mRNA Expression Of DNA Methyltransferases Research Articles

Triphenyl Phosphate Alters Methyltransferase Expression and Induces Genome-Wide Aberrant DNA Methylation in Zebrafish Larvae.

Emerging environmental contaminants, organophosphate flame retardants (OPFRs), pose significant threats to ecosystems and human health. Despite numerous studies reporting the toxic effects of OPFRs, research on their epigenetic alterations remains limited. In this study, we investigated the effects of exposure to 2-ethylhexyl diphenyl phosphate (EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP) on DNA methylation patterns during zebrafish embryonic development. We assessed general toxicity and morphological changes, measured global DNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase (DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven translocation (TET) methylcytosine dioxygenase genes. Additionally, we analyzed genome-wide methylation patterns in zebrafish larvae using reduced-representation bisulfite sequencing. Our morphological assessment revealed no general toxicity, but a statistically significant yet subtle decrease in body length following exposure to TMPP and EHDPP, along with a reduction in head height after TPHP exposure, was observed. Eye diameter and head width were unaffected by any of the OPFRs. There were no significant changes in global DNA methylation levels in any exposure group, and TMPP showed no clear effect on DNMT expression. However, EHDPP significantly decreased only DNMT1 expression, while TPHP exposure reduced the expression of several DNMT orthologues and TETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation. Differential methylation occurred primarily in introns (43%) and intergenic regions (37%), with 9% and 10% occurring in exons and promoter regions, respectively. Pathway enrichment analysis of differentially methylated region-associated genes indicated that TPHP exposure enhanced several biological and molecular functions corresponding to metabolism and neurological development. KEGG enrichment analysis further revealed TPHP-mediated potential effects on several signaling pathways including TGFβ, cytokine, and insulin signaling. This study identifies specific changes in DNA methylation in zebrafish larvae after TPHP exposure and brings novel insights into the epigenetic mode of action of TPHP.

Alterations in DNA methylation machinery in a rat model of osteoarthritis of the hip

BackgroundThis study aimed to validate alterations in the gene expression of DNA methylation-related enzymes and global methylation in the peripheral blood mononuclear cell (PBMC) and synovial tissues of animal hip osteoarthritis (OA) models.MethodsAnimals were assigned to the control (no treatment), sham (25 µL of sterile saline), and OA (25 µL of sterile saline and 2 mg of monoiodoacetate) groups. Microcomputed tomography scan, histopathological assessment and pain threshold measurement were performed after induction. The mRNA expression of the DNA methylation machinery genes and global DNA methylation in the PBMC and hip synovial tissue were evaluated.ResultsThe OA group presented with hip joint OA histopathologically and radiologically and decreased pain threshold. The mRNA expression of DNA methyltransferase (Dnmt 3a), ten–eleven translocation (Tet) 1 and Tet 3 in the synovial tissue of the OA group was significantly upregulated. Global DNA methylation in the synovial tissue of the OA group was significantly higher than that of the control and sham groups.ConclusionsThe intra-articular administration of monoiodoacetate induced hip joint OA and decreased pain threshold. The DNA methylation machinery in the synovial tissues of hip OA was altered.

Effects of Dietary Betaine on the Laying Performance, Antioxidant Capacity, and Uterus and Ovary Function of Laying Hens at the Late Stage of Production.

Betaine has been found to alleviate oxidative stress, inflammation, and apoptosis. However, whether dietary betaine can protect late-laying hens against these adverse effects is unknown. Here, 270 65-week-old Jinghong-1 laying hens were randomly divided into the Control, 0.1% Betaine, and 0.5% Betaine groups and fed a basal diet, 0.1%, and 0.5% betaine supplemented diet, respectively. The trial lasted for seven weeks. Birds that consumed 0.5% betaine laid more eggs with thicker eggshells. Accordingly, uterine reduced glutathione (GSH), glutathione peroxidase (GSH-PX), and ovarian superoxide dismutase (SOD) contents were increased. The uterine calcium ion content and the mRNA expression of ovalbumin, ovotransferrin, and carbonic anhydrase two were increased. Moreover, ovarian IL-1β, Caspase-1, Caspase-8, and Caspase-9 mRNA expressions were decreased; luteinising hormone receptor (LHR) and follicle-stimulating hormone receptor mRNA expressions were increased. Furthermore, dietary betaine decreased the ovaries' mRNA expression of DNA methyltransferase 1 (DNMT)1, DNMT3a, and DNMT3b. The methylation level at the promoter region of ovarian LHR decreased. These results indicated that dietary betaine consumption with a concentration of 0.5% could increase the laying rate and the eggshell thickness during the late-laying period. The underlying mechanism may include antioxidative, anti-apoptosis, and hormone-sensitivity-enhancing properties.

