNK1 and NLRP3 inflammasome activation, especially after cholesterol loading, with reversal by holomycin treatment, indicating human relevance. Hypercholesterolemia and TET2 deficiency converge on a common pathway of NLRP3 inflammasome activation mediated by JNK1 activation and BRCC3-mediated NLRP3 deubiquitylation, with potential therapeutic implications for the prevention of cardiovascular disease in TET2 CH.

LncRNA BRCAT54 is downregulated and inhibits cancer cell proliferation by downregulating miR-130b-3p through methylation in prostate cancer.

BRCAT54 and miR-130b-3p are two recently characterized critical players in cancer biology, while their functions in prostate cancer (PC) are unknown. From preliminary sequencing analysis, we observed altered expression of BRCAT54 and miR-130b-3p in PC and an inverse correlation between them. This study was conducted to explore their involvement in PC. A total of 64 PC patients were enrolled to collect paired PC and nontumor tissues. The expression of BRCAT54 and miR-130b-3p were determined by RT-qPCR. Overexpression of BRCAT54 and miR-130b-3p was achieved in PC cells to explore their roles in regulating the expression of each other. Methylation-specific PCR (MSP) was conducted to explore the role of BRCAT54 in regulating promoter methylation of miR-130b-3p. BrdU assay was used to evaluate the role of BRCAT54 and miR-130b-3p in regulating PC cell proliferation. The results showed that PC tissues exhibited downregulation of BRCAT54 and upreglation of miR-130b-3p compared to that in nontumor tissues. They were inversely correlated across PC tissue samples. Overexpression of BRCAT54 decreased RNA accumulation of miR-130b-3p in PC cells. In addition, overexpression of BRCAT54 increased promoter methylation of miR-130b-3p. Moreover, BRCAT54 suppressed the role of miR-130b-3p in promoting PC cell proliferation. In conclusion, BRCAT54 is downregulated in PC and it may inhibit cancer cell proliferation by downregulating miR-130b-3p through methylation.

Depletion of tet2 results in age-dependent changes in DNA methylation and gene expression in a zebrafish model of myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias, and dysplasia. The gene encoding ten-eleven translocation 2 (tet2), a dioxygenase enzyme that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, is a recurrently mutated tumor suppressor gene in MDS and other myeloid malignancies. Previously, we reported a stable zebrafish line with a loss-of-function mutation in the tet2 gene. The tet2m/m-mutant zebrafish developed a pre-MDS state with kidney marrow dysplasia, but normal circulating blood counts by 11 months of age and accompanying anemia, signifying the onset of MDS, by 24 months of age. In the current study, we collected progenitor cells from the kidney marrows of the adult tet2m/m and tet2wt/wt fish at 4 and 15 months of age and conducted enhanced reduced representation of bisulfite sequencing (ERRBS) and bulk RNA-seq to measure changes in DNA methylation and gene expression of hematopoietic stem and progenitor cells (HSPCs). A global increase in DNA methylation of gene promoter regions and CpG islands was observed in tet2m/m HSPCs at 4 months of age when compared with the wild type. Furthermore, hypermethylated genes were significantly enriched for targets of SUZ12 and the metal-response-element-binding transcription factor 2 (MTF2)-involved in the polycomb repressive complex 2 (PRC2). However, between 4 and 15 months of age, we observed a paradoxical global decrease in DNA methylation in tet2m/m HSPCs. Gene expression analyses identified upregulation of genes associated with mTORC1 signaling and interferon gamma and alpha responses in tet2m/m HSPCs at 4 months of age when compared with the wild type. Downregulated genes in HSPCs of tet2-mutant fish at 4 months of age were enriched for cell cycle regulation, heme metabolism, and interleukin 2 (IL2)/signal transducer and activator of transcription 5 (STAT5) signaling, possibly related to increased self-renewal and clonal advantage in HSPCs with tet2 loss of function. Finally, there was an overall inverse correlation between overall increased promoter methylation and gene expression.

