DNMT1 mRNA Research Articles

DNMT1 driven by mouse amniotic fluid mesenchymal stem cell exosomes improved corneal cryoinjury via inducing microRNA-33 promoter DNA hypermethylation modification in corneal epithelium cells.

Severe corneal cryoinjury can cause permanent corneal swelling and bullous keratopathy, one of the main reason for loss of sight. Mouse amniotic fluid mesenchymal stem cells (mAF-MSCs) can repair corneal damage caused by freezing; however, whether the exosomes derived from mAF-MSCs have the same repair effect is unknown. In this study, the mAF-MSC-exosomes were transplanted into the eyeballs of corneal cryoinjured mice. Histopathological examination showed that the mAF-MSC-exosomes improved the corneal structure and status of corneal epithelial cells in corneal cryoinjured mice. RRBS-sequencing showed that compared with the control group, four genes (Rpl13-ps6, miR-33, Hymai, and Plagl1), underwent DNA hypermethylation modification after mAF-MSC-exosomes treatment. The result of FISH indicated that miR-33-3p hybridization signals were enhanced in corneal epithelial cells from mice treated with mAF-MSC-exosomes. Semi-quantitative PCR and western blotting indicated that mAF-MSC-exosomes contained high levels of DNMT1 mRNA and protein. Additionally, luciferase report assays indicated that miR-33-3p overexpression in NIH-3T3 mouse embryonic fibroblast cells inhibited the activity of luciferase carrying a sequence from the 3' untranslated region of Bcl6. Moreover, BCL6 mRNA and protein levels in corneal tissues from mice treated with mAF-MSC-exosomes were higher than those in the control group. Therefore, our results suggested that mAF-MSC-exosomes could repair corneal cryoinjury by releasing DNMT1, which induced hypermethylation of the miR-33 promoter in corneal epithelial cells. Consequent downregulated miR-33 transcription upregulated Bcl6 expression, ultimately achieving the repair of corneal cryoinjury in mice.

Changes in <i>DNMT1</i> expression as a marker of epigenetic regulation disturbanses in multiple sclerosis patients

BACKGROUND: Multiple sclerosis is a chronic neurodegenerative autoimmune disease characterized by the presence of foci of inflammation and demyelination in the central nervous system. The initiation of pathological processes in multiple sclerosis is caused by a complex interaction of genetic factors, unfavorable environmental factors and epigenetic influences. Progressive neurological symptoms caused by axonal conduction disorders, axonal death and neurodestruction lead to a significant decreased patients’ quality of life and disability. The search for a new markers to improve diagnostic and therapeutic methods, including taking into account the genetic background and epigenetic interactions, is an urgent task.
 AIM: The work was aimed to study the changes in DNMT1 mRNA expression in multiple sclerosis patients with different disease duration, to analyze methylation of DNMT1 promoter, and compare the changes in the level of DNMT1 expression with the homocysteine content in the blood, and the presence of polymorphic variants in genes coding the key folate cycle enzymes.
 MATERIALS AND METHODS: The level of DNMT1 mRNA expression in peripheral mononuclear blood cells was assessed by reversed transcription followed by polymerase chain reaction. Fluorescent polymerase chain reaction followed by methyl-sensitive analysis of high-resolution melting curves was used to analyze methylation of the DNMT1 promoter. The content of homocysteine in the blood was determined by chemiluminescence immunoassay. The real-time polymerase chain reaction was used for genotyping by polymorphism of folate cycle genes; the fluorescent probes with the LNA modifications were used to discriminate alleles.
 RESULTS: It has been shown that in multiple sclerosis patients, including those at the onset of the disease, the level of DNMT1 mRNA expression is significantly lower than in the control group. No relationship was found between the decrease in DNMT1 expression and the level of promoter methylation. Strong positive relationship between the level of DNMT1 mRNA expression and homocysteine content in patients with multiple sclerosis and the combined effects of the genotypes of MTR A2756G and MTHFR C677T polymorphism on the expression of DNMT1 have been shown. These findings suggest that genetically determined features of folate metabolism may contribute to the disruption of epigenetic regulation in multiple sclerosis.
 CONCLUSIONS: The obtained results indicate the promise of research aimed to identifying the factors causing epigenetic changes in multiple sclerosis. Studying the mechanisms of the folate cycle genes polymorphic variants contribution to the pathogenesis of multiple sclerosis could be one of the possible ways to improve diagnostic and therapeutic approaches.

Comprehensive Analysis of DNA Methyltransferases Expression in Primary and Relapsed Ovarian Carcinoma.

