DNA (cytosine-5)-methyltransferase 3A Research Articles

DNMT3A-R882: a mutation with many paradoxes.

Understanding the underlying mechanism of acute myeloid leukemia (AML) has led to the discovery of novel biomarkers to help predict, treat and monitor leukemia. DNA (cytosine-5)-methyltransferase 3A (DNMT3A) is considered a prognostic and therapeutic epigenetic target in AML patients with a hotspot mutation of R882. R882 mutation is associated with impaired differentiation of Hematopoietic stem cells in the bone marrow and disease progression. The prevalence of R882 mutation varied in different ethnicities and countries, and similarly, its prognostic impact differed among numerous studies. Nevertheless, the co-occurrence of mutations in R882 with NPM1 and FLT3 has been reported more frequently and is associated with a worse prognosis. These studies also suggest diverse results regarding bone marrow transplantation response as a treatment, while chemoresistance is reached as a conclusive outcome These findings highlight the crucial need for an in-depth discussion on the significance of the R882 mutation in AML patients. Understanding its impact on leukemic transformation, prognosis, and treatment is vital for advancing clinical implications.

Oncolytic Activity of Canine Distemper Virus in Human Ductal Breast Carcinoma Cells

Introduction: Oncolytic virotherapy is a novel strategy for cancer treatment in humans and companion animals. Canine distemper virus (CDV) is known to induce apoptosis in tumor cells, thus serving as a potential candidate for oncolytic therapy. However, the mechanism of viral oncolytic activity is less studied and varies depending on the type of cancer and cell lines. Methods: In the present study, the susceptibility of the MCF-7 cell line to CDV infection was assessed using the CDV strain, which was confirmed previously through sequence analysis in the Vero cell line. The impact of CDV infection on cell proliferation and apoptosis was studied by evaluating the expression of four target genes including the myeloid cell leukemia 1 (MCL-1), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), transcription factor (SP1), and DNA (cytosine-5)-methyltransferase 3A (DNMT3A). Results: CDV replication in the cells induced cytopathic effect and decreased in the cell proliferation rates compared to the uninfected control. MCL-1, SP1, and PIK3R1 gene expression was down-regulated, while the expression of DNMT3A was up-regulated 3 days post-infection. The expression levels of the target genes suggest that CDV may be inducing the intrinsic apoptotic pathway in the cancer cell line. Conclusion: Overall, the results strongly propose CDV strain as a potential candidate for cancer therapy after detailed studies.

The effect of graphydine oxide (GDYO) nanosheets on disrupting theF-actin filament of anaplastic thyroid carcinoma (ATC) cells

Anaplastic thyroid carcinoma is deadly cancer with high mortality rates. Previous studies have shown that MART-10, ananalog to vitamin D, effectively prevents cancer’s metastatic potential by downregulating N-cadherin and filamentousactin filaments (F-action filament). In more recent studies around AML (acute myeloid leukemia), a novel materialname graphene oxide nanosheets (GDYO) are effective against AML related to DNA(cytosine-5)methyltransferase3A(DNMT3A)-mutated genes. It has also been found that GDYO nanosheets have been able to disrupt the F-actinfilament of AML cells, inducing cell apoptosis which leads to the destruction of the mutated cells. This paper exploresthe further application of this material in the biomedical field by testing the ability of GDYO nanosheets to inducecell apoptosis in other types of cancer, for example, ATC cells. The study will use an 8505C cell line with negativephosphate-buffered saline (PBS) and positive control Nexavar, a clinically tested drug that treats early ATC cell growth.Nexavar, also known as Sorafenib, inhibits tumor growth by a dual mechanism, acting directly on the tumor (throughinhibition of Raf and Kit signaling) and tumor angiogenesis (through inhibition of VEGFR and PDGFR signaling).The paper investigates whether GDYO can successfully disrupt the F-actin filaments of ATC cells, thereby killing themthrough inducing apoptosis and preventing the metastatic potential of ATC cells.