H19 Promoter DNA Methylation is Lower Among Early Abortion Patients Undergoing IVF Embryo Transfer.

H19 is the first long noncoding RNA (lncRNA) found to be associated with gene imprinting. It is highly expressed in the embryonic stage and may have important regulatory effects on human embryonic development. We investigated the differences between the levels of H19 promoter DNA methylation in the chorionic villi of patients who experienced spontaneous abortion (SA) following in vitro fertilization embryo transfer (IVF-ET) and those of patients with a normal early pregnancy (NEP). We also analyzed the associated DNA methyltransferase (DNMT) activity. Chorionic villus tissue from patients with SA and NEP were collected. The DNA methylation levels of two CpG islands in the promoter region of the H19 gene in the two groups were detected by bisulfite sequencing, and the mRNA expression of DNMTs was analyzed by real-time polymerase chain reaction. The sample size of each group was 32, and there were no significant differences in baseline data, including age, parity, and body mass index, between the two groups. Among the 7 CpG islands measured, the methylation rates of 3 CpG islands (CpG 1, 6, and 7) were significantly lower in the SA group than in the NEP group (P < 0.01). The methylation levels of the other 4 CpG islands were not significantly different between the two groups. There were no differences in the expression of DNMT1 between the two groups (P > 0.05), but DNMT3a and DNMT3b RNA levels were significantly lower in SA group than in the NEP group (P < 0.01). The lower H19 promoter DNA methylation levels found in the chorionic villi of patients with SA patients following IVF-ET may be explained by decreased expression of DNMT3a and DNMT3b.

Cyhexatin causes developmental toxic effects by disrupting endocrine system and inducing behavioral inhibition, apoptosis and DNA hypomethylation in zebrafish (Danio rerio) larvae

Cyhexatin (CYT), an organotin acaricide, is extensively utilized in developing countries to mitigate plant diseases caused by mites and minimize agricultural crop losses. However, the comprehensive mechanisms underlying the developmental stage of non-target organisms remain largely unexplored. In this study, zebrafish embryos were firstly exposed to CYT (0.06, 0.12, and 0.20 ng/mL, referred to as CYTL, CYTM, and CYTH, respectively) from 2 hpf (hours post fertilization) to 30 dpf (days post fertilization). No developmental toxicity was observed in the CYTL and CYTM groups, except for induced deformed phenotypes in the CYTM group at 120 hpf. However, exposure to CYTH resulted in significant reductions in spontaneous movement (24 hpf), heart rate (48 hpf), hatching rate (48 and 72 hpf), body weight (30 dpf), whole body length (30 dpf), and locomotion (30 dpf). Additionally, CYTH exposure induced morphological malformations, including spinal curvature, pericardial edema, and tail curvature in zebrafish larvae. Moreover, CYTH treatment induced apoptosis, increased reactive oxygen species (ROS) production, and resulted in significant reductions in free T3, cholesterol, estradiol, and testosterone levels in zebrafish larvae, while free T4 levels were increased. RNA-Seq analysis indicated that CYTH exposure led to significant alterations in the genome-wide gene expression profiles of zebrafish, particularly in the thyroid hormone and steroid biosynthesis signaling pathways, indicating endocrine disruption. Furthermore, CYTH exposure induced global DNA hypomethylation, reduced S-adenosylmethionine (SAM) levels and the SAM/S-adenosylhomocysteine (SAH) ratio, elevated SAH levels, and suppressed the mRNA expression of DNA methyltransferases (DNMTs) while also downregulating DNMT1 at both the gene and protein levels in zebrafish larvae. Overall, this study partially elucidated the developmental toxicity and endocrine disruption caused by CYT in zebrafish, providing evidence of the environmental hazards associated with this acaricide.