Despite recent advances in epithelial ovarian carcinoma (EOC) treatment, its recurrence and mortality rates have not improved significantly. DNA hypermethylation has generally been associated with an ominous prognosis and chemotherapy resistance, but the role of DNA methyltransferases (DNMTs) in EOC remains to be investigated. In the current study, we systematically retrieved gene expression data from patients with EOC and studied the immunohistochemical expression of DNMTs in 108 primary and 26 relapsed tumors. Our results showed that the DNMT1, DNMT3A, DNMT3B and DNMT3L RNA levels were higher and the DNMT2 level was lower in tumors compared to non-neoplastic tissue, and DNMT3A and DNMT2 expression decreased from Stage-II to Stage-IV carcinomas. The proteomic data also suggested that the DNMT1 and DNMT3A levels were increased in the tumors. Similarly, the DNMT1, DNMT3A and DNMT3L protein levels were overexpressed and DNMT2 expression was reduced in high-grade carcinomas compared to non-neoplastic tissue and low-grade tumors. Moreover, DNMT1 and DNMT3L were increased in relapsed tumors compared to their primaries. The DNMT3A, DNMT1 and DNMT3B mRNA levels were correlated with overall survival. Our study demonstrates that DNMT1 and DNMT3L are upregulated in primary high-grade EOC and further increase in relapses, whereas DNMT3A is upregulated only in the earlier stages of cancer progression. DNMT2 downregulation highlights the presumed tumor-suppressor activity of this gene in ovarian carcinoma.

Polyphyllin I induces ferroptosis in castration-resistant prostate cancer cells through the ERK/DNMT1/ACSL4 axis.

Castration-resistant prostate cancer (CRPC) inevitably arises after androgen deprivation therapy (ADT). Therefore, there is an urgent need to search for novel treatment strategies for CRPC. Polyphyllin I (PPI), one of the steroidal saponins in paris polyphylla, has been shown to have an anticancer effect. This study investigated the role and mechanism of PPI in CRPC cell ferroptosis. Protein levels of GPX4, p-extracellular regulated protein kinases (ERK), ERK, DNMT1, and ACSL4 were measured by Western blot. DNMT1 and ACSL4 mRNA expression was analyzed byreverse transcription-quantitative polymerase chain reaction (RT-qPCR). Prostate cancer cells (DU145, PC3) were treated with PPI. Cell viability was assessed utilizing Cell Counting Kit-8 (CCK-8) assay. The role of PPI in regulating ferroptosis was determined by analyzing lipid reactive oxygen species (ROS), malonyl dialdehyde (MDA), iron (Fe2+ ), and glutathione (GSH) content. Chromatin immunoprecipitation (ChIP) assay verified the effect of DNMT1 on the ACSL4 promoter. The methylation level of ACSL4 promoter was assessed utilizing MSP. A nude mice xenograft was adopted to detect the effect of PPI in vivo. PPI inhibited CRPC cell proliferation, reduced levels of GSH and GPX4, and increased levels of MDA, Fe2+ , and ROS, while ERK inhibitor reversed the effect of PPI on ferroptosis. PPI repressed the methylation level of ACSL4 promoter by inhibiting DNMT1. DNMT1 knockdown promoted CRPC cell ferroptosis by regulating ACSL4. PPI induced ferroptosis and suppressed CRPC growth in nude mice. PPI can be used as a ferroptosis inducer to induce ferroptosis in CRPC cells via the ERK/DNMT1/ACSL4 axis, suggesting that PPI may be a new strategy for CRPC treatment.

Epigenetic regulation of collagen in the mare's placenta: putative relationship with maternal age