Using autophagy, apoptosis, cytoskeleton, and epigenetics markers to investigate the origin of infertility in ex-fissiparous freshwater planarian individuals (nomen nudum species) with hyperplasic ovaries

The origin of the sterility observed in ex-fissiparous freshwater planarians with hyperplasic ovaries has yet to be explained. To improve our understanding of this enigmatic phenomenon, immunofluorescence staining and confocal microscopy examination were used the assess autophagy, apoptosis, cytoskeleton, and epigenetics markers in the hyperplasic ovaries of ex-fissiparous individuals and the normal ovaries of sexual individuals. Immunofluorescence positivity for the autophagic marker microtubule-associated protein 1 light chain 3 (LC3) was significantly lower in the hyperplasic ovary than in the normal ovary. Compared with the normal ovary, the hyperplasic ovary exhibited significantly higher immunofluorescence positivity for the apoptotic marker caspase 3, suggesting that autophagy and apoptosis are closely associated in this pathogenicity. Furthermore, the level of global DNA (cytosine-5)-methyltransferase 3A (DNMT3) protein expression was significantly higher in the normal ovary than in the hyperplasic ovary, suggesting that DNA methylation is involved in the infertility phenomenon. The cytoskeleton marker actin also exhibited relatively higher immunofluorescence intensity in the normal ovary than in the hyperplasic ovary, consistent with previous findings on the role of cytoskeleton architecture in oocyte maturation. These results help improve our understanding of the causes of infertility in ex-fissiparous planarians with hyperplasic ovaries and provide new insights that will facilitate future studies on this mysterious pathogenicity.

Examination of the Functional Relationship between PD-L1 DNA Methylation and mRNA Expression in Non-Small-Cell Lung Cancer

Immunotherapy targeting the interaction between programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) is a treatment option for patients with non-small-cell lung cancer (NSCLC). The expression of PD-L1 by the NSCLC cells determines treatment effectiveness, but the relationship between PD-L1 DNA methylation and expression has not been clearly described. We investigated PD-L1 DNA methylation, mRNA expression, and protein expression in NSCLC cell lines and tumor biopsies. We used clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) to modify PD-L1 genetic contexts and endonuclease deficient Cas9 (dCas9) fusions with ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) to manipulate PD-L1 DNA methylation. In NSCLC cell lines, we identified specific PD-L1 CpG sites with methylation levels inversely correlated with PD-L1 mRNA expression. However, inducing PD-L1 mRNA expression with interferon-γ did not decrease the methylation level for these CpG sites, and using CRISPR-Cas9, we found that the CpG sites did not directly confer a negative regulation. dCas9-TET1 and dCas9-DNMT3A could induce PD-L1 hypo- and hyper-methylation, respectively, with the latter conferring a decrease in expression showing the functional impact of methylation. In NSCLC biopsies, the inverse correlation between the methylation and expression of PD-L1 was weak. We conclude that there is a regulatory link between PD-L1 DNA methylation and expression. However, since these measures are weakly associated, this study highlights the need for further research before PD-L1 DNA methylation can be implemented as a biomarker and drug target for measures to improve the effectiveness of PD-1/PD-L1 immunotherapy in NSCLC.

Extremely low-frequency electromagnetic field (ELF-EMF) induces alterations in epigenetic regulation in the myometrium – An in vitro study

Previous research by the authors indicated that an extremely low-frequency electromagnetic field (ELF-EMF) evokes molecular alterations in the porcine myometrium. It was hypothesized that the ELF-EMF could induce alterations in the epigenetic regulation of gene expression in the myometrium. In the current study, slices of the porcine myometrium during the peri-implantation period (n = 4) were used for further in vitro exposition to ELF-EMF (50 Hz, 8 mT, 2 h treatment duration). The study tested whether the ELF-EMF may affect: 1/the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and DNA (cytosine-5)-methyltransferase 3a (DNMT3a), 2/the level of genomic DNA methylation, and 3/the level of amplification of methylated and unmethylated variants of promoter regions of selected genes with altered expression in response to ELF-EMF. It was found that ELF-EMF treatment increased DNMT1, decreased DNMT3a mRNA transcript and protein abundance, and increased the level of genomic DNA methylation. The direction of alterations in the level of amplification of methylated and unmethylated variants of the promoter region of selected genes with altered expression, i.e. prodynorphin (PDYN), interleukin 15 (IL15) signal transducer and activator of transcription 5A (STAT5A), tumor necrosis factor (TNF), and between down-regulated genes were early growth response 2 (EGR2), hyaluronan and proteoglycan link protein 1 (HAPLN1), and uteroferrin associated basic protein-2 (UABP2), mostly involving the direction of changes in their transcriptional activity, which was evaluated in a previous study by the authors. Thus, ELF-EMF radiation disturbs epigenetic mechanisms, which may underlay ELF-EMF-related transcriptomic alterations in the myometrium.

