Rhein suppresses esophageal cancer development by regulating cell cycle through DNMT3B gene.

The mechanism by which DNMT3B facilitates esophageal cancer (ESCA) progression is currently unknown, despite its association with adverse prognoses in several cancer types. To investigate the potential therapeutic effects of the Chinese herbal medicine rhubarb on esophageal cancer (ESCA), we adopted an integrated bioinformatics approach. Gene Set Enrichment Analysis (GSEA) was first utilized to screen active anti-ESCA components in rhubarb. We then employed Weighted Gene Co-expression Network Analysis (WGCNA) to identify key molecular modules and targets related to the active components and ESCA pathogenesis. This system-level strategy integrating multi-omics data provides a powerful means to unravel the molecular mechanisms underlying the anticancer activities of natural products, like rhubarb. To investigate module gene functional enrichment, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. In addition, we evaluated the predictive impact of DNMT3B expression on ESCA patients utilizing the Kaplan-Meier method. Finally, we conducted experiments on cell proliferation and the cell cycle to explore the biological roles of DNMT3B. In this study, we identified Rhein as the main active ingredient of rhubarb that exhibited significant anti-ESCA activity. Rhein markedly suppressed ESCA cell proliferation. Utilizing Weighted Gene Co-expression Network Analysis (WGCNA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, we determined that the blue module was associated with Rhein target genes and the cell cycle. Additionally, DNMT3B was identified as a Rhein target gene. Analysis of The Cancer Genome Atlas (TCGA) database revealed that higher DNMT3B levels were associated with poor prognosis in ESCA patients. Furthermore, Rhein partially reversed the overexpression of DNMT3B to inhibit ESCA cell proliferation. In vitro studies demonstrated that Rhein and DNMT3B inhibition disrupted the S phase of the cell cycle and affected the production of cell cycle-related proteins. In this study, we found that Rhein exerts its anti-proliferative effects in ESCA cells by targeting DNMT3B and regulating the cell cycle.

A pharmacodynamic assay to monitor treatment with the hypomethylating cytosine analogs, decitabine and azacitidine.

Hypomethylating therapies using decitabine or azacitidine are actively investigated to treat acute myeloid leukemia, myelodysplastic syndromes, as maintenance therapy after allogenic stem cell transplant and hemoglobinopathies. The therapeutic mechanism is to de-repress genes that have been turned off through oncogenesis or development via methylation. The therapy can be non-cytotoxic at low dosage, sparing healthy stem cells and operating on committed precursors. Because the methods of determining maximum tolerated dose are not well suited to this paradigm, and because the mechanism of action, which is depletion of DNA methylase 1 (DNMT1), is complex and dependent on passing through a cell cycle, a pharmacodynamic assay that measures DNMT1 can inform clinical trials aimed at establishing and improving therapy. Herein, we provide an assay that measures DNMT1 relative levels in circulating T cells of peripheral blood.

Arsenic disulfide promoted the demethylation of PTPL1 in diffuse large B cell lymphoma cells.

Background: Promoter hypermethylation of the tumor suppressor gene is one of the well-studied causes of cancer development. The drugs that reverse the process by driving demethylation could be a candidate for anticancer therapy. This study was designed to investigate the effects of arsenic disulfide on PTPL1 methylation in diffuse large B cell lymphoma (DLBCL). Methods: We knocked down the expression of PTPL1 in two DLBCL cell lines (i.e., DB and SU-DHL-4 cells) using siRNA. Then the DLBCL proliferation was determined in the presence of PTPL1 knockdown. The methylation of PTPL1 in DLBCL cells was analyzed by methylation specific PCR (MSPCR). The effect of arsenic disulfide on the PTPL1 methylation was determined in DLBCL cell lines in the presence of different concentrations of arsenic disulfide (5 µM, 10 µM and 20 µM), respectively. To investigate the potential mechanism on the arsenic disulfide-mediated methylation, the mRNA expression of DNMT1, DNMT3B and MBD2 was determined. Results: PTPL1 functioned as a tumor suppressor gene in DLBCL cells, which was featured by the fact that PTPL1 knockdown promoted the proliferation of DLBCL cells. PTPL1 was found hypermethylated in DLBCL cells. Arsenic disulfide promoted the PTPL1 demethylation in a dose-dependent manner, which was related to the inhibition of DNMTs and the increase of MBD2. Conclusion: Experimental evidence shows that PTPL1 functions as a tumor suppressor gene in DLBCL progression. PTPL1 hyper-methylation could be reversed by arsenic disulfide in a dose-dependent manner.