Placental plasticity influences fetal development, pregnancy success, and foal growth. Older fertile mares deliver heavier foals and placentas, with increased collagen deposition. In humans, epigenetics regulates placental development and function. Epigenetics, through DNA methylation, also modulates endometrial fibrosis. As such, we hypothesized that as mares get older, collagen deposition in the placenta might be epigenetically modulated. This study assessed if transcription of collagen genes in the equine placenta was epigenetically modulated through DNA methyltransferases (DNMTs), and affected by aging. Immediately after foaling, samples from different chorioallantoic areas (gravid horn, non-gravid horn, and body) were collected from young (n=10; 4-6 years) and older mares (n=10; 12-18 years). Gene transcription of collagen type 1 (COL1A1), collagen type 3 (COL3A1), and DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was determined (qRT-PCR). Unpaired T-test and Mann-Whitney test were used with statistical significance set a p <0.05. In older mares, COL1A1 mRNA levels were higher in the gravid than in the non-gravid horn (p<0.05), and also upregulated in comparison to young mares’ placentas (p<0.05). In young mares, these transcripts were increased in the placenta body in comparison to the gravid (p<0.05) and non-gravid horns (p<0.01). In older mares, COL3A1 transcriptionwas higher in gravid and non-gravid horns, with respect to the same placenta regions in young mares (p<0.05). In older mares, DNMT1 mRNA expression in the body was lower than in younger mares (p<0.05). For all placentas, DNMT1 transcripts in the non-gravid horn were lowest, compared to the gravid horn (p<0.01) and body (p<0.001). In young mares, DNMT1 mRNA expression in the body was also higher than in the gravid horn (p<0.05). With mares aging, there was a downregulation of DNMT3A transcripts in gravid and non-gravid placenta horns, compared to young mares’ placentas (p< 0.05). In older mares, its levels were lowest in the gravid horn, compared to the non-gravid horn (p<0.05) and body (p<0.01). Similarly, in older mares’ placentas, DNMT3B transcripts were downregulated in the gravid horn (p<0.05) and placenta body (p<0.001), compared to young mares. DNMT3B mRNA levels were lowest in the non-gravid horn, both in young mares, in comparison to placenta body (p<0.01) and in older mares, with respect to the gravid horn and body (p<0.01). Considering mares aging, hypomethylation indicated by decreased DNMTs transcripts, in parallel with upregulation of collagen gene expression in gravid and non-gravid horns, may suggest epigenetic involvement on placental plasticity. However, further research is needed to unravel the intricacies of epigenetic modulation of collagen in equine placentas.

GSK-3484862 targets DNMT1 for degradation in cells.

Maintenance of genomic methylation patterns at DNA replication forks by DNMT1 is the key to faithful mitotic inheritance. DNMT1 is often overexpressed in cancer cells and the DNA hypomethylating agents azacytidine and decitabine are currently used in the treatment of hematologic malignancies. However, the toxicity of these cytidine analogs and their ineffectiveness in treating solid tumors have limited wider clinical use. GSK-3484862 is a newly-developed, dicyanopyridine containing, non-nucleoside DNMT1-selective inhibitor with low cellular toxicity. Here, we show that GSK-3484862 targets DNMT1 for protein degradation in both cancer cell lines and murine embryonic stem cells (mESCs). DNMT1 depletion was rapid, taking effect within hours following GSK-3484862 treatment, leading to global hypomethylation. Inhibitor-induced DNMT1 degradation was proteasome-dependent, with no discernible loss of DNMT1 mRNA. In mESCs, GSK-3484862-induced Dnmt1 degradation requires the Dnmt1 accessory factor Uhrf1 and its E3 ubiquitin ligase activity. We also show that Dnmt1 depletion and DNA hypomethylation induced by the compound are reversible after its removal. Together, these results indicate that this DNMT1-selective degrader/inhibitor will be a valuable tool for dissecting coordinated events linking DNA methylation to gene expression and identifying downstream effectors that ultimately regulate cellular response to altered DNA methylation patterns in a tissue/cell-specific manner.

Lenvatinib inhibited HCC cell migration and invasion through regulating the transcription and ubiquitination of UHRF1 and DNMT1

Hepatocellular carcinoma (HCC) is one of the most common causes of malignancy-related deaths. Lenvatinib, as a multi-targeted tyrosine kinase inhibitor, has gained increasing attention for its antitumor activity. However, the effect and mechanisms of Lenvatinib on HCC metastasis are virtually unknown. In this study, we revealed that Lenvatinib inhibited HCC cell motility and epithelial mesenchymal transition (EMT), along with cell adhesion and extension. Concomitant high DNMT1 and UHRF1 mRNA levels were in HCC patients and indicated worse prognosis. On the one hand, Lenvatinib modulated the transcription of UHRF1 and DNMT1via negatively regulation of ERK/MAPK pathway. On the other hand, Lenvatinib downregulated DNMT1 and UHRF1 expression by promoting their protein degradation through ubiquitin–proteasome pathway, consequently, resulting in upregulation of E-Cadherin. Moreover, Lenvatinib attenuated Huh7 cell adhesion and metastasis in vivo. Our findings provided insight into the intriguing molecular mechanisms regarding the anti-metastasis effect of Lenvatinib in HCC.

Epigenetic upregulation of spleen tyrosine kinase in cancer cells through p53-dependent downregulation of DNA methyltransferase

Syk is a tumor suppressor gene in some solid tumors. Currently, it remains unknown how Syk gene hypermethylation is controlled by DNA methyltransferase (DNMT) and p53. In colorectal cancer HCT116 cells, we found that protein and mRNA levels of Syk were much higher in WT than in p53−/− cells. Both p53 inhibitor PFT-α and p53 silencing can reduce the protein and mRNA expression of Syk in WT cells, while DNMT inhibitor 5-Aza-2′-dC can increase Syk expression in p53−/− cells. Interestingly, the DNMT expression in p53−/− HCT116 cells was higher than that in WT cells. PFT-α can not only enhance Syk gene methylation but also increase DNMT1 protein and mRNA levels in WT HCT116 cells. In metastatic lung cancer cell lines A549 and PC9, which express WT p53 and gain function of p53, respectively, PFT-α can also downregulate Syk mRNA and protein expression. However, the Syk methylation level was increased by PFT-α in A549 but not in PC9 cells. Likewise, 5-Aza-2′-dC transcriptionally increased Syk gene expression in A549 cells, but not in PC9 cells. In summary methylation of Syk promoter requires DNMT1, and p53 can upregulate Syk expression via downregulation of DNMT1 at the transcriptional level.

