Downregulation Of DNA Methyltransferase Research Articles

Evaluating the role of DNA methyltransferases in trophoblast fusion and preeclampsia development: Insights from methylation-regulated genes.

The etiology of preeclampsia (PE), a complex and multifactorial condition, remains incompletely understood. DNA methylation, which is primarily regulated by three DNA methyltransferases (DNMTs), DNMT1, DNMT3A, and DNMT3B, plays a vital role in early embryonic development and trophectoderm differentiation. Yet, how DNMTs modulate trophoblast fusion and PE development remains unclear. In this study, we found that the DNMTs expression was downregulated during trophoblast cells fusion. Downregulation of DNMTs was observed during the reconstruction of the denuded syncytiotrophoblast (STB) layer of placental explants. Additionally, overexpression of DNMTs inhibited trophoblast fusion. Conversely, treatment with the DNA methylation inhibitor 5-aza-CdR decreased the expression of DNMTs and promoted trophoblast fusion. A combined analysis of DNA methylation data and gene transcriptome data obtained from the primary cytotrophoblasts (CTBs) fusion process identified 104 potential methylation-regulated differentially expressed genes (MeDEGs) with upregulated expression due to DNA demethylation, including CD59, TNFAIP3, SDC1, and CDK6. The transcription regulation region (TRR) of TNFAIP3 showed a hypomethylation with induction of 5-aza-CdR, which facilitated CREB recruitment and thereby participated in regulating trophoblast fusion. More importantly, clinical correlation analysis of PE showed that the abnormal increase in DNMTs may be involved in the development of PE. This study identified placental DNA methylation-regulated genes that may contribute to PE, offering a novel perspective on the role of epigenetics in trophoblast fusion and its implication in PE development.

PRMT1 Inhibition Activates the Interferon Pathway to Potentiate Antitumor Immunity and Enhance Checkpoint Blockade Efficacy in Melanoma.

Targeting PRMT1 stimulates interferon signaling by increasing expression of endogenous retroviral elements and double-stranded RNA through repression of DNMT1, which induces antitumor immunity and synergizes with immunotherapy to suppress tumor progression.

DNMT1-mediated DNA methylation in toll-like receptor 4 (TLR4) inactivates NF-κB signal pathway-triggered pyroptotic cell death and cellular inflammation to ameliorate lipopolysaccharides (LPS)-induced osteomyelitis

Toll-like receptor 4 (TLR4) plays a critical role in various human diseases, and was associated with pyroptotic cell death and inflammatory responses. DNA methylation, which has stable and reversible properties, has been reported to alter the expression of target genes, including TLR4. However, the role of methylated TLR4 in osteomyelitis (OM) and the underlying molecular mechanisms remain unclear. RNA sequencing was used to identify differentially expressed genes and associated signaling pathways. RT-qPCR, Western blot, emzyme-linked immunosorbent assay (ELISA), cell counting kit-8 (CCK-8) and LDH assay kit were used to detect mRNA and protein expression of relevant genes, cell viability and the LDH activity, respectively. TLR4 methylation was detected by methylation-specific PCR (MSP) and verified by Chromatin immunoprecipitation (ChIP). Here, we found that DNA methyltransferase-1 (DNMT1)-mediated TLR4 demethylation significantly suppressed lipopolysaccharides (LPS)-induced pyroptosis and inflammatory response by inhibiting the TLR4/nuclear transcription factor-kappa B (NF-κB) axis. First, we confirmed TLR4 as the study target by mRNA transcriptome sequencing analysis, and TLR4 was observably high-expressed in both OM patients and LPS-treated osteoblastic MC3T3-E1. Then, we found that downregulation of DNMT1 blocked TLR4 promoter methylation modification, resulting in upregulation of TLR4. Simultaneously, functional experiments indicated that suppression of TLR4 or overexpression of DNMT1 promoted cell proliferation and inhibited cell pyroptosis and inflammation in LPS-induced MC3T3-E1, while upregulation of TLR4 restored the effects of DNMT1 silencing on OM progression. In addition, TLR4 elevated phosphorylation of IκB-α and NF-κB p65 in the NF-κB signal pathway, and inhibition of TLR4 or the NF-κB inhibitor PDTC reversed the influence of inhibition of DNMT1. In conclusion, our study demonstrated that DNMT1-mediated TLR4 DNA methylation alleviated LPS-induced OM by inhibiting the NF-κB signaling pathway.