Oridonin inhibits DNMT3A R882 mutation-driven clonal hematopoiesis and leukemia by inducing apoptosis and necroptosis

DNA (cytosine-5)-methyltransferase 3A (DNMT3A) mutations occur in ~20% of de novo acute myeloid leukemia (AML) patients, and >50% of these mutations in AML samples are heterozygous missense alterations within the methyltransferase domain at residue R882. DNMT3A R882 mutations in AML patients promote resistance to anthracycline chemotherapy and drive relapse. In this study, we performed high-throughput screening and identified that oridonin, an ent-kaurene diterpenoid extracted from the Chinese herb Rabdosia rubescens, inhibits DNMT3A R882 mutant leukemic cells at a low-micromolar concentration (IC50 = 2.1 µM) by activating both RIPK1-Caspase-8-Caspase-3-mediated apoptosis and RIPK1-RIPK3-MLKL-mediated necroptosis. The inhibitory effect of oridonin against DNMT3A R882 mutant leukemia cells can also be observed in vivo. Furthermore, oridonin inhibits clonal hematopoiesis of hematopoietic stem cells (HSCs) with Dnmt3a R878H mutation comparing to normal HSCs by inducing apoptosis and necroptosis. Overall, oridonin is a potential and promising drug candidate or lead compound targeting DNMT3A R882 mutation-driven clonal hematopoiesis and leukemia.

Abstract 2181: Obesity and the consensus molecular subtypes of colorectal cancer

Abstract Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer-related deaths in the U.S. Obesity - a worldwide public health concern - is a known risk factor for cancer including CRC. However, the mechanisms underlying the link between CRC and obesity have yet to be elucidated in part because of the molecular heterogeneity of CRC. In 2015, an international consortium classified CRC into four consensus molecular subtypes (CMSs). However, the molecular links between the CMSs and obesity have not been investigated. Here, we preformed a transcriptomic analysis to elucidate links between the distinct CMSs and obesity to better understand the pathophysiology of CRC. RNA-seq data and associated tumor and patient characteristics including body weight and height data for 231 patients were obtained from The Cancer Genomic Atlas - Colon Adenocarcinoma (TCGA-COAD) database. Tumor samples were classified into the four CMSs with the CMScaller R package; Body mass index (BMI) was calculated and categorized as normal, overweight, and obese. No significant differences were observed in sex, age, ethnicity, or anatomical location of patient tumors and stage of colon cancer across BMI categories. In contrast, a significant difference in CMS categorization between BMI categories was observed; more CMS3 tumors were observed in the obese than the normal BMI category. Gene Set Enrichment Analysis (GSEA) was conducted to elucidate CRC enriched pathways with a false discovery rate of P < 0.05 in the obese compared to normal BMI category for each CMS. Inflammation-related Hallmark get sets were enriched in CMS1 (8 gene sets), CMS2 (2 set sets), and CMS4 (8 gene sets), but not in CMS3 tumors. Obesity-linked CMS specific Hallmark gene set enrichment not related to inflammation was observed in CMS1 (KRAS signaling up) and CMS4 (E2F targets, Myc targets v1, DNA repair, MTORC signaling, G2M checkpoint) but not in CMS2 and CMS3. Differentially expressed genes (DEGs) between the obese and normal BMI patient tumor tissues for each CMS were identified with the DESeq2 R package and used to construct a protein-protein interaction network and determine 10 hub nodes (genes) with the STRING and CytoHubba apps, respectively, in Cytoscape. Nine of the 10 obesity-linked hub genes differed by CMS; the top ranked obesity-linked hub genes were Bassoon Presynaptic Cytomatrix Protein (BSN), DNA (cytosine-5)-methyltransferase 3A (DNMT3A), Melanoma-Associated Antigen 6 (MAGEA6) and UDP Glucuronosyltransferase Family 1 Member A6 (UGT1A6), for CMS1-4, respectively. Kaplan-Meier analysis for overall and relapse free survival was performed using the top 20 upregulated obesity-linked DEGs in each CMS. Higher expression of the DEGs in the CRC cohort GSE17536 was associated with significantly worse overall survival for CMS4 and relapse free survival for CMS2 and CMS3. Taken together, our data suggests that obesity is linked to CRC through CMS-dependent pathways. Citation Format: Michael W. Greene, Peter Abraham, Elizabeth A. Lipke, Peyton C. Kuhlers. Obesity and the consensus molecular subtypes of colorectal 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 2181.