Hepatitis B virus X protein induces p16 gene promoter methylation through upregulation of DNA methylation transferases DNMT1 and DNMT3A.

As a tumor suppressor, p16 can competitively block the cyclin D1-CDK4/6 complex to arrest the cell cycle in the G1 phase. Lack of p16 gene expression can lead to infinite cell proliferation and malignant transformation. To determine whether the hepatitis B virus X protein (HBx) regulates the methylation and expression of the p16 gene. We constructed a eukaryotic expression vector carrying the HBx gene and green fluorescent protein (GFP), and transfected it into HepG2 cells to build cell lines stably expressing GFP and GFP-HBx. The p16 protein level and p16 gene methylation were measured in these cell lines. We further detected the mRNA expression of DNA methyltransferases (DNMTs) 1, 3A and 3B. The DNMT1, DNMT3A and DNMT3B gene promoter sequences were inserted into a reporter vector (pGL3-Basic) to build recombinant vectors, which were then transiently transfected into different cell lines. After 48 h, the luciferase activity was measured. The level of p16 protein in GFP-HBx/HepG2 cells was significantly lower than in HepG2 and GFP/HepG2 cells. The CpG methylation was present in the p16 gene promoter region of GFP-HBx/HepG2 cells. The DNMT1 and DNMT3A mRNA levels in GFP-HBx/HepG2 cells were significantly elevated compared to that in the HepG2 cells (p = 0.0495). The luciferase activity in GFP-HBx/HepG2 cells transfected with the pGL3-DNMT1/3A pro plasmid was significantly higher than in HepG2 and GFP/HepG2 cells (both p < 0.05). The HBx can induce p16 gene promoter methylation and inhibit the expression of p16 in HepG2 cells. This occurs due to HBx activation of DNMT1 and DNMT3A promoters and the induction of p16 promoter methylation, which downregulates the expression of p16 protein.

Транскрипционная активность генов ДНК-метилтрансфераз у жителей Уральского региона, подвергшихся хроническому радиационному воздействию

In addition to damaging the genetic apparatus of the cell, ionizing radiation can cause epigenetic alterations. DNA methylation that plays a vital part in regulation of cellular processes is a common epigenetic modification. DNA methylation ensured by DNA methyltransferases occurs in the CpG-rich sequences. The study was aimed to assess mRNA expression of genes encoding DNA methyltransferases (DNMT1, DNMT3A, DNMT3B) in the chronically exposed individuals who live along the River Techa over a long-term period. A total of 112 people were examined more than 65 years after the beginning of chronic exposure. The average accumulated dose to red bone marrow (RBM) was 782.0 ± 82.3 mGy, and the average accumulated dose to thymus and peripheral lymphoid organs was 93.2 ± 13.6 mGy. The subjects' age at the time of examination was 67.9 ± 0.8 years (54–83 years). The relative mRNA levels for the studied genes were assessed by realtime polymerase chain reaction (real-time PCR). mRNA expression of DNMT1 correlated positively with the dose to RBM (p = 0.04), thymus and peripheral lymphoid organs (p = 0.02), as well as with the dose rate in these organs (p = 0.05, p = 0.04, respectively) during the period of the highest levels of radiation exposure. In individuals exposed in the high dose range (over 1000 mGy) there was a significant increase in the expression of DNMT1 mRNA compared to the comparison group (p = 0.02). The findings may indicate the DNMT1 gene involvement in epigenetic alterations that occur in the chronically exposed people in the long term.

Altered methyltransferase gene expression, mitochondrial copy number and 4977-bp common deletion in subfertile men with variable sperm parameters.