Pharmacokinetics and pharmacodynamics of an oral formulation of decitabine and tetrahydrouridine.

Sickle cell disease (SCD) is caused by an inherited structural abnormality of adult hemoglobin causing polymerization. Fetal hemoglobin interferes with polymerization but is epigenetically silenced by DNA methyltransferase 1 (DNMT1) in adult erythropoiesis. Decitabine depletes DNMT1 and increases fetal and total hemoglobin in SCD patients, but is rapidly catabolized by cytidine deaminase (CDA) in vivo. Tetrahydrouridine (THU) inhibits CDA, safeguarding decitabine. The pharmacokinetics and pharmacodynamics of three oral combination formulations of THU and decitabine, with different coatings producing different delays in decitabine release, were investigated in healthy participants. Tetrahydrouridine and decitabine were rapidly absorbed into the systemic circulation after a single combination oral dose, with relative bioavailability of decitabine ≥74% in fasted males compared with separate oral administration of THU followed by decitabine 1 h later. THU and decitabine Cmax and area under the plasma concentration versus time curve were higher in females versus males, and fasted versus fed states. Despite sex and food effect on pharmacokinetics, the pharmacodynamic effect of DNMT1 downregulation was comparable in males and females and fasted and fed states. Treatments were well tolerated. Combination oral formulations of THU with decitabine produced pharmacokinetics and pharmacodynamics suitable for oral DNMT1-targeted therapy.

Ginseng mediates its anticancer activity by inhibiting the expression of DNMTs and reactivating methylation-silenced genes in colorectal cancer.

Developing safe and effective therapeutic modalities remains a critical challenge for improving the prognosis of patients with colorectal cancer (CRC). In this regard, targeting epigenetic regulation in cancers has recently emerged as a promising therapeutic approach. Since several natural compounds have recently been shown to be important epigenetic modulators, we hypothesized that Ginseng might exert its anticancer activity by regulating DNA methylation alterations in CRC. In this study, a series of cell culture studies were conducted, followed by their interrogation in patient-derived 3D organoid models to evaluate Ginseng's anticancer activity in CRC. Genome-wide methylation alterations were interrogated by undertaking MethylationEpic BeadChip microarrays. First, 50% inhibitory concentrations (IC50) were determined by cell viability assays, and subsequent Ginseng treatment demonstrated a significant anticancer effect on clonogenicity and cellular migration in CRC cells. Treatment with Ginseng potentiated cellular apoptosis through regulation of apoptosis-related genes in CRC cells. Furthermore, Ginseng treatment downregulated the expression of DNA methyltransferases (DNMTs) and decreased the global DNA methylation levels in CRC cells. The genome-wide methylation profiling identified Ginseng-induced hypomethylation of transcriptionally silenced tumor suppressor genes. Finally, cell culture-based findings were successfully validated in patient-derived 3D organoids. In conclusion, we demonstrate that Ginseng exerts its antitumorigenic potential by regulating cellular apoptosis via the downregulation of DNMTs and reversing the methylation status of transcriptionally silenced genes in CRC.

DNMT1 mediates proliferation, migration and invasion of extravillous trophoblasts by regulating the methylation level of APLNR