Role of MLH1 and MSH2 deficiency in the development of tumorigenesis and chemo-tolerance of cervical Carcinoma: Clinical implications

Cervical cancer (CACX) is one of the top causes of cancer death in women globally. The involvement of several cellular pathways in carcinogenesis is still poorly understood. Here, we focused to evaluate the contributory role of Mismatch Repair (MMR) pathway genes-MLH1 and MSH2 in CACX and their association with chemo-tolerance of the disease. For this purpose, molecular profiles (expression/promoter methylation/deletion) of the genes were analysed in both normal cervical epithelium and tumour tissue, also validated in in-silico dataset as well. Later on, prognostic importance of the genes was identified through analysis of their methylation/expression status in plasma DNA of circulating tumour cells (CTCs) and cisplatin-tolerant CACX cell lines respectively. It was found that the expression profile of MLH1 and MSH2 genes was considerably reduced from undifferentiated basal-parabasal layers of normal cervical epithelium towards progression of the disease. Further analysis showed that frequent deletion [34–48%] and promoter methylation events [28–46%] of the genes were the plausible reasons for their reduced expression during tumorigenesis. Incidentally, the prevalence of MLH1 [32%] and MSH2 [27%] promoter methylation found in CTCs of plasma of the clinically advanced CACX patients implicated their prognostic importance of the disease. In addition, the patients having high alterations of those genes resulted in poor patient outcomes even after the therapy. In in-depth analysis of this result in cisplatin-tolerant CACX cell lines, we discovered that increased promoter methylation frequency of those genes at higher concentrations of cisplatin and gradual accumulation of the cells in the G2/M phase of the cell cycle were the rational causes for their reduced expression and MMR deficiency in the system. Hence, it is possible to conclude that the gradual down-regulation of MLH1 and MSH2 proteins may be a key event for MMR pathway inactivation in CACX. This might also be associated with chemo-tolerance and overall poor survival among the patients.

Light-Dependent Expression and Promoter Methylation of the Genes Encoding Succinate Dehydrogenase, Fumarase, and NAD-Malate Dehydrogenase in Maize (Zea mays L.) Leaves.

The expression and methylation of promoters of the genes encoding succinate dehydrogenase, fumarase, and NAD-malate dehydrogenase in maize (Zea mays L.) leaves depending on the light regime were studied. The genes encoding the catalytic subunits of succinate dehydrogenase showed suppression of expression upon irradiation by red light, which was abolished by far-red light. This was accompanied by an increase in promoter methylation of the gene Sdh1-2 encoding the flavoprotein subunit A, while methylation was low for Sdh2-3 encoding the iron-sulfur subunit B under all conditions. The expression of Sdh3-1 and Sdh4 encoding the anchoring subunits C and D was not affected by red light. The expression of Fum1 encoding the mitochondrial form of fumarase was regulated by red and far-red light via methylation of its promoter. Only one gene encoding the mitochondrial NAD-malate dehydrogenase gene (mMdh1) was regulated by red and far-red light, while the second gene (mMdh2) did not respond to irradiation, and neither gene was controlled by promoter methylation. It is concluded that the dicarboxylic branch of the tricarboxylic acid cycle is regulated by light via the phytochrome mechanism, and promoter methylation is involved with the flavoprotein subunit of succinate dehydrogenase and the mitochondrial fumarase.

Pro- and anti-tumour activities of CD146/MCAM in breast cancer result from its heterogeneous expression and association with epithelial to mesenchymal transition.

CD146, also known as melanoma cell adhesion molecule (MCAM), is expressed in numerous cancers and has been implicated in the regulation of metastasis. We show that CD146 negatively regulates transendothelial migration (TEM) in breast cancer. This inhibitory activity is reflected by a reduction in MCAM gene expression and increased promoter methylation in tumour tissue compared to normal breast tissue. However, increased CD146/MCAM expression is associated with poor prognosis in breast cancer, a characteristic that is difficult to reconcile with inhibition of TEM by CD146 and its epigenetic silencing. Single cell transcriptome data revealed MCAM expression in multiple cell types, including the malignant cells, tumour vasculature and normal epithelium. MCAM expressing malignant cells were in the minority and expression was associated with epithelial to mesenchymal transition (EMT). Furthermore, gene expression signatures defining invasiveness and a stem cell-like phenotype were most strongly associated with mesenchymal-like tumour cells with low levels of MCAM mRNA, likely to represent a hybrid epithelial/mesenchymal (E/M) state. Our results show that high levels of MCAM gene expression are associated with poor prognosis in breast cancer because they reflect tumour vascularisation and high levels of EMT. We suggest that high levels of mesenchymal-like malignant cells reflect large populations of hybrid E/M cells and that low CD146 expression on these hybrid cells is permissive for TEM, aiding metastasis.