Semen parameters have been found to predict reproductive success poorly and are the most prevalent diagnostic tool for male infertility. There are few conflicting reports regarding the correlation of DNMT genes expression, mitochondrial DNA copy number (mtDNAcn) and deletion (mtDNAdel) with different sperm parameters. To investigate DNMT mRNA level, mtDNAcn and deletion in infertile men, with different sperm parameters, compared with fertile men, semen samples from 30 men with unknown male infertility and normal sperm parameters (experimental group I), 30 infertile patients with at least two abnormal sperm parameters (experimental group II) and 30 fertile normozoospermic men (control group) were collected. After semen analysis, total RNA and DNA were extracted. The isolated DNA was used for assessing the respective mtDNAcn and the presence of common 4977 bp deletion in mtDNA by applying real-time quantitative PCR and multiplex PCR, respectively. Synthesized cDNA from total RNAs was used to quantify DNMT1, DNMT3A and DNMT3B transcripts in study groups by using real-time quantitative reverse-transcription PCR. Significantly higher proportions of mtDNAcn were found in experimental group II. DNMT1 was significantly downregulated in both experimental groups and 4977 bp deletion was not detected. Progressive motility and normal morphology were significantly and negatively correlated with mtDNAcn. A significant positive correlation was detected between sperm parameters and DNMT1 mRNA levels. In conclusion, infertile men with different sperm parameter qualities showed elevated mtDNA content. Abnormal sperm parameters associated with DNMT1 gene expression indicate the possibility of changes in some epigenetic aspects of spermatogenesis in subfertile men with different sperm parameters.

Changes in Expression of DNA-Methyltransferase and Cannabinoid Receptor mRNAs in Blood Lymphocytes After Acute Cannabis Smoking.

BackgroundCannabis use is a component risk factor for the manifestation of schizophrenia. The biological effects of cannabis include effects on epigenetic systems, immunological parameters, in addition to changes in cannabinoid receptors 1 and 2, that may be associated with this risk. However, there has been limited study of the effects of smoked cannabis on these biological effects in human peripheral blood cells. We analyzed the effects of two concentrations of tetrahydrocannabinol (THC) vs. placebo in lymphocytes of a subset of participants who enrolled in a double-blind study of the effects of cannabis on driving performance (outcome not the focus of this study).MethodsTwenty four participants who regularly use cannabis participated in an experiment in which they smoked cannabis cigarettes (5.9 or 13.4% THC) or placebo (0.02%) ad libitum. Blood samples were drawn at baseline and several times after smoking. Lymphocytes were separated and stored at –80°C for further analysis. Samples were analyzed for mRNA content for cannabinoid receptors 1 (CB1) and 2 (CB2), methylation and demethylating enzymes (DNMT, TET), glucocorticoid receptor (NRC3) and immunological markers (IL1B, TNFα) by qPCR using TaqMan probes. The results were correlated with THC whole blood levels during the course of the day, as well as THCCOOH baseline levels. Statistical analyses used analysis of variance and covariance and t-tests, or non-parametric equivalents for those values which were not normally distributed.ResultsThere were no differences in background baseline characteristics of the participants except that the higher concentration THC group was older than the low concentration and placebo groups, and the low concentration THC group had higher baseline CB2 mRNA levels. Both the 5.9 and 13.4% THC groups showed increased THC blood levels that then decreased toward baseline within the first hour. However, there were no significant differences between THC blood levels between the 5.9 and 13.4% groups at any time point. At the 4-h time point after drug administration the 13.4% THC group had higher CB2 (P = 0.021) and DNMT3A (P = 0.027) mRNA levels than the placebo group. DNMT1 mRNA levels showed a trend in the same direction (P = 0.056). The higher 13.4% THC group had significantly increased CB2 mRNA levels than the 5.9% concentration group at several post drug administration time points and showed trends for difference in effects for between 5.9 and 13.4% THC groups for other mRNAs. TET3 mRNA levels were higher in the 13.4% THC group at 55 min post-cannabis ingestion. When the high and lower concentration THC groups were combined, none of the differences in mRNA levels from placebo remained statistically significant. Changes in THC blood levels were not related to changes in mRNA levels.ConclusionOver the time course of this study, CB2 mRNA increased in blood lymphocytes in the high concentration THC group but were not accompanied by changes in immunological markers. The changes in DNMT and TET mRNAs suggest potential epigenetic effects of THC in human lymphocytes. Increases in DNMT methylating enzymes have been linked to some of the pathophysiological processes in schizophrenia and, therefore, should be further explored in a larger sample population, as one of the potential mechanisms linking cannabis use as a trigger for schizophrenia in vulnerable individuals. Since the two THC groups did not differ in post-smoking blood THC concentrations, the relationship between lymphocytic changes and the THC content of the cigarettes remains to be determined.

PLAGL1 Gene Demethylation Induced by Epigallocatechin Gallate Promotes Pheochromocytoma Cell Apoptosis Via Wnt/β-catenin Signaling Pathway.