IntroductionProliferation, migration and invasion of extravillous trophoblasts (EVTs) play an important role in the progression of preeclampsia (PE). The purpose of this study was to investigate the molecular mechanism by which DNA methylase regulates the transcription level of APLNR and affects the phenotypic function of EVTs. Materials and methodsPE mice model and H/R model in HTR8/Svneo cells were constructed. Clinical samples of normal pregnant women and PE patients were collected. Expression and methylation level of APLNR in vivo and in vitro were detected. ChIP-qPCR was used to detect the binding of DNA methyltransferase at the APLNR promoter. The expression of DNA methyltransferase 1 (DNMT1), NO and eNOS in vitro were detected. EVTs proliferation, migration and invasion in vitro were detected. ResultsIn placental tissues or HTR8/Svneo cells of the PE model group, the expression of APLNR was reduced and APLNR methylation level was up-regulated. There was no significant difference in the APLNR expression in placental tissues between normal pregnant women and PE patients. H/R conditions only promote the binding of DNMT1 at the APLNR promoter. DNMT1 interference decreased the enrichment degree of DNMT1 in APLNR promoter region and up-regulated the mRNA and protein levels of APLNR in vivo and in vitro. The activation of APLNR by Elabela (ELA) can promote eNOS transcription, thereby promoting cell proliferation and NO level, while eNOS inhibitor can reverse this effect. DNMT1 down-regulation inhibted APLNR methylation level, promoted eNOS transcription, and promoted EVTs proliferation, migration and invasion, which could be revised by the interference of APLNR. DiscussionDNMT1 promotes eNOS transcription by inhibting APLNR methylation level, and promotes EVTs proliferation, migration and invasion, thus providing a new and broad application prospect for PE treatment.

5613127 PHARMACOKINETICS AND PHARMACODYNAMICS OF AN INNOVATIVE ORAL FORMULATION OF DECITABINE AND TETRAHYDROURIDINE IN HEALTHY PEOPLE

Background: Sickle cell disease (SCD) pathophysiology emerges from an inherited structural abnormality of adult hemoglobin that causes it to polymerize when deoxygenated, forcing red blood cells (RBCs) into a sickled conformation.1 Sickled RBCs are rigid, damage vessel endothelium, are less able to flow, and are more likely to hemolyze than healthy RBCs.1 Fetal hemoglobin interferes with polymerization but is epigenetically silenced by DNA methyltransferase 1 (DNMT1) in adult (post-fetal) erythropoiesis.2 Decitabine, a DNMT1 inhibitor, can increase fetal and total hemoglobin in people with SCD; however in vivo it is rapidly catabolized by cytidine deaminase (CDA).3 Tetrahydrouridine (THU) inhibits CDA thus safeguarding and thereby increasing the bioavailability of decitabine.3–5 Aims: Building on previous work examining oral administration of THU and decitabine separated by 1 hour,3 this study evaluated the pharmacokinetics, pharmacodynamics, and safety of three test formulations of a combination capsule with delayed-release decitabine and THU. This will inform development of an optimum oral combination formulation that is more practical for evaluation, adherence, and clinical use. Methods: The pharmacokinetics and pharmacodynamics of three test formulations of a combination capsule with delayed-release decitabine and THU were investigated in two phase 1 studies: one in healthy adult males (n=16) under fasting conditions (NCT03828084) vs a reference treatment of THU followed 1 hour (±5 min) later by decitabine; and one in healthy adult males and females (n=48) under fed and fasting conditions (NCT04086238). The three combined formulations (designated A, B, C) differed by composition of the coating, producing different delays in decitabine release: treatment A provided the least delay in decitabine release and treatment C the greatest. The reference treatment was designated D. Results: A single, combined oral dose led to rapid absorption of THU and decitabine into systemic circulation (Figure). The relative bioavailability of decitabine in plasma was ≥74% in fasted males, compared with separate oral administration of THU followed by decitabine 1 hour later. Higher THU and decitabine maximum concentration (Cmax) and area under the plasma concentration–time curve (AUC) were observed in females vs males, and fasted vs fed states. In the fed state, decitabine Cmax and AUC were comparable between males and females for formulations A and B. Despite a food effect on pharmacokinetics, the pharmacodynamic effect of DNMT1 downregulation post-dose was comparable in both fasted and fed states. The combination formulations and the reference treatment were well tolerated. Treatment-emergent adverse events were mild or moderate with no serious adverse events. Platelet and neutrophil counts did not change significantly. Conclusion: The combination formulation of THU with decitabine produced pharmacokinetics and pharmacodynamics suitable for oral DNMT1-targeted therapy, the goal of which is to increase fetal hemoglobin in people with SCD. The safety and efficacy of a combination formulation developed based on these results is being evaluated in the phase 2 trial ASCENT1 (NCT05405114).