iChIP-SILAC analysis identifies epigenetic regulators of CpG methylation of the p16INK4A gene.

Allele-specific epigenetic events regulate the expression of specific genes such as tumor suppressor genes. Methods to biochemically identify epigenetic regulators remain limited. Here, we used insertional chromatin immunoprecipitation (iChIP) to address this issue. iChIP combined with quantitative mass spectrometry identified DNA methyltransferase 1 (DNMT1) and epigenetic regulators as proteins that potentially interact with a region of the p16INK4A gene that is CpG-methylated in one allele in HCT116 cells. Some of the identified proteins are involved in the CpG methylation of this region, and of these, DEAD-box helicase 24 (DDX24) contributes to CpG methylation by regulating the protein levels of DNMT1. Thus, iChIP is a useful method to identify proteins which bind to a target locus of interest.

Epigenetic Activation of Cytochrome P450 1A2 Sensitizes Hepatocellular Carcinoma Cells to Sorafenib.

Cytochrome P450 1A2 (CYP1A2) is a known tumor suppressor in hepatocellular carcinoma (HCC), but its expression is repressed in HCC and the underlying mechanism is unclear. In this study, we investigated the epigenetic mechanisms of CYP1A2 repression and potential therapeutic implications. In HCC tumor tissues, the methylation rates of CYP1A2 CpG island (CGI) and DNA methyltransferase (DNMT) 3A protein levels were significantly higher, and there was a clear negative correlation between DNMT3A and CYP1A2 protein expression. Knockdown of DNMT3A by siRNA significantly increased CYP1A2 expression in HCC cells. Additionally, treating HCC cells with decitabine (DAC) resulted in a dose-dependent upregulation of CYP1A2 expression by reducing the methylation level of CYP1A2 CGI. Furthermore, we observed a decreased enrichment of H3K27Ac in the promoter region of CYP1A2 in HCC tissues. Treatment with the trichostatin A (TSA) restored CYP1A2 expression in HCC cells by increasing H3K27Ac levels in the CYP1A2 promoter region. Importantly, combination treatment of sorafenib with DAC or TSA resulted in a leftward shift of the dose-response curve, lower IC50 values, and reduced colony numbers in HCC cells. Our findings suggest that hypermethylation of the CGI at the promoter, mediated by the high expression of DNMT3A, and hypoacetylation of H3K27 in the CYP1A2 promoter region, leads to CYP1A2 repression in HCC. Epigenetic drugs DAC and TSA increase HCC cell sensitivity to sorafenib by restoring CYP1A2 expression. Our study provides new insights into the epigenetic regulation of CYP1A2 in HCC and highlights the potential of epigenetic drugs as a therapeutic approach for HCC. SIGNIFICANCE STATEMENT: This study marks the first exploration of the epigenetic mechanisms underlying cytochrome P450 (CYP) 1A2 suppression in hepatocellular carcinoma (HCC). Our findings reveal that heightened DNA methyltransferase expression induces hypermethylation of the CpG island at the promoter, coupled with diminished H3K27Ac levels, resulting in the repression of CYP1A2 in HCC. The use of epigenetic drugs such as decitabine and trichostatin A emerges as a novel therapeutic avenue, demonstrating their potential to restore CYP1A2 expression and enhance sorafenib sensitivity in HCC cells.