Altered expression of DNA methyltransferases and methylation status of the TLR4 and TNF-α promoters in COVID-19

Epigenetic modifications play a significant role in the host's immune response to viral infection. Two epigenetic events, DNA methylation and histone acetylation, are crucial for modifying the chromatin architecture and the location of regulatory elements such as promoters and enhancers. In this case-control study, we evaluated the expression of genes involved in epigenetic machinery (DNMT1, DNMT3A, DNMT3B, HDAC2, and HDAC3) and the degree of methylation of promoters of immune response genes (IFITM1/2/3, TLR3/4, TNF-α, NF-κB, and MYD88) as well as global methylation (LINE-1 and global 5–mC) in blood samples from 120 COVID-19 patients (30 mild, 30 moderate, 30 severe, and 30 critical) and 30 healthy subjects without COVID-19. In contrast to previous reports, DNMT3A and DNMT3B expression was found to be significantly downregulated in COVID-19 cases, whereas DNMT1, HDAC2, and HDAC3 expression did not change. DNMT1 and DNMT3A were negatively correlated with COVID-19 severity. Critically ill patients had lower HDAC3 expression levels. TLR4 and TNF-α had increased promoter methylation, whereas IFITM1/2/3, TLR3, NF-κB, MYD88, and LINE-1 did not differ between cases and controls. Methylation of the TNF-α promoter increased as disease severity increased. Significantly less methylation of the TLR3 promoter was observed in patients with a positive outcome (recovery). We also found a correlation between the expression of DNMT3B and the methylation level of the TLR4 promoter. In milder cases, the global 5–mC levels were lower than that in more severe cases. Our findings suggest the exclusion of DNMTs inhibitors previously recommended for COVID-19 treatment and the need for additional research in this area.

Long noncoding RNA HOTAIR facilitates pulmonary vascular endothelial cell apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in COPD

BackgroundTo study the regulatory effect of Long non-coding RNA (LncRNA) HOX transcript antisense RNA (HOTAIR) on pulmonary vascular endothelial cell (HPVEC) apoptosis and determine whether the HOTAIR facilitate HPVEC apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in chronic obstructive pulmonary disease (COPD).MethodsLncRNA array was used to measure the differentially expressed lncRNAs in COPD and non-COPD lung tissues. Expression of HOTAIR in COPD patient lungs and cigarette smoke extract (CSE)-induced HPVEC was assessed by qRT-PCR. The location of HOTAIR was determined in COPD patient lungs and HPVEC by RNA in situ hybridization (RNA-ISH). The emphysema mouse model and HOTAIR knockdown mice were each established by inhaling cigarette smoke or intratracheal lentiviral vectors instillation. The dysregulation of DNA methyltransferase enzyme 1 (DNMT1), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and Cleaved-caspase 3 protein expression were detected by Western blotting. HOTAIR, DNMT1, Bcl-2 and Bax mRNA expression were measured by quantitative real-time polymerase chain reaction. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays were used to assess apoptotic ratio in mice and CSE-induced HPVEC. Methylation-specific PCR (MSP) assay was conducted to observe the alterations in the methylation of the Bcl-2 promoter in specimens. RNA pull-down assay was used for analysis of the correlation between HOTAIR and DNMT1.ResultsThe expression levels of the HOTAIR were up-regulated in COPD patient lungs and CSE-induced HPVEC. HPVEC apoptosis with down-regulated Bcl-2 expression, increased promoter methylation, DNMT1, Bax and Cleaved-caspase 3 expression was found in emphysema mouse model and CSE-induced HPVEC. Knockdown HOTAIR can attenuate cell apoptosis and emphysema via DNMT1 mediated hypermethylation of Bcl-2 promoter in mice. In vitro, HOTAIR can aggravate the apoptosis of CSE-exposed HPVEC. DNMT1 was a target of HOTAIR and had a positive correlation with HOTAIR.ConclusionHOTAIR facilitates HPVEC apoptosis via DNMT1 mediated hypermethylation of Bcl-2 promoter in COPD, and attenuating the expression of HOTAIR may be a new therapy to prevent COPD.