Objective:The aim of this study was to investigate the expression and the promoter methylation level of PLAGL1 gene and the mechanism of epigallocatechin gallate (EGCG) that induces PLAGL1 gene demethylation and promotes the apoptosis of pheochromocytoma (PCC) in PC12 cell line. Methods: The PC12 cells were treated with 25, 50, 75, 100, and 150 μg/mL EGCG for 48 hours. MSP was used to examine PLAGL1 gene methylation and an MTT assay was performed to detect the cell proliferation. The cell apoptosis was detected using flow cytometry. The mRNA and protein expressions of DNMT1, PLAGL1, Wnt, and β-catenin were detected using RT-quantitative PCR and Western blot.Results:EGCG dose-dependently reduced the cell viability and reversed PLAGL1 gene hypermethylation in PC12 cells (P<0.05). The cell apoptosis was significantly increased in PC12 cells treated with EGCG. The EGCG treatment restored the expressions of PLAGL1 and downregulated the expression of DNMT1, Wnt, and β-catenin in PC12 cells (P<0.05). Conclusion:The EGCG induces the demethylation process of PLAGL1 gene through down-regulating DNMT1 and restores the PLAGL1 mRNA and protein expression. The Wnt/β-catenin signaling pathway is involved in the regulation of PCC cell apoptosis promoted by EGCG inducing PLAGL1 gene demethylation.

A study on DNA methylation modifying natural compounds identified EGCG for induction of IFI16 gene expression related to the innate immune response in cancer cells.

Innate immune sensor IFN-induced protein 16 (IFI16) exhibits anti-inflammatory effects via IFNβ and IFN-stimulated gene (ISG)15 induction in cancer cells. Epigallocatechin gallate (EGCG) is a potent natural DNA methyltransferase inhibitor (DNMTi). Previous studies revealed that conventional DNMTis, such as 5-azacytidine (5-aza-dc), induce IFI16 expression and EGCG decreases DNMT mRNA expression and global methylation (5mC) level via promoter demethylation of tumor suppressor genes in cancer cell lines. To the best of our knowledge, however, EGCG-mediated IFI16 promoter methylation status has been overlooked. Here, initial screening was performed to determine IFI16 expression and its correlation with DNMTs in cancer cell lines from various databases. Following treatment of breast cancer cell lines with 5-aza-dc, vitamin C and EGCG, expression levels of IFI16 and its downstream transcription targets IFNβ1 and ISG15 were assessed using RT-qPCR, and the 5mC level was assessed using ELISA. In silico molecular docking simulation was performed for all DNMTs to predict the mode of ligands binding with proteins. Finally, promoter methylation level in IFI16 gene was assessed following EGCG treatment. EGCG treatment induced IFI16 expression, interacted with certain amino acids residues in DNMT proteins and decreased 5mC level and promoter methylation of IFI16. The present results may provide a basis for targeting IFI16 expression as a therapeutic option in breast cancer cell lines.

Human pulmonary artery smooth muscle cell dysfunction is regulated by miR-509-5p in hypoxic environment

ABSTRACT Reportedly, dysfunction of human pulmonary arterial smooth muscle cells (PASMCs) is associated with the pathogenesis of pulmonary arterial hypertension (PAH). Herein, the role of miR-509-5p in hypoxia-induced PASMCs and the underlying mechanism were explored. PASMCs were cultured under both normoxia and hypoxia conditions. Quantitative real-time polymerase-chain reaction (qPCR) was employed for quantifying the expressions of miR-509-5p and DNMT1 mRNA in the serum of PAH patients and PASMCs. MiR-509-5p mimics and inhibitors were then, respectively, transfected into PAMSCs, and CCK-8 and Transwell assays were utilized to detect PASMCs’ proliferation and migration. Flow cytometry was executed for evaluating PASMCs’ apoptosis. Interrelation between miR-509-5p and DNMT1 was determined utilizing bioinformatics analysis and dual-luciferase reporter assay. Western blot assay was used to detect the expression of DNMT1 or SOD2. MiR-509-5p in serum samples of patients with PAH as well as hypoxia-induced PASMCs was significantly down-regulated, whereas DNMT1 was markedly up-regulated. MiR-509-5p mimics reduces the proliferation and migration of PASMCs, but promotes the apoptosis; conversely, miR-509-5p inhibitors exerted opposite effects. DNMT1 was identified as a target gene of miR-509-5p, and overexpression of DNMT1 reversed the biological functions of miR-509-5p in regulating the phenotypes of PAMSCs. MiR-509-5p up-regulated the expression of SOD2 by down-regulating DNMT1. MiR-509-5p regulates the proliferation, migration and apoptosis of PASMCs, and restoration of miR-509-5p may be a promising strategy to treat PAH.