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.

Exosomal Transfer of DNA Methyl-Transferase mRNA Induces an Immunosuppressive Phenotype in Human Monocytes.

Survivors of sepsis exhibit persistent immunosuppression. Epigenetic events may be responsible for some of these immunosuppressive changes. During sepsis circulating exosomes contain large quantities of DNA methyltransferase (DNMT) mRNAs. We hypothesized that exosomes directly transfer DNMT mRNAs to recipient monocytes with resultant methylation events and immunosuppression. Exosomes containing DNMT mRNA were generated by stimulating monocytes with LPS. Confocal microscopy was used to determine uptake kinetics in the presence of pharmacologic inhibition. Expression and packaging of specific DNMT mRNA was controlled using DNMT siRNAs. Whole genome and gene specific methylation was assessed using bisulfite sequencing. Ingenuity pathway analysis was performed to determine the biological function of significance of differentially methylated regions. Exosomes effectively transferred DNMT mRNA to recipient monocytes. Pharmacologic inhibition of exosome uptake prevented this increase in DNMT mRNA expression. Recipient monocytes exhibited hypermethylation changes and gene suppression. siRNAs decreased the packaging of DNMT mRNAs and prevented TNFα gene suppression, restoring immunocompetence. These data support a role for exosome-mediated transfer of DNMT mRNA with resultant methylation and gene silencing. Pharmacologic uptake inhibition or targeted siRNA mediated DNMT gene silencing prevented DNMT mRNA transfer and maintained the cell's ability to express TNFα in response to LPS. This highlights the potential therapeutic value of targeting these exosome-mediated epigenetic events to maintain the host immune response during sepsis.

Levels of epigenetic regulators TET2 and DNMT1 correlate with virus-stimulated pDC differentiation from IFN-α producing cells to antigen presenting cells

Abstract Plasmacytoid dendritic cells (pDC) are potent producers of IFN-α in response to DNA and RNA viruses and subsequently mature into antigen presenting cells. We investigated the role of DNA methyltransferase 1 (DNMT1) and Ten-eleven translocation methyl-cytosine dioxygenase 2 (TET2), which are generally known as gene silencers and activators, respectively, in pDC activation. PBMC from healthy donors were stimulated with Herpes Simplex Virus (HSV) or Influenza-A virus (IAV) for 6- and 24h. PBMC were surface-stained for pDC markers CD123, HLA-DR and CD11C and co-stimulatory markers CD80 and CD86, then intracellularly for DNMT1, TET2 and IFN-α, and acquired by flow cytometry. mRNA expression of DNMT1 in purified pDC was determined by qRT-PCR. At 6h, DNMT1 protein was expressed at equivalent levels in pDC with and without stimulation and in IFN-α+ and IFN-α-pDC, and in unstimulated controls. In contrast, TET2 proteins were elevated in IFN-α+ but not IFN-α-pDC and controls at 6h. At 24h, DNMT1 increased while TET2 was reduced in stimulated pDC, correlating with high IFN-α levels at 6h and low levels at 24h. There was no change in DNMT1 gene expression in virus-stimulated vs. unstimulated controls. CD80 and CD86 were elevated at 24h compared to 6h in stimulated pDC, confirming pDC differentiation from IFN-α producing to antigen presenting cells. At the point of pDC differentiation, TET2 levels were decreased and DNMT1 increased. These results suggest that TET2 is demethylating and causing activation of genes associated with pDC IFN-α production, while DNMT1 methylates and silences genes in this pathway. This results in an initial induction of TET2 and IFN-α production followed by upregulation of co-stimulatory markers, DNMT1 and loss of IFN-α. Supported by 1. AAI trainee fellowship Careers in Immunology Fellowship Program AAI EIN #: 52-2317193 2. R01Al106125

The Impact of Sorghum Polyphenols on DNA Methylation and Signaling Pathways in Colon Cancer