Microvesicle-Mediated Transfer of DNA Methyltransferase Proteins Results in Recipient Cell Immunosuppression

IntroductionPatients with sepsis exhibit significant, persistent immunologic dysfunction. Evidence supports the hypothesis that epigenetic regulation of key cytokines plays an important role in this dysfunction. In sepsis, circulating microvesicles (MVs) containing elevated levels of DNA methyltransferase (DNMT) mRNA cause gene methylation and silencing in recipient cells. We sought to examine the functional role of MV DNMT proteins in this immunologic dysfunction. MethodsIn total, 33 patients were enrolled within 24 h of sepsis diagnosis (23 sepsis, 10 critically ill controls). Blood and MVs were collected on days 1, 3, and 5 of sepsis, and protein was isolated from the MVs. Levels of DNMT protein and activity were quantified. MVs were produced in vitro by stimulating naïve monocytes with lipopolysaccharide. Methylation was assessed using bisulfate site-specific qualitative real-time polymerase chain reaction. ResultsThe size of MVs in the patients with sepsis decreased from days 1 to 5 compared to the control group. Circulating MVs contained significantly higher levels of DNMT 1 and 3A, protein. We recapitulated the production of these DNMT-containing MVs in vitro by treating monocytes with lipopolysaccharide. We found that exposing naïve monocytes to these MVs resulted in increased promoter methylation of tumor necrosis factor alpha. ConclusionsAn analysis of the isolated MVs revealed higher levels of DNMT proteins in septic patients than those in nonseptic patients. Exposing naïve monocytes to DNMT-containing MVs produced in vitro resulted in hypermethylation of tumor necrosis factor alpha, a key cytokine implicated in postsepsis immunosuppression. These results suggest that DNMT-containing MVs cause epigenetic changes in recipient cells. This study highlights a novel role for MVs in the immune dysfunction of patients with sepsis.

USP7 accelerates FMR1-mediated ferroptosis by facilitating TBK1 ubiquitination and DNMT1 deubiquitination after renal ischemia-reperfusion injury.

Renal ischemia/reperfusion (I/R) leads to acute kidney injury and is associated with cell ferroptosis, an oxidative programmed cell death. This study aims to explore whether USP7 regulates ferroptosis in rat kidneys suffered I/R and the underlying mechanisms. Human renal tubular epithelial cells HK-2 were treated with hypoxia/reoxygenation (H/R) to establish a cell model. The expression of ubiquitin specific peptidase 7 (USP7) in H/R-treated cells was determined. USP7 siRNA was transfected into H/R-treated cells, followed by the detection of cell proliferation, iron ion concentration, oxidative stress levels and glutathione peroxidase 4 (GPX4) and solute carrier family 7-member 11 (SLC7A11) protein levels. Western blotting and immunoprecipitation analyses were performed to detect the effects of USP7 on the ubiquitination of TANK-binding kinase 1 (TBK1) and DNA methyltransferase 1 (DNMT1). Then, H/R-treated cells were transfected with USP7 siRNA alone or together with TBK1 siRNA. Co-immunoprecipitation was used to detect binding relationship between TBK1 and FMRP translational regulator 1 (FMR1). The level of DNMT1 and methylation ratio of the FMR1 promoter region were determined with chromatin immunoprecipitation and methylation specific PCR assays, respectively. Furthermore, USP7 siRNA and FMR1 siRNA were transfected alone or together into H/R-treated cells, followed by the detection of cell functions. An I/R rat model was constructed to analyze the effects of USP7 on renal function in rats. USP7 was significantly upregulated in H/R-treated cells. USP7 interference markedly increased HK-2 cell proliferation and the protein levels of GPX4 and SLC7A11, restrained the iron ion concentration, and ameliorated oxidative stress. USP7 promoted TRIM27-mediated TBK1 ubiquitination and degradation. USP7 inhibition resulted in increased ubiquitination and decreased stability of DNMT1. USP7 was able to recruit DNMT1 to the FMR1 promoter region, which increased promoter methylation rates and suppressed FMR1 expression. TBK1 or FMR1 overexpression could reverse the effects of USP7 on cell functions. Inhibition of USP7 alleviated renal ischemia-reperfusion injury in rats. USP7 inhibition attenuated I/R-induced renal injury by inhibiting ferroptosis through decreasing ubiquitination of TBK1 and promoting DNMT1-mediated methylation of FMR1.