Curcumin regulates pulmonary extracellular matrix remodeling and mitochondrial function to attenuate pulmonary fibrosis by regulating the miR-29a-3p/DNMT3A axis.

Epigenetic regulation and mitochondrial dysfunction are essential to the progression of idiopathic pulmonary fibrosis (IPF). Curcumin (CCM) in inhibits the progression of pulmonary fibrosis by regulating the expression of specific miRNAs and pulmonary fibroblast mitochondrial function; however, the underlying mechanism is unclear. C57BL/6 mice were intratracheally injected with bleomycin (5 mg/kg) and treated with CCM (25 mg/kg body weight/3 times per week, intraperitoneal injection) for 28 days. Verhoeff-Van Gieson, Picro sirius red, and Masson's trichrome staining were used to examine the expression and distribution of collagen and elastic fibers in the lung tissue. Pulmonary fibrosis was determined using micro-computed tomography and transmission electron microscopy. Human pulmonary fibroblasts were transfected with miR-29a-3p, and RT-qPCR, immunostaining, and western blotting were performed to determine the expression of DNMT3A and extracellular matrix collagen-1 (COL1A1) and fibronectin-1 (FN1) levels. The expression of mitochondrial electron transport chain complex (MRC) and mitochondrial function were detected using western blotting and Seahorse XFp Technology. CCM in increased the expression of miR-29a-3p in the lung tissue and inhibited the DNMT3A to reduce the COL1A1 and FN1 levels leading to pulmonary extracellular matrix remodeling. In addition, CCM inhibited pulmonary fibroblasts MRC and mitochondrial function via the miR-29a-3p/DNMT3A pathway. CCM attenuates pulmonary fibrosis via the miR-29a-3p/DNMT3A axis to regulate extracellular matrix remodeling and mitochondrial function and may provide a new therapeutic intervention for preventing pulmonary fibrosis.

Structure-guided functional suppression of AML-associated DNMT3A hotspot mutations.

DNA methyltransferases DNMT3A- and DNMT3B-mediated DNA methylation critically regulate epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote polymerization, leading to aberrant DNA methylation that may contribute to the pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the mutation-induced functional misregulation of DNMT3A remains unclear. Here, we report the crystal structures of the DNMT3A methyltransferase domain, revealing a molecular basis for its oligomerization behavior distinct to DNMT3B, and the enhanced intermolecular contacts caused by the R882H or R882C mutation. Our biochemical, cellular, and genomic DNA methylation analyses demonstrate that introducing the DNMT3B-converting mutations inhibits the R882H-/R882C-triggered DNMT3A polymerization and enhances substrate access, thereby eliminating the dominant-negative effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutations-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy.

Downregulation of UBB potentiates SP1/VEGFA-dependent angiogenesis in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) presents a unique profile characterized by high levels of angiogenesis and robust vascularization. Understanding the underlying mechanisms driving this heterogeneity is essential for developing effective therapeutic strategies. This study revealed that ubiquitin B (UBB) is downregulated in ccRCC, which adversely affects the survival of ccRCC patients. UBB exerts regulatory control over vascular endothelial growth factor A (VEGFA) by directly interacting with specificity protein 1 (SP1), consequently exerting significant influence on angiogenic processes. Subsequently, we validated that DNA methyltransferase 3 alpha (DNMT3A) is located in the promoter of UBB to epigenetically inhibit UBB transcription. Additionally, we found that an unharmonious UBB/VEGFA ratio mediates pazopanib resistance in ccRCC. These findings underscore the critical involvement of UBB in antiangiogenic therapy and unveil a novel therapeutic strategy for ccRCC.

DNMT3A Cooperates with YAP/TAZ to Drive Gallbladder Cancer Metastasis.