Colorectal cancer (CRC) is a major public health issue, accounting for nearly 150,000 cases annually. Because CRC correlates with inflammation and oxidative stress, exploiting the anti‐oxidative and anti‐inflammatory nature of bioactive components in our diet, such as polyphenols, could potentially contribute to the inhibition of cancer initiation. Polyphenols are an immense family of naturally occurring secondary plant metabolites found in fruits, vegetables, and cereal grains, and have been demonstrated across many in vitro and in vivo studies to protect against carcinogenesis. Not only are polyphenols implicated to impact cancer development but they may also be involved in epigenetic regulation. Abnormalities in epigenetic modifications can lead to aberrant regulation of gene expression, and depending on the genes in question, this may contribute to the cell’s ability to acquire hallmarks of cancer. New varietals of the grain Sorghum bicolor provided by the USDA contain high amounts of polyphenols, which may contribute to CRC prevention due to their strong antioxidative and anti‐carcinogenic properties. The expression of genes involved in the promotion or prevention of CRC, like the Wnt/β‐catenin signaling pathway, may be regulated via epigenetic mechanisms. We hypothesize that the high polyphenol sorghum extracts may decrease DNA methylation, thus impacting the expression genes involved in the Wnt/β‐catenin signaling pathway. For our current study, we treated HCT116 and SW480 human colon cancer cells with novel high‐polyphenol S. bicolor extracts (HP and SC) or an extract of low‐polyphenol Macia‐Sorghum for up to 48 hours. Controls included cells treated with solvent or 5 µM epigallocatechin gallate as a benchmark. Total RNA was harvested, and the mRNA expression of selected genes was quantitated via qPCR. Relative to solvent control, the high‐polyphenol S. bicolorextract HP increased mRNA expression of DNA methyltransferase 1 (DNMT1), the key enzyme that upholds methylation patterns after DNA synthesis. Furthermore, mRNA expression of DNMT3A, the enzyme responsible for de novo DNA methylation, was also increased. The SC extract significantly decreased the expression of β‐catenin while increasing expression of transcription factor 4 (TCF4), a gene that mediates the expression of pro‐tumorigenic Wnt target genes such as VEGF and cMYC. Encouraged by these preliminary findings, we will investigate mRNA and protein expression of genes that are involved in epigenetic mechanisms, as well as those of downstream signaling pathways. Our study may help elucidate the question, whether consumption of high‐polyphenol S. bicolor as part of the human diet may lead to beneficial changes in DNA methylation. Subsequently, the expression of specific genes involved in the promotion or prevention of colorectal cancer may be modified, providing a dietary intervention to CRC.

The Potential Role of Nigella sativa Seed Oil as Epigenetic Therapy of Cancer.

Nigella sativa oil, commonly known as black seed oil (BSO), is a well-known Mediterranean food, and its consumption is associated with beneficial effects on human health. A large number of BSO’s therapeutic properties is attributed to its pharmacologically active compound, thymoquinone (TQ), which inhibits cell proliferation and induces apoptosis by targeting several epigenetic players, including the ubiquitin-like, containing plant homeodomain (PHD) and an interesting new gene, RING finger domains 1 (UHRF1), and its partners, DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1). This study was designed to compare the effects of locally sourced BSO with those of pure TQ on the expression of the epigenetic complex UHRF1/DNMT1/HDAC1 and the related events in several cancer cells. The gas chromatographs obtained from GC-MS analyses of extracted BSO showed that TQ was the major volatile compound. BSO significantly inhibited the proliferation of MCF-7, HeLa and Jurkat cells in a dose-dependent manner, and it induced apoptosis in these cell lines. BSO-induced inhibitory effects were associated with a significant decrease in mRNA expression of UHRF1, DNMT1 and HDAC1. Molecular docking and MD simulation showed that TQ had good binding affinity to UHRF1 and HDAC1. Of note, TQ formed a stable metal coordinate bond with zinc tom, found in the active site of the HDAC1 protein. These findings suggest that the use of TQ-rich BSO represents a promising strategy for epigenetic therapy for both solid and blood tumors through direct targeting of the trimeric epigenetic complex UHRF1/DNMT1/ HDAC1.