The long non-coding RNA lncMYOZ2 mediates an AHCY/MYOZ2 axis to promote adipogenic differentiation in porcine preadipocytes

Long non-coding RNAs (lncRNAs) play a vital role in regulating adipogenesis. However, the associated regulatory mechanisms have yet to be described in detail in pig. In this study, we demonstrate a critical role for lncMYOZ2 in adipogenesis from porcine preadipocytes. Specifically, lncMYOZ2 was more abundant in the adipose tissue of Mashen (fat-type) pigs than for Large White (lean-type) pigs, and knockdown of this lncRNA significantly inhibited the differentiation of porcine preadipocytes into adipocytes. Mechanistically, we used RNA pull-down and RIP assays to establish that lncMYOZ2 interacts with adenosylhomocysteinase (AHCY). Moreover, lncMYOZ2 knockdown increased promoter methylation of the target gene MYOZ2 and lowered its expression. Finally, we describe a positive regulatory role for MYOZ2 in adipogenesis. Collectively, these findings establish lncMYOZ2 as an important epigenetic regulator of adipogenesis via the aforementioned AHCY/MYOZ2 pathway, and provide insights into the role of lncRNAs in porcine adipose development.

AQP5-1364A/C Polymorphism Affects AQP5 Promoter Methylation.

The quantity of aquaporin 5 protein in neutrophil granulocytes is associated with human sepsis-survival. The C-allele of the aquaporin (AQP5)-1364A/C polymorphism was shown to be associated with decreased AQP5 expression, which was shown to be relevant in this context leading towards improved outcomes in sepsis. To date, the underlying mechanism of the C-allele—leading to lower AQP5 expression—has been unknown. Knowing the detailed mechanism depicts a crucial step with a target to further interventions. Genotype-dependent regulation of AQP5 expression might be mediated by the epigenetic mechanism of promoter methylation and treatment with epigenetic-drugs could maybe provide benefit. Hence, we tested the hypothesis that AQP5 promoter methylation differs between genotypes in specific types of immune cells.: AQP5 promoter methylation was quantified in cells of septic patients and controls by methylation-specific polymerase chain reaction and quantified by a standard curve. In cell-line models, AQP5 expression was analyzed after demethylation to determine the impact of promoter methylation on AQP5 expression. C-allele of AQP5-1364 A/C promoter polymorphism is associated with a five-fold increased promoter methylation in neutrophils (p = 0.0055) and a four-fold increase in monocytes (p = 0.0005) and lymphocytes (p = 0.0184) in septic patients and healthy controls as well. In addition, a decreased AQP5 promoter methylation was accompanied by an increased AQP5 expression in HL-60 (p = 0.0102) and REH cells (p = 0.0102). The C-allele which is associated with lower gene expression in sepsis is accompanied by a higher methylation level of the AQP5 promoter. Hence, AQP5 promoter methylation could depict a key mechanism in genotype-dependent expression.

Promoter methylation analysis of DKK2 may be a potential biomarker for early detection of cervical cancer.

Dickkopf 2 (DKK2) plays an important role in multiple cancers. Its potential value in the clinical diagnosis of cervical cancer has remained unclear. To investigate the expression and promoter methylation levels of DKK2 in cervical cancer and their clinicopathological associations. We used the Gene Expression Omnibus, Oncomine, Cancer Genome Atlas, and University of ALabama at Birmingham CANcer data analysis databases, reverse transcription-PCR, and methylation-specific PCR analysis to predict and examine the expression of DKK2 mRNA and DKK2 methylation levels in cell lines and cervical cancer tissues from 79 patients with cervical cancer and 63 with cervical precancerous lesions including 25 with low-grade squamous intraepithelial lesions (LSIL) and 38 patients with high-grade squamous intraepithelial lesions (HSIL). DKK2 mRNA expression was downregulated in all cancer cell lines and cervical cancer tissues, whereas hypermethylation of DKK2 was higher in cervical cancer tissue samples. DKK2 methylation in cervical cancer was significantly higher than that in HSIL (χ2 = 8.346, P = 0.004), whereas DKK2 methylation in HSIL was significantly higher than that in normal cervical samples (χ2 = 7.934, P = 0.005) and in LSIL samples (χ2 = 4.375, P = 0.037). DKK2 silencing caused by its promoter hypermethylation was confirmed by treatment with the methyltransferase inhibitor 5-Aza-dC in cell lines. Patients with lymph node metastasis exhibited increased promoter methylation frequency (χ2 = 5.239, P = 0.022) and low DKK2 mRNA expression (χ2 = 3.958, P = 0.047) compared with patients with no lymph node metastasis. Patients with high-risk human papillomavirus infection exhibited increased promoter methylation frequency (χ2 = 6.279, P = 0.015). DKK2 epigenetic changes of DKK2 may play a key role in the development of cervical cancer, suggesting that DKK2 hypermethylation could be used as a triage test for screening, early diagnosis, or risk prediction of cervical cancer.