Aflatoxin B1 impairs in vitro early developmental competence of ovine oocytes

Aflatoxin is considered as one of the most harmful mycotoxins in the world to be found in human food and animal feed. Previous reports have revealed that Aflatoxin B1 (AFB1) may disrupt gamete development through epigenetic modifications as well as promotion of oxidative stress, excessive autophagy, and apoptosis. Therefore, in this study we aimed to address the effects of AFB1 on the meiotic and cytoplasmic maturation, internal reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), blastocyst formation as well as mRNA alterations for the apoptotic (BAX and Caspase3), anti-apoptotic (BCL2), and DNA methyltransferase (DNMTs) genes in ovine oocytes. To accomplish this, maturation of oocytes was performed in presence of increasing AFB1 concentrations (0, 10, 50, and 100 μM). Meiotic and cytoplasmic maturation, intracellular ROS level, and ΔΨm were evaluated following 24 h of IVM. Embryonic cleavage and blastocyst formation following fertilization were also assessed. We also investigated alterations of BAX, BCL2,Caspase3, DNMT1, DNMT3a, and DMT3b mRNA levels in mature oocytes. In the presence of 50 and 100 μM AFB1, the number of oocytes reaching the metaphase II stage decreased and the oocytes presented with lower intracellular levels of GSH (P < 0.05). Furthermore, intracellular ROS production in matured oocytes reached the highest-level following exposure to 50 and 100 μM of AFB1 (P < 0.05). Reduction of ΔΨm was clearly evident in the AFB1-treated groups (P < 0.05). Rates of cleavage and blastocyst formation decreased in the presence of AFB1 as compared with those recorded in the Control group (P < 0.05). Apoptosis-related gene analysis in AFB1 treated groups (10 and 50 μM) revealed a higher abundance of the BAX and Caspase3 genes, and a lower abundance of the BCL2 gene as compared with the Control group (P < 0.05). Additionally, our data showed that relative abundances of DNMT3b gene decreased in the 10 μM group when compared to the Control group (P < 0.05). We showed that exposure of oocytes to AFB1 leads to a reduced nuclear and cytoplasmic maturation that may ultimately impair the embryonic development in the sheep oocyte. Furthermore, alterations in DNA methylation and initiation of apoptosis through excessive ROS generation could be a prime molecular mechanism responsible for the disruption of oocyte developmental competence in the presence of AFB1 in the ovine model.

The tyrosine kinase inhibitor LPM4870108 impairs learning and memory and induces transcriptomic and gene‑specific DNA methylation changes in rats.

Tyrosine kinase inhibitors (TKIs), which have been developed and approved for cancer treatment in the last few years, are involved in synaptic plasticity of learning and memory. Epigenetic modifications also play crucial roles in the process of learning and memory, but its relationship with TKI-induced learning and memory impairment has not been investigated. We hypothesized that LPM4870108, an effective anti-cancer Trk inhibitor, might affect the learning and memory via epigenetic modifications. In this study, rats were orally administered with LPM4870108 (0, 1.25, 2.5, or 5.0mg/kg) twice daily for 28days, after which animals were subjected to a Morris water maze test. LPM4870108 exposure caused learning and memory impairments in this test in a dose-dependent manner and reduced the spine densities. Whole-genome transcriptomic analysis revealed significant differences in the patterns of hippocampal gene expression in LPM4870108-treated rats. These transcriptomic data were combined with next-generation bisulfite sequencing analysis, after which RT-PCR and pyrosequencing were conducted, revealing epigenetic alterations associated with genes (Snx8, Fgfr1, Dusp4, Vav2, and Satb2) known to regulate learning and memory. Increased mRNA and protein expression levels of hippocampal Dnmt1 and Dnmt3a were also observed in these rats. Overall, these data suggest that gene-specific alterations in patterns of DNA methylation can potentially contribute to the incidence of learning and memory deficits associated with exposure to LPM4870108.

ZNF191 alters DNA methylation and activates the PI3K-AKT pathway in hepatoma cells via transcriptional regulation of DNMT1.