Synergistic Interactions between the Hypomethylating Agent Thio-Deoxycytidine and Venetoclax in Myelodysplastic Syndrome Cells

Interactions between the novel hypomethylating agent (HMA) thio-deoxycytidine (T-dCyd) and the BCL-2 antagonist ABT-199 (venetoclax) have been examined in human myelodysplastic syndrome (MDS) cells. The cells were exposed to agents alone or in combination, after which apoptosis was assessed, and a Western blot analysis was performed. Co-administration of T-dCyd and ABT-199 was associated with the down-regulation of DNA methyltransferase 1 (DNMT1) and synergistic interactions documented by a Median Dose Effect analysis in multiple MDS-derived lines (e.g., MOLM-13, SKM-1, and F-36P). Inducible BCL-2 knock-down significantly increased T-dCyd's lethality in MOLM-13 cells. Similar interactions were observed in the primary MDS cells, but not in the normal cord blood CD34+ cells. Enhanced killing by the T-dCyd/ABT-199 regimen was associated with increased reactive oxygen species (ROS) generation and the down-regulation of the anti-oxidant proteins Nrf2 and HO-1, as well as BCL-2. Moreover, ROS scavengers (e.g., NAC) reduced lethality. Collectively, these data suggest that combining T-dCyd with ABT-199 kills MDS cells through an ROS-dependent mechanism, and we argue that this strategy warrants consideration in MDS therapy.

NRF2 modulation in TRAMP mice: an in vivo model of prostate cancer.

Prostate cancer (PCa) is one of the most common cancers worldwide and oxidative stress is involved in its occurrence, development and progression. In fact, in transgenic adenocarcinoma of mouse prostate (TRAMP) mice, prostate cancer onset is associated with the methylation of the first five CpG in the nuclear factor erythroid 2-related factor 2 (NRF2) promoter, a key regulator of oxidative stress response, leading to its downregulation and accumulation of reactive oxygen species (ROS). It has been demonstrated that both natural and synthetic compounds can reactivate NRF2 expression inhibiting the methylation status of its promoter by downregulation of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs). Interestingly, NRF2 re-expression significantly reduced prostate cancer onset in TRAMP mice highlighting an important role of NRF2 in prostate tumorigenesis. We analysed the current literature regarding the role of natural and synthetic compounds in modulating NRF2 pathway in TRAMP mice, an in vivo model of prostate cancer, to give an overview on prostate carcinogenesis and its possible prevention. We can conclude that specific natural and synthetic compounds can downregulate DNMTs and/or HDACs inhibiting the methylation status of NRF2 promoter, then reactivating the expression of NRF2 protecting normal prostatic cells from ROS damage and tumorigenesis.

Dynamicity of histone H3K27ac and H3K27me3 modifications regulate the cold-responsive gene expression in Oryza sativa L. ssp. indica

Cultivated in tropical and subtropical regions, Oryza sativa L. ssp. indica is largely affected by cold-stress, especially at the seedling stage. The present model of the stress-responsive regulatory network in plants entails the role of genetic and epigenetic factors in stress-responsive gene expression. Despite extensive transcriptomic studies, the regulation of various epigenetic factors in plants cold-stress response is less explored. The present study addresses the effect of genome-wide changes of H3K27 modifications on gene expression in IR64 rice, during cold-stress. Our results suggest a positive correlation between the changes in H3K27 modifications and stress-responsive gene activation in indica rice. Cold-induced enrichment of H3K27 acetylation promotes nucleosomal rearrangement, thereby facilitating the accessibility of the transcriptional machinery at the stress-responsive loci for transcription activation. Although H3K27ac exhibits uniform distribution throughout the loci of enriched genes; occupancy of H3K27me3 is biased to intergenic regions. Integration of the ChIP-seq data with transcriptome indicated that upregulation of stress-responsive TFs, photosynthesis-TCA-related, water-deficit genes, redox and JA signalling components, was associated with differential changes of H3K27ac and H3K27me3 levels. Furthermore, cold-induced upregulation of histone acetyltransferases and downregulation of DNA methyltransferases was noted through the antagonistic switch of H3K27ac and H3K27me3. Moreover, motif analysis of H3K27ac and H3K27me3 enriched regions are associated with putative stress responsive transcription factors binding sites, GAGA element and histone H3K27demethylase. Collectively our analysis suggests that differential expression of various chromatin and DNA modifiers coupled with increased H3K27ac and depleted H3K27me3 increases DNA accessibility, thereby promoting transcription of the cold-responsive genes in indica rice.