Epigenetically-regulated miR-30a/c-5p directly target TWF1 and hamper ccRCC cell aggressiveness

Clear cell renal cell carcinoma (ccRCC) is highly prone to metastasize and displays an extremely low 5-year survival rate. Not only miRNAs (miRs) are key gene expression regulators but can also be epigenetically modified. Abnormal miR expression has been linked with epithelial-mesenchymal transition (EMT)-driven ccRCC progression. MiR-30a/c-5p were found downregulated in ccRCC and associated with aggressiveness. Herein, we sought to unravel miR-30a/c-5p mechanistic role in ccRCC. RNA sequencing and genome-wide methylome data of ccRCC and normal tissue samples from The Cancer Genome Atlas database were integrated to identify candidate miRs cytosine-phosphate-guanine (CpG) loci deregulated in ccRCC. TargetScan was searched to identify miR putative targets. MiR-30a/c-5p expression and promoter methylation was evaluated in vitro, by PCR. Western blot, functional and luciferase assays were performed after cell transfection with either pre-miR, antimiR, or siRNA against twinfilin-1 (TWF1). Immunohistochemistry (IHC) was performed in ccRCC tissues. We found miR-30c-5p downregulation and aberrant promoter methylation in ccRCC tissues. In vitro studies revealed concomitant miR-30a/c-5p downregulation and increased promoter methylation, as well as a significant re-expression following decitabine treatment. Functional assays demonstrated that both miRs significantly decreased cell aggressiveness and the protein levels of EMT-promoting players, while upregulating epithelial markers, namely Claudin-1 and ZO-1. Importantly, we confirmed TWF1 as a direct target of both miRs, and its potential involvement in epithelial-mesenchymal transition/mesenchymal-epithelial transition regulation. IHC analysis revealed higher TWF1 expression in primary tissues from patients that developed metastases, after surgical treatment. Our results implicate miR-30a/c-5p in ccRCC cells' aggressiveness attenuation by directly targeting TWF1 and hampering EMT.

SAM and folic acid prevent arsenic-induced oxidative and nitrative DNA damage in human lymphoblast cells by modulating expression of inflammatory and DNA repair genes

Growing evidence suggests that arsenic exposure increases the risk of developing a variety of inflammation-associated chronic diseases and cancers. Our previous study revealed that increased transcript levels of inflammatory genes (i.e. COX2, EGR1, and SOCS3) coupled with hypomethylation of the promoter regions of these genes was associated with increased DNA damage in arsenic-exposed newborns through their early childhood. This study further investigated the ability of the methyl group donors, S-adenosyl methionine (SAM) and folic acid, to prevent promoter hypomethylation that results in decreased mRNA expression of inflammatory genes (COX2, EGR1, and SOCS3), and a reduction in arsenic-induced oxidative and nitrative DNA damage in human lymphoblast cells. Pretreatment with SAM (100 nM, 2 days) increased promoter methylation, reduced the mRNA levels of these inflammatory genes, and decreased both 8-hydroxydeoxyguanosine (8-OHdG) and 8-nitroguanine levels by 50% (p < 0.01) in arsenic-treated cells. In addition, pretreatment with folic acid (10 μM, 7 days), a micronutrient, led to a significant increase in promoter methylation associated with the reduction in mRNA levels of these inflammatory genes and decreased levels of 8-OHdG and 8-nitroguanine by 80% and 90% (p < 0.01), respectively, compared with arsenic treatment alone. Moreover, pretreatments with these methyl group donors increased mRNA expression of an antioxidant defense regulator (Nrf2) and DNA repair genes (hOGG1, XRCC1, and PARP1). This study shows for the first time that SAM or folic acid supplementation can prevent arsenic-induced oxidative and nitrative DNA damage. This suggests the potential use of SAM or folic acid for prevention of arsenic toxicity in human populations.