Exposure to carbon black nanoparticles during pregnancy aggravates lipopolysaccharide-induced lung injury in offspring: an intergenerational effect

Carbon black nanoparticles (CBNPs) are one of the most frequently used nanoparticles. Exposure to CBNPs during pregnancy (PrE to CBNPs) can directly induce inflammation, lung injury, and genotoxicity in dams and results in abnormalities in offspring. However, whether exposure to CBNPs during pregnancy enhances the susceptibility of offspring to environmental stimuli remains unknown. To address this issue, in this study, we intranasally treated pregnant mice with mock or CBNPs from gestational day (GD) 9 to GD18, and F1 and F2 offspring were normally obtained. By intratracheal instillation of mice with lipopolysaccharide (LPS) to trigger a classic animal model for acute lung injury, we intriguingly found that after LPS treatment, F1 and F2 offspring after exposure during pregnancy to CBNPs both exhibited more pronounced lung injury symptoms, including more degenerative histopathological changes, vascular leakage, elevated MPO activity, and activation of inflammation-related signaling transduction, compared with F1 and F2 offspring in the mock group, suggesting PrE to CBNPs would aggravate LPS-induced lung injury in offspring, and this effect was intergenerational. We also observed that PrE to CBNPs upregulated the mRNA expression of DNA methyltransferases (Dnmt) 1/3a/3b and DNA hypermethylation in both F1 and F2 offspring, which might partially account for the intergenerational effect. Together, our study demonstrates for the first time that PrE to CBNPs can enhance sensitivity to LPS in both F1 and F2 offspring, and this intergenerational effect may be related to DNA hypermethylation caused by CBNPs.

Abstract 2088: The effect of sorghum on epigenetic modifications in colon cancer

Abstract Colorectal cancer is a major public health issue, accounting for nearly 150,000 cases annually. Because colorectal cancer is correlated with inflammation and oxidative cellular stress, it would be highly beneficial to exploit bioactive components in our diet that could potentially inhibit cancer promotion. Certain varieties of the dietary grain sorghum (S. bicolor) contain high amounts bioactive polyphenols, which may contribute to the prevention of colon cancer due to their antioxidant properties, anti-inflammatory, and anti-carcinogenesis effects, some of which may be mediated by epigenetic mechanisms. Tumor suppressor genes are often hyper-methylated in tumors. Therefore, DNA methylation and histone acetylation patterns may affect the expression of genes that play a role in the promotion or prevention of colon cancer. Our objective is to investigate how high-polyphenol sorghum varietals impact epigenetic mechanisms in human colon cancer cells, including quantitating global DNA methylation and histone acetylation. We treated HCT116 human colon cancer cells with high-polyphenol sorghum extracts or solvent control, or 5 µM epigallocatechin gallate as a positive control, as it's known to decrease DNA methylation. Total RNA, protein, and DNA were harvested after 24 and 48 hours. mRNA expression of DNA methyltransferases (DNMT) were quantitated with qPCR. Our preliminary results suggest that high polyphenol sorghum may decrease the mRNA expression of DNMT1, the key enzyme that upholds methylation patterns after DNA synthesis. Furthermore, mRNA expression of DNMT 3A, which is the enzyme responsible for de novo DNA methylation, also indicated a decrease. We further plan to assess protein expression and catalytic activity of DNMTs and histone acetyltransferases in addition to global DNA methylation and histone acetylation to investigate the impact of these high polyphenol sorghum varietals on relevant epigenetic mechanisms in both HCT116 and CACO-2 cells. Consumption of high-polyphenol sorghum as part of the human diet may lead to global or gene-specific hypo-methylation or hyper-acetylation, and this may be of importance in the prevention of colon cancer. Research supported by USDA ARS Kansas. Citation Format: Zeguela Kamagate, Dmitriy Smolensky, Petra Tsuji. The effect of sorghum on epigenetic modifications in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2088.

8a, a New Acridine Antiproliferative and Pro-Apoptotic Agent Targeting HDAC1/DNMT1.