Combinatorial epigenetic mechanisms of sulforaphane, genistein and sodium butyrate in breast cancer inhibition

Dietary phytochemicals are currently being studied with great interest due to their ability to regulate the epigenome resulting in prevention of cancer. Some natural botanicals have been reported to have enhanced and synergistic impact on cancer suppression when administered at optimum concentrations and in-conjunction. Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables and sodium butyrate (NaB) is a short-chain fatty acid produced by gut microbiota. They have been intensively explored due to numerous anti-cancerous properties and ability to modulate epigenetic machinery by inhibition of histone deacetylase (HDAC). Genistein (GE), present in soy, is a known DNA methyltransferase (DNMT) inhibitor. While combined chemoprotective epigenetic effects induced by SFN and GE have been investigated, the key impact of combinatorial SFN-NaB, GE-NaB, and SFN-GE-NaB bioactive components in regulation of various mechanisms are poorly defined. In the present study, we found that combinations of dietary compounds had synergistic effects in decreasing cellular viability at lower dosages than their single dosages in breast cancer cell lines. The respective combinations limited growth and increased apoptosis and necrosis in cancerous cells among which the tri-combination displayed the most significant impact. Additionally, the respective combinations of compounds arrested MDA-MB-231 and MCF-7 breast cancer cells at G2/M phase. Our further mechanistic evaluation revealed that respective di-combinations and tri-combination had higher impact in down-regulation of DNMTs (DNMT3A and DNMT3B), HDACs (HDAC1, HDAC6 and HDAC11), histone methyltransferases (EZH2 and SUV39H1) and histone acetyltransferases (GCN5, PCAF, P300 and CBP) levels as compared to singly administered compounds. We also found that these combinations exhibited global epigenetic changes by inhibition of DNMT and HDAC activity, histone H3 at lysine 27 methylation (H3K27me) and histone H3 at lysine 9 methylation (H3K9me) levels, and by induction of histone acetyltransferases activity. Collectively, our investigation indicates that combined SFN, GE and NaB is highly effective in inhibiting breast cancer genesis by, at least in part, regulating epigenetic modifications, which may have implications in breast cancer therapy.

Epigallocatechin Gallate Induces the Demethylation of Actinin Alpha 4 to Inhibit Diabetic Nephropathy Renal Fibrosis via the NF-KB Signaling Pathway In Vitro.

Actinin alpha 4 (ACTN4) is expressed in the kidney podocytes. ACTN4 gene methylation in patients with diabetic nephropathy (DN) remains high. Underlying mechanism of epigallocatechin-3-gallate (EGCG) inducing ACTN4 demethylation, and its inhibitory effect on DN renal fibrosis remains unclear.Methods:Human podocyte cell line, HPC, was treated with high glucose to establish model of DN. The levels of cytokines, vascular endothelial growth factor (VEGF) and interleukin (IL)-8, and fibrosis markers, alpha smooth muscle actin (α-SMA) and fibronectin (FN), were determined using enzyme-linked immunosorbent assay. HPC cells were treated with EGCG, and cell viability was determined by MTT assay, ACTN4 gene methylation was analyzed by MSP. mRNA and protein expression levels were measured using RT-qPCR and Western blotting, respectively.Results:Actinin alpha 4 gene promoter was hypermethylated in the high glucose-treated groups. EGCG reversed the hypermethylated status of ACTN4, along with the upregulation of ACTN4 levels and downregulation of DNA methyltransferase 1 (DNMT1), NF-κB p65, p-NF-κB p65, IκB-α, VEGF, IL-8, α-SMA, and FN levels (P<.05).Conclusion:Epigallocatechin-3-gallate reduced hypermethylation of ACTN4 in HPC cells by downregulating DNMT1 expression and restoring ACTN4 expression, contributing to the upregulation of the NF-KB p65, p-NF-KB p65, IKB-α, VEGF, IL-8, α-SMA, and FN levels (P<.05).