Study on the Immune Escape Mechanism of Acute Myeloid Leukemia With DNMT3A Mutation.

DNA (cytosine-5)-methyltransferase 3A (DNMT3A)-mutated acute myeloid leukemia (AML) has a poor prognosis, but the exact mechanism is still unclear. Here, we aimed to explore the mechanism of immune escape in AML with DNMT3A mutation. We constructed a DNMT3A knockout clone and DNMT3A-R882H-mutated clones. RNA-seq results showed that transcription factors and macrophage inflammatory proteins were significantly downregulated in the DNMT3A mutant clones. KEGG enrichment and gene set enrichment analysis (GSEA) showed that a large number of genes were enriched in inflammatory immune-related pathways, such as the toll-like receptor signaling pathway. Therefore, we co-cultured AML cells with macrophages. The DNMT3A-mutated AML cells attenuated M1 macrophage polarization and resisted its killing effect in vitro and in vivo. In xenografts, the tumor volumes in the experimental group were significantly larger than those in the control group, and the proportion of M2 macrophages was significantly higher. After the co-culture, the increase in pro-inflammatory cytokine expression in the mutant cells was significantly lower than that in the control group, while that in immunosuppressive factors was not significantly different. In co-cultivated supernatants, the concentration of inflammatory factors in the experimental group was significantly lower than that in the control group, while that of immunosuppressive factors was significantly higher. Resistin significantly promoted the expression of inflammatory proteins in AML cells. It relieved the inhibitory effect of DNMT3A mutation, promoted the phenotypic recovery of the co-cultured macrophages, eliminated resistance, and regulated the immune microenvironment. Thus, resistin may serve as an ancillary drug for patients with DNMT3A-mutated AML.

Methylation-Induced Silencing of ALDH2 Facilitates Lung Adenocarcinoma Bone Metastasis by Activating the MAPK Pathway.

Bone metastasis (BM) dramatically reduces the quality of life and life expectancy in lung adenocarcinoma (LUAD) patients. There is an urgent need to identify potential biomarkers for application in the treatment of this deadly disease. We compared patient BM, LUAD, and para-LUAD tissues using proteomic analysis and identified aldehyde dehydrogenase 2 (ALDH2), which can detoxify acetaldehyde to acetic acid, as one of the key regulators in lung tumor metastasis. Both the mRNA and protein levels of ALDH2 were significantly lower in tumor tissues than in normal tissues and were lowest in BM tissues with increased migratory capacity. Also, ALDH2 was upregulated following treatment with 5-azacitidine, a DNA methyltransferase inhibitor, in H1299, H460, and HCC827 cells. Further, we identified a potential methylated CpG island 3, with the longest methylated CpG island area in ALDH2, and performed bisulfite genomic sequencing of these sites. An average of 78.18% of the sites may be methylated in CpG island 3. Knockdown of DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and methylated CpG binding protein 4 (MBD4) upregulated ALDH2 expression. ALDH2 functions as a mitogen-activated protein kinase (MAPK) upstream to inhibit cell proliferation and migration, promote cell apoptosis, and alter the epithelial–mesenchymal transition (EMT) by elevating E-cadherin and attenuating vimentin. Cell proliferation and migration were inhibited after the addition of the JNK inhibitor SP600125. In the multivariate analysis, M stage (p = 0.003), ALDH2 (p = 0.008), and phospho-c-Jun N-terminal kinase (p-JNK) (p = 0.027) expression were independent prognostic factors for overall survival in patients with BM. In vivo experiments also showed that ALDH2 expression could suppress tumor formation. In summary, we found that ALDH2 expression is a prognostic factor for BM in LUAD and that DNMT3A and MBD4 repression of ALDH2 via a MAPK-dependent pathway alters the EMT process, indicating that these proteins could act as potential biomarkers or therapeutic targets for LUAD metastasis.