Epigenetic therapy using histone deacetylase (HDAC) inhibitors has become an attractive project in new drug development. However, DNA methylation and histone acetylation are important epigenetic ways to regulate the occurrence and development of leukemia. Given previous studies, N-(2-aminophenyl)benzamide acridine (8a), as a histone deacetylase 1 (HDAC1) inhibitor, induces apoptosis and shows significant anti-proliferative activity against histiocytic lymphoma U937 cells. HDAC1 plays a role in the nucleus, which we confirmed by finding that 8a entered the nucleus. Subsequently, we verified that 8a mainly passes through the endogenous (mitochondrial) pathway to induce cell apoptosis. From the protein interaction data, we found that 8a also affected the expression of DNA methyltransferase 1 (DNMT1). Therefore, an experiment was performed to assess the binding of 8a to DNMT1 at the molecular and cellular levels. We found that the binding strength of 8a to DNMT1 enhanced in a dose-dependent manner. Additionally, 8a inhibits the expression of DNMT1 mRNA and its protein. These findings suggested that the anti-proliferative and pro-apoptotic activities of 8a against leukemia cells were achieved by targeting HDAC1 and DNMT1.

The Retrotransposition of L1 is Involved in the Reconsolidation of Contextual Fear Memory in Mice.

The long interspersed element-1 (L1) participates in memory formation, and DNA methylation patterns of L1 may suggest resilience or vulnerability factors for Post-Traumatic Stress Disorder (PTSD), of which the principal manifestation is a pathological exacerbation of fear memory. However, the unique roles of L1 in the reconsolidation of fear memory remain poorly understood. The study aimed to investigate the role of L1 in the reconsolidation of context-dependent fear memory. Mice underwent fear conditioning and fear recall in the observation chambers. Fear memory was assessed by calculating the percentage of time spent freezing in 5 min. The medial prefrontal cortex (mPFC) and hippocampus were removed for further analysis. Open Reading Frame 1 (ORF1) mRNA and ORF2 mRNA of L1 were analyzed by real-time quantitative polymerase chain reaction. After reactivation of fear memory, lamivudine was administered and its effects on fear memory reconsolidation were observed. ORF1 and ORF2 mRNA expressions in the mPFC and hippocampus after recent (24 h) and remote (14 days) fear memory recall exhibited augmentation via different temporal and spatial patterns. Reconsolidation of fear memory was markedly inhibited in mice treated with lamivudine, which could block L1. DNA methyltransferase mRNA expression declined following lamivudine treatment in remote fear memory recall. The retrotransposition of L1 participated in the reconsolidation of fear memory after reactivation of fear memory, and with lamivudine treatment, spontaneous recovery decreased with time after recent and remote fear memory recall, providing clues for understanding the roles of L1 in fear memory.

Continuous gibberellin A3 exposure from weaning to sexual maturity induces ovarian granulosa cell apoptosis by activating Fas-mediated death receptor signaling pathways and changing methylation patterns on caspase-3 gene promoters

Information on the effects of gibberellic acid (gibberellin A3, GA3) on ovarian follicle development is limited. In our present study, 21-day-old female Wistar rats were exposed to GA3 by gavage (25, 50, and 100 mg/kg body weight, once per day) for eight weeks to evaluate the influence of GA3 on ovarian follicle development. After treatment, significant (P < 0.05) increases (to 40.17 % and 44.5 %, respectively) in atretic follicle proportions and significant decreases (to 19.49 % and 17.86 %, respectively) in corpus luteum proportions were observed in the 50 and 100 mg/kg treatment groups compared to the control group. Significant (P < 0.05) increases (to 31.3 % and 42.0 %, respectively) in follicle apoptosis were observed in the 50 and 100 mg/kg treatment groups by transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Significantly increased expression of caspase-3, caspase-8, caspase-9 and Fas was observed by real-time PCR and Western blotting. Bisulfite sequencing PCR (BSP) revealed obviously decreased total methylation percentages of the caspase-3 promoter region in the two treatment groups. Real-time quantitative PCR also showed significantly decreased mRNA expression of DNA methyltransferase (Dnmt) 3a and Dnmt3b. Further in vitro studies showed that a DNA methylation inhibitor could enhance the GA3-induced increase in the mRNA expression of caspase-3. Overall, our present study indicates that GA3 administration from weaning until sexual maturity can affect ovarian follicle development by inducing apoptosis and suggests that signaling through the Fas-mediated apoptotic pathway may be an important underlying mechanism of this apoptosis. In addition, GA3-induced aberrant DNA methylation patterns might be partly responsible for upregulation of caspase-3 gene expression.