Gallbladder cancer (GBC) is an extremely lethal malignancy with aggressive behaviors, including liver or distant metastasis; however, the underlying mechanisms driving the metastasis of GBC remain poorly understood. In this study, it is found that DNA methyltransferase DNMT3A is highly expressed in GBC tumor tissues compared to matched adjacent normal tissues. Clinicopathological analysis shows that DNMT3A is positively correlated with liver metastasis and poor overall survival outcomes in patients with GBC. Functional analysis confirms that DNMT3A promotes the metastasis of GBC cells in a manner dependent on its DNA methyltransferase activity. Mechanistically, DNMT3A interacts with and is recruited by YAP/TAZ to recognize and access the CpG island within the CDH1 promoter and generates hypermethylation of the CDH1 promoter, which leads to transcriptional silencing of CDH1 and accelerated epithelial-to-mesenchymal transition. Using tissue microarrays, the association between the expression of DNMT3A, YAP/TAZ, and CDH1 is confirmed, which affects the metastatic ability of GBC. These results reveal a novel mechanism through which DNMT3A recruitment by YAP/TAZ guides DNA methylation to drive GBC metastasis and provide insights into the treatment of GBC metastasis by targeting the functional connection between DNMT3A and YAP/TAZ.

DNMT3L inhibits hepatocellular carcinoma progression through DNA methylation of CDO1: insights from big data to basic research.

BACKGROUND: DNMT3L is a crucial DNA methylation regulatory factor, yet its function and mechanism in hepatocellular carcinoma (HCC) remain poorly understood. Bioinformatics-based big data analysis has increasingly gained significance in cancer research. Therefore, this study aims to elucidate the role of DNMT3L in HCC by integrating big data analysis with experimental validation. METHODS: Dozens of HCC datasets were collected to analyze the expression of DNMT3L and its relationship with prognostic indicators, and were used for molecular regulatory relationship evaluation. The effects of DNMT3L on the malignant phenotypes of hepatoma cells were confirmed in vitro and in vivo. The regulatory mechanisms of DNMT3L were explored through MSP, western blot, and dual-luciferase assays. RESULTS: DNMT3L was found to be downregulated in HCC tissues and associated with better prognosis. Overexpression of DNMT3L inhibits cell proliferation and metastasis. Additionally, CDO1 was identified as a target gene of DNMT3L and also exhibits anti-cancer effects. DNMT3L upregulates CDO1 expression by competitively inhibiting DNMT3A-mediated methylation of CDO1 promoter. CONCLUSIONS: Our study revealed the role and epi-transcriptomic regulatory mechanism of DNMT3L in HCC, and underscored the essential role and applicability of big data analysis in elucidating complex biological processes.

DNMT3B PWWP mutations cause hypermethylation of heterochromatin.

The correct establishment of DNA methylation patterns is vital for mammalian development and is achieved by the de novo DNA methyltransferases DNMT3A and DNMT3B. DNMT3B localises to H3K36me3 at actively transcribing gene bodies via its PWWP domain. It also functions at heterochromatin through an unknown recruitment mechanism. Here, we find that knockout of DNMT3B causes loss of methylation predominantly at H3K9me3-marked heterochromatin and that DNMT3B PWWP domain mutations or deletion result in striking increases of methylation in H3K9me3-marked heterochromatin. Removal of the N-terminal region of DNMT3B affects its ability to methylate H3K9me3-marked regions. This region of DNMT3B directly interacts with HP1α and facilitates the bridging of DNMT3B with H3K9me3-marked nucleosomes in vitro. Our results suggest that DNMT3B is recruited to H3K9me3-marked heterochromatin in a PWWP-independent manner that is facilitated by the protein's N-terminal region through an interaction with a key heterochromatin protein. More generally, we suggest that DNMT3B plays a role in DNA methylation homeostasis at heterochromatin, a process which is disrupted in cancer, aging and Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome.

DNMT1/DNMT3a-mediated promoter hypermethylation and transcription activation of ICAM5 augments thyroid carcinoma progression.