Crosstalk between histone modification and DNA methylation orchestrates the epigenetic regulation of the costimulatory factors, Tim‑3 and galectin‑9, in cervical cancer.

Persistent infection with high-risk human papillomavirus is known to cause cervical cancer. The binding of the costimulatory factors, Tim-3 and galectin-9, can cause immune tolerance and lead to immune escape during carcinogenesis. Epigenetic regulation is essential for Tim-3/galectin-9 expression, which affects the outcome of local cervical cancer infection. Hence, exploring the epigenetic regulatory mechanisms of costimulatory signaling by Tim-3/galectin-9 is of great interest for investigating the mechanisms through which these proteins are regulated in cervical cancer tumorigenesis. In this study, we report that E2F-1 and FOXM1 mediated by HPV18 E6 and E7 can enhance the transcriptional activity of Enhancer of zeste homolog 2 (EZH2) by binding to its promoter region, resulting in the induced expression of the EZH2-specific target protein, H3K27me3, which consequently reduces the expression of the downstream target gene, DNA (cytosine-5)-methyltransferase 3A (DNMT3A). EZH2 and H3K27me3 directly interact with the DNMT3A promoter region to negatively regulate its expression in HeLa cells. Moreover, the downregulated DNMT3A and the decreased methylation levels in HAVCR2/LGALS9 promoter regions in HeLa cells promoted the expression of Tim-3/galectin-9. Furthermore, the high expression of Tim-3/galectin-9 was associated with HPV positivity among patients with cervical cancer. Moreover, HAVCR2/LGALS9 promoter regions were hypermethylated in normal cervical tissues, and this hypermethylated status inhibited gene expression. On the whole, these findings suggest that EZH2, H3K27me3 and DNMT3A mediate the epigenetic regulation of the negative stimulatory molecules, Tim-3 and galectin-9 in cervical cancer which is associated with HPV18 infection.

Selective cytochrome P450 1A1 but not 1B1 promoterCpG island DNA methylation by 6-formylindolo[3,2-b]carbazole (FICZ).

Epigenetic alterations are essential for normal mammalian development and regulation of gene expression. In this study, we aimed to determine if an enigmatic endogenous ligand of the aryl hydrocarbon receptor (AHR), 6-formylindolo[3,2-b]carbazole (FICZ), and methionine (Meth) have an epigenetic impact on AHR-regulated cytochrome P450 1A1 and B1 (CYP1A1 and CYP1B1) gene expression. Human hepatoma (HepG2-XRE-Luc and huh7) cells were exposed to FICZ in a medium with and without Meth supplementation. Selective and transient silencing of CYP1A1 but not CYP1B1 were seen by FICZ. Here we found that FICZ transiently represses CYP1A1 by targeting DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and concomitant DNA methylation of the CYP1A1 promoter gene. Treatments with 5-aza-dC augmented CYP1A1 transcription activity. Our results reveal a new mechanism for transient activation of AHR by FICZ that can negatively and positively influence gene expression, and highlight the regulatory role of Meth on the CYP1A1 gene expression.

Male-Specific Long Noncoding RNA TTTY15 Inhibits Non-Small Cell Lung Cancer Proliferation and Metastasis via TBX4.