Effects of β-carotene on Expression of Selected MicroRNAs, Histone Acetylation, and DNA Methylation in Colon Cancer Stem Cells.

BackgroundBeta-carotene (BC) is a carotenoid which exerts anti-cancer effects in several types of cancer, including colorectal cancer. Epigenetic modifications of genes, such as histone deacetylation and DNA hypermethylation, have also been detected in various types of cancer. To understand the molecular mechanism underlying cancer preventive and therapeutic effects of BC, microRNAs (miRNAs), histone acetylation, and global DNA methylation in colon cancer stem cells (CSCs) were investigated.MethodsHCT116 colon cancer cells positive for expression of CD44 and CD133 were sorted by flow cytometry and used in subsequent experiments. Cell proliferation was examined by the MTT assay and self-renewal capacity was analyzed by the sphere formation assay. The miRNA sequencing array was used to detect miRNAs regulated by BC. Histone acetylation levels were measured by the Western blot analysis. mRNA expression of DNA methyltransferases (DNMTs) was examined by qPCR and global DNA methylation levels were determined by enzyme-linked immunosorbent assay.ResultsTreatment of CD44+CD133+ colon CSCs with BC caused a reduction in both cell proliferation and sphere formation. Analysis of the miRNA sequencing array showed that BC regulated expression of miRNAs associated with histone acetylation. Histone H3 and H4 acetylation levels were elevated by BC treatment. In addition, BC treatment down-regulated DNMT3A mRNA expression and global DNA methylation in colon CSCs.ConclusionsThese results suggest that BC regulates epigenetic modifications for its anti-cancer effects in colon CSCs.

Sulforaphane-induced epigenetic regulation of Nrf2 expression by DNA methyltransferase in human Caco-2 cells.

The present study aimed to investigate the mechanism underlying sulforaphane-mediated epigenetic regulation of nuclear factor-erythroid derived 2-like 2 (Nrf2) expression in human colon cancer. Proteins were extracted from normal Caco-2 cells using sulforaphane and 5-aza-2'-deoxycytidine (5-Aza) combined with trichostatin A (TSA). The mRNA and protein expression levels and activity of DNA methyltransferase 1 (DNMT1) were determined. Methylation-specific polymerase chain reaction and bisulfite genomic sequencing were also used to measure the methylation levels of CpG sites in the Nrf2 promoter region. Nrf2 expression was measured using reverse transcription-quantitative PCR and western blot analysis. The results demonstrated that sulforaphane did not affect DNMT1 mRNA expression levels. DNMT1 protein expression was inhibited by sulforaphane and 5-Aza co-treatment with TSA. Nrf2 promoter methylation decreased significantly in the sulforaphane group compared with the control group. Nrf2 promoter methylation level in the 5-Aza+TSA group was the lowest among all groups. Nrf2 mRNA levels exhibited significant differences between the sulforaphane-treated and control groups, as well as between the 5-Aza+TSA and control groups, and the sulforaphane-treated and 5-Aza+TSA groups. Nrf2 protein expression was also inhibited by sulforaphane, as well as 5-Aza co-treatment with TSA. The results revealed that sulforaphane may promote demethylation of the Nrf2 promoter region to increase activation of Nrf2, which induces chemoprevention of colon cancer.

Correlation Between Altered DNA Methylation of Intergenic Regions of ITPR3 and Development of Delayed Cerebral Ischemia in Patients with Subarachnoid Hemorrhage