Role of ascorbic acid in the regulation of epigenetic processes induced by Porphyromonas gingivalis in endothelial-committed oral stem cells

Periodontitis is a common inflammatory disease that affects the teeth-supporting tissue and causes bone and tooth loss. Moreover, in a worldwide population, periodontal disease is often associated with cardiovascular diseases. Emerging studies have reported that one of the major pathogens related to periodontitis is Porphyromonas gingivalis (P. gingivalis), which triggers the inflammatory intracellular cascade. Here, we hypothesized a possible protective effect of ascorbic acid (AA) in the restoration of the physiological molecular pathway after exposure to lipopolysaccharide derived from P. gingivalis (LPS-G). In particular, human gingiva-derived mesenchymal stem cells (hGMSCs) and endothelial-differentiated hGMSCs (e-hGMSCs) exposed to LPS-G showed upregulation of p300 and downregulation of DNA methyltransferase 1 (DNMT1), proteins associated with DNA methylation and histone acetylation. The co-treatment of AA and LPS-G showed a physiological expression of p300 and DNMT1 in hGMSCs and e-hGMSCs. Moreover, the inflammatory process triggered by LPS-G was demonstrated by evaluation of reactive oxygen species (ROS) and their intracellular localization. AA exposure re-established the physiological ROS levels. Despite the limitations of in vitro study, these findings collectively expand our knowledge regarding the molecular pathways involved in periodontal disease, and suggest the involvement of epigenetic modifications in the development of periodontitis.

Epigenetic landscape in blood leukocytes following ketosis and weight loss induced by a very low calorie ketogenic diet (VLCKD) in patients with obesity

The molecular mechanisms underlying the potential health benefits of a ketogenic diet are unknown and could be mediated by epigenetic mechanisms. To identify the changes in the obesity-related methylome that are mediated by the induced weight loss or are dependent on ketosis in subjects with obesity underwent a very-low calorie ketogenic diet (VLCKD). Twenty-one patients with obesity (n=12 women, 47.9±1.02yr, 33.0±0.2kg/m2) after 6 months on a VLCKD and 12 normal weight volunteers (n=6 women, 50.3±6.2yrs, 22.7±1.5kg/m2) were studied. Data from the Infinium MethylationEPIC BeadChip methylomes of blood leukocytes were obtained at time points of ketotic phases (basal, maximum ketosis, and out of ketosis) during VLCKD (n=10) and at baseline in volunteers (n=12). Results were further validated by pyrosequencing in representative cohort of patients on a VLCKD (n=18) and correlated with gene expression. After weight reduction by VLCKD, differences were found at 988 CpG sites (786 unique genes). The VLCKD altered methylation levels in patients with obesity had high resemblance with those from normal weight volunteers and was concomitant with a downregulation of DNA methyltransferases (DNMT)1, 3a and 3b. Most of the encoded genes were involved in metabolic processes, protein metabolism, and muscle, organ, and skeletal system development. Novel genes representing the top scoring associated events were identified, including ZNF331, FGFRL1 (VLCKD-induced weight loss) and CBFA2T3, C3orf38, JSRP1, and LRFN4 (VLCKD-induced ketosis). Interestingly, ZNF331 and FGFRL1 were validated in an independent cohort and inversely correlated with gene expression. The beneficial effects of VLCKD therapy on obesity involve a methylome more suggestive of normal weight that could be mainly mediated by the VLCKD-induced ketosis rather than weight loss.

DNA Methylation may be a testicular plateau adaptation in Tibetan pig

ABSTRACT DNA methylation plays an important role in reproduction. Tibetan pigs maintain high sperm motility, even under extreme conditions at high altitudes. However, the DNA methylation mechanisms underlying testicular plateau adaptations in Tibetan pigs remain unclear. We determined the genomic DNA methylation level in the testicular tissues of Tibetan pigs living at high altitudes (TP) and of Yorkshire pigs that migrated to high altitudes (YP) using the fluorescence method. Quantitative real-time PCR and western blots were used to measure the gene and protein expression levels, respectively, of three DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and hypoxia-inducible factor 2α (HIF2α). We found that the genomic DNA methylation level and the mRNA and protein expression levels of the four genes were all significantly lower in the testicular tissues of TP than YP. In short, compared with YP, the downregulation of DNA methyltransferases in the TP testes led to lower levels of genomic DNA methylation, which would facilitate higher sperm motility. Meanwhile, HIF2α expression in the TP testes was down-regulated, which might contributes to TP testes’ adaption to the plateau environment. This study provided new insights into the DNA methylation mechanisms underlying plateau adaptations in TP testicles.