Gender affects cancer susceptibility. Currently, there are only a few studies on Y chromosome-linked long noncoding RNAs (lncRNAs), and the potential association between lncRNAs and cancers in males has not been fully elucidated. Here, we examined the expression of testis-specific transcript Y-linked 15 (TTTY15) in 37 males with non-small cell lung cancer (NSCLC), and performed circular chromosome conformation capture with next-generation sequencing to determine the genomic interaction regions of the TTTY15 gene. Our results showed that the expression levels of TTTY15 were lower in NSCLC tissues. Lower TTTY15 expression levels were associated with Tumor-Node-Metastasis (TNM) stage. A TTTY15 knockdown promoted malignant transformation of NSCLC cells. Based on the bioinformatics analysis of circular chromosome conformation capture data, we found that T-box transcription factor 4 (TBX4) may be a potential target gene of TTTY15. The RNA immunoprecipitation and chromatin immunoprecipitation results showed that TTTY15 may interact with DNA (cytosine-5)-methyltransferase 3A (DNMT3A), and the TTTY15 knockdown increased the binding of DNMT3A to the TBX4 promoter. We concluded that low TTTY15 expression correlates with worse prognosis among patients with NSCLC. TTTY15 promotes TBX4 expression via DNMT3A-mediated regulation. The identification of lncRNAs encoded by male-specific genes may help to identify potential targets for NSCLC therapy.

Overexpression of DNMT3A promotes proliferation and inhibits differentiation of porcine intramuscular preadipocytes by methylating p21 and PPARg promoters

Intramuscular fat (IMF), which is modulated by the number and size of intramuscular preadipocytes, plays a key role in pork quality. DNA (cytosine-5)-methyltransferase 3A (DNMT3A), an enzyme that catalyzes the transfer of methyl groups to specific CpG structures in DNA, is involved in the management of diverse intracellular processes. However, the physiological functions of DNMT3A in proliferation and differentiation of porcine intramuscular preadipocytes have not been clearly established. Here, we found that DNMT3A significantly promoted the proliferation, while inhibited the differentiation of intramuscular preadipocytes. We demonstrated that overexpression of DNMT3A promoted the expression of cell proliferation markers but significantly decreased the expression of p21 to repress cell proliferation by the methylation of p21 promoter. Moreover, overexpression of DNMT3A decreased lipid accumulation and significantly down-regulated the levels of adipogenic marker genes including PPARg (Peroxisome proliferator-activated receptor gamma), SREBP-1c (Sterol regulatory element-binding protein 1c), and aP2 (FABP4, fatty acid binding protein 4) through the methylation of PPARg promoter. The blocking effect of DNMT3A on adipogenesis can be rescued by rosiglitazone treatment. Collectively, these findings illustrated the essential role of DNMT3A in the proliferation and differentiation of porcine intramuscular preadipocytes, and provide a potential target to improve pork quality.

Understanding the R882H mutation effects of DNA methyltransferase DNMT3A: a combination of molecular dynamics simulations and QM/MM calculations.

DNA (cytosine-5)-methyltransferase 3A (DNMT3A), a key enzyme for de novo epigenetic methylation in human beings, was reported to undergo an R882H mutation in approximately 25% of M4/M5 subtype acute myeloid leukemia (AML) patients. In this work, a combination of classical molecular dynamics (MD) simulations and QM/MM calculation methods was utilized to reveal the molecular mechanism behind the activity attenuation caused by R882H mutation. We found that R882H mutation induces a “folded” conformation in the methyl donor S-adenosylmethionine (SAM) through different types of hydrogen bond formation at the terminal carbonyl oxygen atom and the hydroxyl O3′ atom of the ribose ring on SAM, with Arg891 as a mediator. Energetically, both the pre-reaction state (PRS) and transition state (TS) were stabilized in the R882H mutant. However, the energy barrier of the rate-determining step from the PRS to the TS was calculated to be roughly 1.0 kcal mol−1 larger in the R882H mutant than the WT. Also, a dynamic transformation occurred along the helix where R882H was located, tending to manifest in a quasi-“Newton's cradle” manner from the mutational site to the active site residues of DNMT3A. Our computational results provided molecular insights into the pathogenic R882H mutation and advanced the understanding of its mechanism.

Overexpression of DNA (Cytosine-5)-Methyltransferase 1 (DNMT1) And DNA (Cytosine-5)-Methyltransferase 3A (DNMT3A) Is Associated with Aggressive Behavior and Hypermethylation of Tumor Suppressor Genes in Human Pituitary Adenomas.