SNCA correlates with immune infiltration and serves as a prognostic biomarker in lung adenocarcinoma

BackgroundThe SNCA gene is a critical gene in Parkinson’s disease (PD) pathology. Accumulating evidence indicates that SNCA is involved in tumorigenesis; however, the role of SNCA in lung adenocarcinoma (LUAD) remains unclear. This study aimed to explore the potential value of SNCA as a prognostic and diagnostic molecular marker in LUAD.MethodsIn this study, we explored the expression pattern, prognostic value, and promoter methylation status of SNCA in LUAD based on Oncomine, UALCAN, and Kaplan–Meier Plotter. Then, using TIMER, we investigated the correlation between SNCA expression and immune infiltration. And cBioPortal were used to analysis the correlation between SNCA expression and immune checkpoint. The transcriptome data of A549 cells overexpressing SNCA were used to further study the potential immune role of SNCA in LUAD. The effect of SNCA on proliferation of A549 cells were evaluated by CCK-8, EdU and colony formation. Finally, LUAD cell lines treated with 5-aza-dC were used to explore the correlation between increased promoter methylation and downregulated mRNA expression of SNCA.ResultsIn general, the expression level of SNCA in LUAD tissue was lower than that in normal tissue, and high expression of SNCA was related to better prognosis.There were significant positive correlations between SNCA expression and immune infiltrations, including CD8+ T cells, macrophages, neutrophils, dendritic cells, B cells, and CD4+ T cells, and immune checkpoints, suggesting that immune infiltration was one of the reasons for the influence of SNCA on prognosis in LUAD. The transcriptome data of A549 cells overexpressing SNCA were further used to screen the relevant immune-related genes regulated by SNCA. Enrichment analysis confirmed that SNCA participates in the PI3K-AKT signaling pathway and other key tumor signaling pathways and regulates the expression of MAPK3, SRC, PLCG1, and SHC1. Cellular proliferation assay showed that SNCA could inhabit the growth of A549 cells via inhibiting activity of PI3K/AKT/ mTOR pathway. Finally, analysis of the methylation level of SNCA promoter showed that the promoter methylation negatively correlated with mRNA level. The expression of SNCA in LUAD cell lines was significantly upregulated by treatment with 5-aza-dC.ConclusionHigh methylation of SNCA promoter in LUAD is one of the reasons for the downregulation of SNCA mRNA level. Given that SNCA could inhibit the proliferation of A549 cells and correlates with immune infiltrates, it may serve as a prognostic biomarker in LUAD.

DNMT1: catalytic and non-catalytic roles in different biological processes.

DNMT1 is the main enzyme that uses the information on DNA methylation patterns in the parent strand and methylates the daughter strand in freshly replicated hemimethylated DNA. It is widely known that DNMT1 is a component of the epigenetic machinery mediating gene repression via increased promoter methylation. However, recent data suggest that DNMT1 can also modulate gene expression independent of its catalytic activity and participates in multiple processes including the cell cycle, DNA damage repair and stem cell function. This review summarizes the noncanonical functions of DNMT1, some of which are clearly independent of maintenance methylation. Finally, phenotypic data on altered DNMT1 levels suggesting that maintenance of optimal levels of DNMT1 is vital for normal development and health is presented.

Loss of Integrin α9β1 on Tumor Keratinocytes Enhances the Stromal Vasculature and Growth of Cutaneous Tumors

Angiogenesis is critical to tumor progression, and the function of integrins in tumor angiogenesis is complex. In this study, we report that loss of integrin α9β1 expression from epidermal tumor cells is critical to maintaining persistent stromal vessel density. Forced expression of α9 in transformed mouse keratinocytes dramatically reduces vessel density in allograft tumors invivo compared with that in the same cells lacking α9β1. Moreover, α9 mRNA expression is dramatically reduced in mouse and human epidermal tumors as is α9β1-dependent gene regulation. Loss of tumor cell α9β1 occurs through at least two mechanisms: (i) ITGA9 gene copy number loss in human tumors and (ii) epigenetic silencing in mouse and human tumors. Importantly, we show that reversal of epigenetic silencing of Itga9 restores α9 expression in mouse keratinocytes and that human tumors without ITGA9 copy number loss have increased promoter methylation. Our data suggest that for epidermal tumorigenesis to occur, tumor cells must avoid the tumor and angiogenic suppressive effects of α9β1 by repressing its expression through deletion and/or epigenetic silencing, thereby promoting stromal development and tumor growth.