Sustained expression of MCP-1 induced low wall shear stress loading in conjunction with turbulent flow on endothelial cells of intracranial aneurysm.

Shear stress was reported to regulate the expression of AC007362, but its underlying mechanisms remain to be explored. In this study, to isolate endothelial cells of blood vessels, unruptured and ruptured intracranial aneurysm (IA) tissues were collected from IA patients. Subsequently, quantitative real‐time PCR (qRT‐PCR), Western blot and luciferase assay were performed to investigate the relationships between AC007362, miRNAs‐493 and monocyte chemoattractant protein‐1 (MCP‐1) in human umbilical vein endothelial cells (HUVECs) exposed to shear stress. Reduced representation bisulphite sequencing (RRBS) was performed to assess the level of DNA methylation in AC007362 promoter. Accordingly, AC007362 and MCP‐1 were significantly up‐regulated while miR‐493 was significantly down‐regulated in HUVECs exposed to shear stress. AC007362 could suppress the miR‐493 expression and elevate the MCP‐1 expression, and miR‐493 was shown to respectively target AC007362 and MCP‐1. Moreover, shear stress in HUVECs led to the down‐regulated DNA methyltransferase 1 (DNMT1), as well as the decreased DNA methylation level of AC007362 promoter. Similar results were also observed in ruptured IA tissues when compared with unruptured IA tissues. In conclusion, this study presented a deep insight into the operation of the regulatory network of AC007362, miR‐493 and MCP‐1 upon shear stress. Under shear stress, the expression of AC007362 was enhanced by the inhibited promoter DNA methylation, while the expression of MCP‐1 was enhanced by sponging the expression of miR‐493.

Social defeat stress exacerbates atopic dermatitis through downregulation of DNA methyltransferase 1 and upregulation of C–C motif chemokine receptor 7 in skin dendritic cells

DNA methylation is an epigenetic modification that regulates gene transcription. DNA methyltransferase 1 (DNMT1) plays an important role in DNA methylation. However, the involvement of DNMT1 and DNA methylation in the pathogenesis of atopic dermatitis (AD) remains unclear. In this study, microarray analysis revealed that peripheral blood mononuclear cells of AD patients with low DNMT1 expression (DNMT1-low) highly expressed dendritic cell (DC) activation-related genes. Also, DNMT1-low AD patients exhibited a higher itch score compared to AD patients with high DNMT1 expression (DNMT1-high). By using an AD-like mouse model induced by the application of Dermatophagoides farinae body ointment, we found that Dnmt1 expression was decreased, while the expression of C–C chemokine receptor type 7 (Ccr7) was upregulated in mouse skin DCs. Furthermore, mice exposed to social defeat stress exhibited Dnmt1 downregulation and Ccr7 upregulation in skin DCs. Additionally, dermatitis and itch-related scratching behavior were exacerbated in AD mice exposed to stress. The relationship between low DNMT1 and itch induction was found in both human AD patients and AD mice. In mouse bone marrow-derived DCs, Ccr7 expression was inhibited by 5-aza-2-deoxycytidine, a methylation inhibitor. Furthermore, in mouse skin DCs, methylation of CpG sites in Ccr7 was modified by either AD induction or social defeat stress. Collectively, these findings suggest that social defeat stress exacerbates AD pathology through Dnmt1 downregulation and Ccr7 upregulation in mouse skin DCs. The data also suggest a role of DNMT1 downregulation in the exacerbation of AD pathology.

Epidrug Modulated Expression of MiR--152 and MiR-148a Reverse Cisplatin Resistance in Ovarian CancerCells: An Experimental In-vitro Study.