BackgroundAlteration of DNA methylation of tumor suppressor genes (TSGs) is one of the most consistent epigenetic changes in human cancers. DNMTs play several important roles in DNA methylation and development of cancers. Regarding DNMTs protein expressions, little is known about the clinical significance and correlation with promoter methylation status of TSGs in human pituitary adenomas.Material/MethodsWe analyzed the protein expression of 3 DNMTs using immunohistochemistry and assessed DNA hypermethylation of RASSF1A, CDH13, CDH1, and CDKN2A (p16) in 63 pituitary adenomas. We examined associations between DNMTs expression and clinicopathological features or promoter methylation status of TSGs.ResultsOverexpression of DNMTs was detected in pituitary adenomas. Frequencies of DNMT1 overexpression were significantly higher in macroadenomas, invasive tumors, and grade III and IV tumors. DNMT3A was frequently detected in invasive tumors and grade IV tumors. In addition, DNMT1 and DNMT3A were frequently detected in high-methylation tumors. Furthermore, in multivariate logistic regression, the significant association between DNMT1 or DNMT3A and high-methylation status persisted after adjusting for clinicopathological features.ConclusionsOur findings suggested that tumor overexpression of DNMT1 and DNMT3A is associated with tumor aggressive behavior and high-methylation status in pituitary adenomas. Our data support a possible role of DNMT1 and DNMT3A in TSG promoter methylation leading to pituitary adenoma invasion and suggest that inhibition of DNMTs has the potential to become a new therapeutic approach for invasive pituitary adenoma.

Regulation of DNA methylation and tumor suppression gene expression by miR-29b in leukemia patients and related mechanisms.

Leukemia is characterized as a kind of malignant clonal disease in hematological stem cells. The study showed an abnormal level of DNA methylation in leukemia cells, which further led to an abnormal expression of hematological genes. This study investigated the role of miR-29b on the modulation of DNA methylation and tumor suppressor gene expression in leukemia patients. A total of 21 leukemia patients were recruited for the collection of monocytes. Methylation levels of promoter sequence of ESR1 and p15 genes were analyzed by methylation assay kit combined with DHPLC. DNA microarray and qRT-PCR were used to measure microRNA expressional profile, and bioinformatics plus luciferase reporter assay confirmed target gene of miR-29b. After transfection with miR-29b, promoter methylation levels of ESR1 and p15 gene were measured. Protein expressions of DNMT1 DNA (cytosine-5)-methyltransferase 1 (DNMT1), DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and DNA (cytosine-5)-methyltransferase 3B (DNMT3B) were quantified. The methylation levels of the promoter region of ESR1 and p15 genes in monocytes of leukemia patient were significantly elevated (p < 0.05). DNA microarray and qRT-PCR confirmed the down-regulation of miR-29b (p < 0.05). Luciferase reporter assay revealed DNMT1, DNMT3A and DNMT3B as target genes of miR-29b. MiR-29b transfection inhibited the expressions of DNMT3A and DNMT3B in Kasumi-1 cells (p < 0.05), and promoter methylation levels of estrogen Receptor 1 (ESR1) and p15 gene were decreased (p < 0.05). In leukemia cells, hyper- methylation existed in the promoter region of tumor suppressor gene. The methylation was enhanced in gene DNMT1, DNMT3A and DNMT3B via the reduction of miR-29b in leukemia tumor cells.

Oral propylene glycol modifies follicular fluid and gene expression profiles in cumulus-oocyte complexes and embryos in feed-restricted heifers.