Delayed cerebral ischemia (DCI) is related to the major causes of morbidity and mortality in patients following subarachnoid hemorrhage (SAH); however, little is known about the role of epigenetics in the pathogenesis of DCI. We investigated the specific DNA methylation profile that may affect the expression of inositol 1-,4-,5-trisphosphate receptor (ITPR3) responsible for cerebral vasospasm following SAH. We prospectively studied patients with SAH between March 2015 and October 2018. The degree of methylation in the distal intergenic region (IGR) located on ITPR3 and gene expression were measured using methylation-specific polymerase chain reaction (MSP) and quantitative real-time polymerase chain reaction (qPCR). To investigate the regulatory mechanims of DNA hypermethylation, we further analyzed the mRNA expression of DNA methyltransferase (DNMT1) and ten-eleven translocation enzymes (TET1, TET2, and TET3). A total of 42 patients were included in our analysis. Patients with SAH and DCI had significantly higher levels of methylation intensity of distal IGR upstream of ITPR3 than those without DCI (median, 0.941 [interquartile range (IQR), 0.857-0.984] versus (0.670 [IQR, 0.543-0.761]; P < 0.001). In addition, patients with DCI showed decreased mRNA expression of ITPR3 compared with patients without DCI (median, 0.039 [IQR, 0.030-0.045] vs. 0.047 [IQR, 0.038-0.064]; P= 0.0328). Patients with DCI had higher DNMT1 expression (P < 0.001) and lower TET1 expression (P= 0.040) than those without DCI; however, differences in TET2 and TET3 levels between the 2 groups were not statistically significant. Hypermethylation of the distal IGR located upstream of ITPR3 is related to greater DCI development in patients with SAH. Further studies of the precise mechanisms of methylation degree and DCI development using invitro and invivo models are needed.

Relation between sperm protamine transcripts with global sperm DNA methylation and sperm DNA methyltransferases mRNA in men with severe sperm abnormalities

This study aimed to evaluate the relationship between mRNA expression of DNA methyltransferases (DNMTs) such as DNMT1, DNMT3A and DNMT3B mRNA and sperm global DNA methylation with protamine transcripts in the sperm from men with severe sperm abnormalities. Sperm from each semen sample were isolated using a standard gradient isolation procedure by layering 1 mL of 40% (v/v) density gradient medium over 1 mL of 80% (v/v). A total of 30 oligoasthenoteratozoospermic ejaculates (OAT) and 30 normozoospermic ejaculates as controls were compared using real-time quantitative reverse transcriptase polymerase chain reaction for mRNA expression of DNMT1, 3A, 3B, protamine1 (P1) and protamine2 (P2). The enzyme-linked immunosorbent assay was used to detect global DNA methylation in sperm. A p-value of <0.05 was considered statistically significant. In OAT ejaculates, the increased level of DNMT3A, 3B mRNA, sperm global methylation, P1 plus P2 mRNA and decrease of P1–P2 ratio were significantly different. Also the content of protamine transcript was not correlated with sperm parameters. The increased total protamine transcript levels were associated with increased mRNA methyltransferases. The increase of DNMT1 may lead to an increased level of global methylation.

Downregulation of nucleolar and spindle-associated protein 1 expression suppresses liver cancer cell function.

The aim of the present study was to determine the role of nucleolar and spindle-associated protein 1 (NuSAP1) in human liver cancer. NuSAP1 expression was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and immunohistochemistry in hepatocellular carcinoma (HCC) and adjacent tissues. The expression of NuSAP1 gene was detected by RT-qPCR in liver cancer cell lines. Expression information for NuSAP1 was determined using the UALCAN and Oncomine databases. The Kaplan-Meier plotter and The Cancer Genome Atlas databases were used to obtain overall survival data for liver cancer. Liver cancer cell lines HepG2 and Huh-7 were transfected with lentiviral particles to silence the endogenous NuSAP1 gene expression. RT-qPCR and western blotting were performed to confirm the silencing efficiency. Cell Counting Kit-8 was used to estimate the effects of NuSAP1 silencing on HepG2 and Huh-7 cell proliferation. Cell cycle and apoptosis analyses were performed using flow cytometry. Cell invasion was assessed using the Transwell assay with microscopy imaging. The results revealed that the NuSAP1 expression levels in HCC tissues were significantly increased compared with the adjacent tissues. The survival time of patients with HCC with a high NuSAP1 expression was markedly decreased compared with that of patients with HCC with a low expression level of NuSAP1. Functional studies revealed that NuSAP1 silencing significantly reduced HepG2 and Huh-7 cell proliferation and invasion. Increased apoptosis and cell cycle arrest at the G1 phase were observed following NuSAP1 knockdown. NuSAP1 silencing significantly inhibited the mRNA expression of DNA methyltransferase but not glioma-associated oncogene. NuSAP1 contributed to liver cancer development by reducing apoptosis and promoting cell cycle progression. The abnormal expression level of NuSAP1 may serve a role in promoting liver cancer progression.