Cisplatin is a common agent which is used to treat Epithelial Ovarian Cancer (EOC), but cisplatin resistance is a major obstacle in successful treatment of ovarian cancer. Aberration in epigenetic changes play an important role in disregulation of gene expression. MiR-152 and miR-148a are frequently down-regulated in EOC due to promoter hyper-methylation. DNA methyltransferase1 (DNMT1), the main enzyme in maintenance of the pattern of DNA methylation, is one of the targets of miR-152 and miR-148a. Aberrantly up-regulation of DNMT1 is responsible for silencing of tumor suppressor genes in carcinogenesis. We hypothesized that re-expression of miR-152 and miR-148a and consequently down-regulation of DNMT1 may resensitize cancerous cells to chemotherapeutics agents. The aim of the present study is to investigate the effect of 5-azacytidine (5-Aza) and Trichostatin A on miR-152 and miR-148a expression in A2780CP ovarian cancer cell line. Optimal doses of 5-Azacitidine and TSA were measured by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A2780CP cell line was treated by each drugs, alone or in combination and the expression of miR-148a, miR-152 and DNMT1 was evaluated by Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-qPCR). The results revealed that TSA and 5-Azacytidine are able to revive the expression of miR-148a and miR-152 genes and mediate growth inhibition of epithelial ovarian cancer cells. The present study suggests that re-expression of miR-148a and miR-152 by epigenetic therapy aiming to DNMT1 suppression might resensitize resistant ovarian tumors to conventional chemotherapy.

Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning.

Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice, accompanied by the downregulation of DNA methyltransferases (DNMTs) in the brain. However, the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood. This study aimed to establish an in vitro model of hypoxic preconditioning, using a cultured mouse hippocampal neuronal cell line (HT22 cells), to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning. HT22 cells were divided into a control group, which received no exposure to hypoxia, a hypoxia group, which was exposed to hypoxia once, and a hypoxic preconditioning group, which was exposed to four cycles of hypoxia. To test the ability of hypoxic preadaptation to induce hypoxic tolerance, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay. Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group. The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by western blot assay and flow cytometry. Compared with the hypoxia group, the expression levels of caspase-3 and spectrin, which are markers of early apoptosis and S-phase arrest, respectively, noticeably reduced in the hypoxic preconditioning group. Finally, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning. The results showed that compared with the control group, hypoxic preconditioning downregulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B. In conclusion, hypoxic preconditioning may exert anti-hypoxic neuroprotective effects, maintaining HT22 cell viability and inhibiting cell apoptosis. These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.

Effect of 5-aza-2'-deoxycytidine on Estrogen Receptor Alpha/Beta and DNA Methyltransferase 1 Genes Expression, Apoptosis Induction, and Cell Growth Prevention of the Colon Cancer HT 29 Cell Line.

Background:Cellular activity such as gene expression is regulated by epigenetic mechanisms and modifications. In mammals, DNA methylation is an essential component of the epigenetic machinery of the cells. DNA hypermethylation of the several tumor suppressor genes (TSGs) is associated with transcriptional gene silencing resulting in colon tumorigenesis. Overexpression of DNA methyltransferase 1 (DNMT1) in colon cancer has been reported in several studies. The methylation of estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) have been demonstrated in various cancers. Previously, we indicated that genistein can reactivate ERα in hepatocellular carcinoma (HCC). The present study was designed to investigate the effect of 5-aza-2′-deoxycytidine (5-aza-CdR) on ERα/ERβ and DNMT1 gene expression, apoptosis induction, and cell viability inhibition of the colon carcinoma HT 29 cell line.Methods:The effect of 5-Aza-CdR on the colon carcinoma HT 29 cell viability was measured by MTT assay. To determine the apoptotic cells, the cells were assessed using the Annexin V-FITC/PI detection kit. The expression of ERα, ERβ, and DNMT1 genes was determined using real-time quantitative RT-PCR.Results:The results indicated that 5-Aza-CdR can inhibit cell growth significantly versus control groups, induce significant apoptosis, down-regulate DNMT1, and up-regulate ERα and ERβ genes expression at different time periods. The percentage of apoptotic cells was 85.83% and 86.84% after 24 and 48 h, respectively (P < 0.01). The IC50 value for 5-Aza-CdR was obtained at 2.5 μM.Conclusions:5-Aza-CdR can up-regulate ERα and ERβ genes expression through DNMT1 down-regulation resulting in apoptosis induction and cell growth prevention.