Promoter methylation is one of the most studied epigenetic modifications and it is highly relevant to the onset and progression of thyroid carcinoma (THCA). This study investigates the promoter methylation and expression pattern of intercellular adhesion molecule 5 (ICAM5) in THCA. CpG islands with aberrant methylation pattern in THCA, and the expression profiles of the corresponding genes in THCA, were analyzed using bioinformatics. ICAM5 was suggested to have a hypermethylation status, and it was highly expressed in THCA tissues and cells. Its overexpression promoted proliferation, mobility, and tumorigenic activity of THCA cells. As for the downstream signaling, ICAM5 was found to activate the MAPK/ERK and MAPK/JNK signaling pathways. Either inhibition of ERK or JNK blocked the oncogenic effects of ICAM5. DNA methyltransferases 1 (DNMT1) and DNMT3a were found to induce promoter hypermethylation of ICAM5 in THCA cells. Knockdown of DNMT1 or DNMT3a decreased the ICAM5 expression and suppressed malignant properties of THCA cells in vitro and in vivo, which were, however, restored by further artificial ICAM5 overexpression. Collectively, this study reveals that DNMT1 and DNMT3a mediates promoter hypermethylation and transcription activation of ICAM5 in THCA, which promotes malignant progression of THCA through the MAPK signaling pathway.

A review on recent advances in assays for DNMT1: a promising diagnostic biomarker for multiple human cancers.

The abnormal expression of human DNA methyltransferases (DNMTs) is closely related with the occurrence and development of a wide range of human cancers. DNA (cytosine-5)-methyltransferase-1 (DNMT1) is the most abundant human DNA methyltransferase and is mainly responsible for genomic DNA methylation patterns. Abnormal expression of DNMT1 has been found in many kinds of tumors, and DNMT1 has become a valuable target for the diagnosis and drug therapy of diseases. Nowadays, DNMT1 has been found to be involved in multiple cancers such as pancreatic cancer, breast cancer, bladder cancer, lung cancer, gastric cancer and other cancers. In order to achieve early diagnosis and for scientific research, various analytical methods have been developed for qualitative or quantitative detection of low-abundance DNMT1 in biological samples and human tumor cells. Herein, we provide a brief explication of the research progress of DNMT1 involved in various cancer types. In addition, this review focuses on the types, principles, and applications of DNMT1 detection methods, and discusses the challenges and potential future directions of DNMT1 detection.

Suppression of DNMT2/3 by proinflammatory cytokines inhibits CtBP1/2-dependent genes to promote the occurrence of atrophic nonunion.

Failure of bone healing after fracture often results in nonunion, but the underlying mechanism of nonunion pathogenesis is poorly understood. Herein, we provide evidence to clarify that the inflammatory microenvironment of atrophic nonunion (AN) mice suppresses the expression levels of DNA methyltransferases 2 (DNMT2) and 3A (DNMT3a), preventing the methylation of CpG islands on the promoters of C-terminal binding protein 1/2 (CtBP1/2) and resulting in their overexpression. Increased CtBP1/2 acts as transcriptional corepressors that, along with histone acetyltransferase p300 and Runt-related transcription factor 2 (Runx2), suppress the expression levels of six genes involved in bone healing: BGLAP (bone gamma-carboxyglutamate protein), ALPL (alkaline phosphatase), SPP1 (secreted phosphoprotein 1), COL1A1 (collagen 1a1), IBSP (integrin binding sialoprotein), and MMP13 (matrix metallopeptidase 13). We also observe a similar phenomenon in osteoblast cells treated with proinflammatory cytokines or treated with a DNMT inhibitor (5-azacytidine). Forced expression of DNMT2/3a or blockage of CtBP1/2 with their inhibitors can reverse the expression levels of BGLAP/ALPL/SPP1/COL1A1/IBSP/MMP13 in the presence of proinflammatory cytokines. Administration of CtBP1/2 inhibitors in fractured mice can prevent the incidence of AN. Thus, we demonstrate that the downregulation of bone healing genes dependent on proinflammatory cytokines/DNMT2/3a/CtBP1/2-p300-Runx2 axis signaling plays a critical role in the pathogenesis of AN. Disruption of this signaling may represent a new therapeutic strategy to prevent AN incidence after bone fracture.