BackgroundAlteration of DNA methylation is an important event in pathogenesis and progression of hepatocellular carcinoma (HCC). DNA methyltransferase (DNMT) 1, the foremost contributor in DNA methylation machinery, was revealed elevated in HCC and significantly correlates with poor prognosis. However, the transcriptional regulation of DNMT1 in HCC remains unknown.MethodsReal‐time PCR and immunohistochemistry were performed to detect DNMT1 and zinc finger transcription factor 191 (ZNF191) expressions in HCCs. Transcription activity of DNMT1promoter was analyzed with Luciferase reporter activity assay. The binding capacity of ZNF191 protein to DNMT1 promoter was examined with chromatin immunoprecipitation‐qPCR (ChIP‐qPCR) and electrophoretic mobility shift assay (EMSA). DNA methylation level of hepatoma cells was detected with Methylation array.ResultsZNF191 can regulate DNMT1 mRNA and protein expression positively, and increase the transcription activity of the DNMT1 promoter. ChIP‐qPCR and EMSA revealed that ZNF191 protein directly binds to the DNMT1 promoter at nt‐240 AT(TCAT)3TC. Moreover, DNMT1 and ZNF191 expression correlate positively in human HCCs. With methylation array, DNA methylation alteration was observed in hepatoma cells with ZNF191 knockdown, and the differential methylation sites are enriched in the PI3K‐AKT pathway. Furthermore, we proved DNMT1 contributes the effect of ZNF191 on hepatoma cell growth via the PI3K‐AKT pathway.ConclusionZNF191 is a novel transcription regulator for DNMT1, and the pro‐proliferation effect of ZNF191/DNMT1/p‐AKT axis in hepatoma cells implies that ZNF191 status in HCCs may affect the therapeutic effect of DNMTs inhibitors and PI3K inhibitors for precise treatment of the disease.

Ginkgo biloba Extract Attenuates the Disruption of Pro- and Anti-inflammatory Balance of Peripheral Blood in Arsenism Patients by Decreasing Hypermethylation of the Foxp3 Promoter Region.

Coal-burning type of arsenism, a chronic arsenism caused by environmental arsenic pollution, found firstly at Guizhou Province of China, manifested as the disruption of pro- and anti-inflammatory T cell balance and multiple organ damage, while no specific treatment for the arsenism patients. The effect of methylation of the forkhead box P3 (Foxp3) promoter region on arsenic-induced disruption of pro- and anti-inflammatory T cell balance was first evaluated in this study, between the control and arsenism groups. The results show that arsenic can induce the hypermethylation of 6 sites in the Foxp3 promoter by upregulating the expression of recombinant DNA Methyltransferase 1 (Dnmt1) mRNA, leading to the downregulation of Foxp3 mRNA, Tregs, and interleukin 10 (IL-10, anti-inflammatory cytokine) levels, and increased the levels of interleukin 17 (IL-17, pro-inflammatory cytokine) in the peripheral blood of patients with arsenic poisoning. Further randomized controlled double-blind experiments (including the placebo control groups and the Ginkgo biloba extract (GBE) intervention groups) showed that compared to the placebo control group or before GBE intervention, the levels of Dnmt1 mRNA, Foxp3 methylation, and IL-17 in the peripheral blood of the GBE intervention group were significantly decreased after intervention (P < 0.05), but the levels of regulatory T cells (Tregs) and IL-10 were significantly increased (P < 0.05). Our study provides some limited evidence that GBE can attenuate the disruption of pro- and anti-inflammatory balance of peripheral blood in arsenism patients by decreasing hypermethylation of the Foxp3 promoter region. This study provides scientific basis for further understanding a possible natural medicinal plant, GBE, as a more effective measure to prevent and control the disruption of pro- and anti-inflammatory balance caused by coal-burning type of arsenism.

Low-level EFCAB1 promoted progress by upregulated DNMT3B and could be as a potential biomarker in lung adenocarcinoma.

BackgroundLung adenocarcinoma (LUAD) incidence is on the rise. We found that EFCAB1 (EF‐Hand Calcium Binding Domain 1) was significantly downregulated in LUAD tissues, but the mechanism of EFCAB1 is unknown.MethodsOne hundred and two LUAD samples and corresponding NT samples were prospectively collected from patients at the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, from August 2018 to August 2021.EFCAB1 expression was estimated in LUAD cells and tissues by qPCR. In‐vitro cytology assays were used to detect the role of EFCAB1 in LUAD cells.ResultsEFCAB1 expression level of LUAD was significantly lower than it's adjacent cancer tissues and that of LUAD with big tumor size (>2 cm) was significantly lower than that of small tumor size (≤2 cm) group. It shown that expression levels of EFCAB1 from A549, HCC827, PC9 were lowly expressed. The cell migration, invasion, colony formation, proliferation ability of EFCAB1 OE A549, PC9 were lower than that of EFCAB1 OE A549, PC9 NC group, while the apoptotic cells percentage of the EFCAB1 OE A549, PC9 group were significantly increased. We found that DNMT1 mRNA expression level of PC9 was higher than that of BEAS‐2B, while these of A549, HCC827 decreased. Compared with BEAS‐2B, DNMT3A mRNA expression level of PC9 increased. DNMT3B mRNA expression level of PC9, HCC827 were higher than these of BEAS‐2B.ConclusionThe EFCAB1 mRNA in LUAD patients and cell lines were downregulated; EFCAB1 overexpression inhibited cell proliferation, migration, invasion, while promoted apoptosis. EFCAB1 was expected to become a biomarker of LUAD.