Dietary supplementation with propylene glycol (PG) increases in vitro production of high-quality embryos in feed-restricted heifers. The aim of the present study was to evaluate the effects of PG in feed-restricted heifers on follicular fluid insulin and insulin-like growth factor (IGF) 1 concentrations, expression of IGF system genes in oocytes and cumulus cells and the expression of selected genes in blastocysts. Feed-restricted (R) heifers were drenched with water or PG during induced oestrous cycles (400mL of PG or water/drench, daily drenching at 1600 hours for the first 9 days of the oestrous cycle). Ovum pick-up (OPU) was performed after superovulation to produce in vitro embryos and without superovulation to recover oocytes, cumulus cells and follicular fluid. OPU was also performed in a control group (not feed restricted and no drenching). Follicular fluid IGF1 concentrations were reduced by R, and PG restored IGF1 concentrations to those seen in the control group. In cumulus cells, expression of IGF1, IGF1 receptor (IGF1R) and IGF binding protein 4 (IGFBP4) was decreased in the R group, and fully (IGF1 and IGF1R) or partially (IGFBP4) restored to control levels by PG. Blastocyst perilipin 2 (PLIN2; also known as adipophilin), Bcl-2-associated X protein (BAX), SCL2A1 (facilitated glucose/fructose transporter GLUT1), aquaporin 3 (AQP3), DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and heat shock 70-kDa protein 9 (HSPA9B) expression were decreased in R heifers; PG restored the expression of the last four genes to control levels. In conclusion, these results suggest that, during follicular growth, PG exerts epigenetic regulatory effects on gene expression in blastocyst stage embryos.

Downregulation of DNA methyltransferase 3 alpha promotes cell proliferation and invasion of ectopic endometrial stromal cells in adenomyosis

Adenomyosis is a common benign gynecological condition in female reproductive tract and the detailed molecular etiology remains largely elusive. Previous studies implicated that deregulated expression of DNA (cytosine-5)-methyltransferase 3A (DNMT3A), a de novo DNA methyltransferase, might be involved in the pathogenesis of adenomyosis. Meanwhile, ectopic endometrial stromal cells (EESCs) were suggested to play crucial roles in adenomyosis. Herein, we evaluated the expression of DNMT3A protein in 36 ectopic endometriums with adenomyosis and 37 eutopic endometriums in controls with Western blotting (WB) or immunohistochemistry (IHC), we found that the expression of DNMT3A was significantly decreased in the ectopic endometriums and EESCs in adenomyosis relative to that of eutopic endometriums and EESCs in control samples, respectively. In addition, our functional assays revealed that overexpression of DNMT3A suppressed cell proliferation and invasion, while knockdown of DNMT3A enhanced cell proliferation and invasion in EESCs. Taken together, our results suggested that DNMT3A expression was decreased in ectopic endometriums and EESCs in adenomyosis, and we provided the first evidence that decreased DNMT3A expression in EESCs facilitated the development of adenomyosis via enhanced cell growth and invasion.

The Expression of DNA Methyltransferase DNMT3a in Classical and Fibrolamellar Hepatocellular Carcinoma

The epigenetic regulation of DNA-templated processes has been studied extensively during the last 15 years. As was revealed, DNA modification such as methylation possess great impact on cell fate and can result in abnormal protein expression patterns what can lead to the induction of carcinogenesis. DNA (cytosine-5)-methyltransferases are enzymes that catalyses the transfer of methyl groups to specific CpG structures in DNA. The methylation of these sequences can lead to inappropriate gene expression such as the silencing of tumor suppressor genes in cancer cells. DNA (cytosine-5)-methyltransferase 3A (DNMT3a) gene encodes a DNA methyltransferase that is thought to function mainly in de novo methylation. In the normal liver DNMT3a is usually expressed on the medium level, as was described in literature. Hepatocellular carcinoma (HCC) still remains one of the most common cause of death among patients with cancer. Fibrolamellar hepatocellular carcinoma (FL) represents rare subtype, which affects usually young people (an onset between 20-30 years) and its etiology is poorly understood. In our study we compared the presence of DNMT3a protein between two different types of HCC-common type and fibrolamellar one. We performed immunohistochemical staining of formalin fixed paraffin embedded tissue sections obtained from 30 patients (22 HCC and 8 FL). We found that DNMT3a immunoreactivity is significantly more pronounced in the non-fibrolamellar variant of HCC than in the fibrolamellar one. The DNMT3a immunoreactivity was predominantly localized in cancer cell nuclei in a form of separate large granules spotted in proximity to heterochromatin region. The reduced presence of DNMT3a in the fibrolamellar variant of HCC may suggest that different epigenetic mechanisms are involved in development of this particular type of liver cancer. Improving our understanding of the roles of DNMT proteins in hepatocarcinogenesis can benefit in the development of epigenetic –based therapy designed for specific HCC subtype.