Ectoine Globally Hypomethylates DNA in Skin Cells and Suppresses Cancer Proliferation.

Epigenetic modifications, mainly aberrant DNA methylation, have been shown to silence the expression of genes involved in epigenetic diseases, including cancer suppression genes. Almost all conventional cancer therapeutic agents, such as the DNA hypomethylation drug 5-aza-2-deoxycytidine, have insurmountable side effects. To investigate the role of the well-known DNA protectant (ectoine) in skin cell DNA methylation and cancer cell proliferation, comprehensive methylome sequence analysis, 5-methyl cytosine (5mC) analysis, proliferation and tumorigenicity assays, and DNA epigenetic modifications-related gene analysis were performed. The results showed that extended ectoine treatment globally hypomethylated DNA in skin cells, especially in the CpG island (CGIs) element, and 5mC percentage was significantly reduced. Moreover, ectoine mildly inhibited skin cell proliferation and did not induce tumorigenicity in HaCaT cells injected into athymic nude mice. HaCaT cells treated with ectoine for 24 weeks modulated the mRNA expression levels of Dnmt1, Dnmt3a, Dnmt3b, Dnmt3l, Hdac1, Hdac2, Kdm3a, Mettl3, Mettl14, Snrpn, and Mest. Overall, ectoine mildly demethylates DNA in skin cells, modulates the expression of epigenetic modification-related genes, and reduces cell proliferation. This evidence suggests that ectoine is a potential anti-aging agent that prevents DNA hypermethylation and subsequently activates cancer-suppressing genes.

Determinants of DNMT2/TRDMT1 preference for substrates tRNA and DNA during the evolution.

RNA methyltransferase DNMT2/TRDMT1 is the most conserved member of the DNMT family from bacteria to plants and mammals. In previous studies, we found some determinants for tRNA recognition of DNMT2/TRDMT1, but the preference mechanism of this enzyme for substrates tRNA and DNA remains to be explored. In the present study, CFT-containing target recognition domain (TRD) and target recognition extension domain (TRED) in DNMT2/TRDMT1 play a crucial role in the substrate DNA and RNA selection during the evolution. Moreover, the classical substrate tRNA for DNMT2/TRDMT1 had a characteristic sequence CUXXCAC in the anticodon loop. Position 35 was occupied by U, making cytosine-38 (C38) twist into the loop, whereas C, G or A was located at position 35, keeping the C38-flipping state. Hence, the substrate preference could be modulated by the easily flipped state of target cytosine in tRNA, as well as TRD and TRED. Additionally, DNMT2/TRDMT1 cancer mutant activity was collectively mediated by five enzymatic characteristics, which might impact gene expressions. Importantly, G155C, G155V and G155S mutations reduced enzymatic activities and showed significant associations with diseases using seven prediction methods. Altogether, these findings will assist in illustrating the substrate preference mechanism of DNMT2/TRDMT1 and provide a promising therapeutic strategy for cancer.

The Role of Clonal Hematopoiesis of Indeterminant Potential and DNA (Cytosine-5)-Methyltransferase Dysregulation in Pulmonary Arterial Hypertension and Other Cardiovascular Diseases.

DNA methylation is an epigenetic mechanism that regulates gene expression without altering gene sequences in health and disease. DNA methyltransferases (DNMTs) are enzymes responsible for DNA methylation, and their dysregulation is both a pathogenic mechanism of disease and a therapeutic target. DNMTs change gene expression by methylating CpG islands within exonic and intergenic DNA regions, which typically reduces gene transcription. Initially, mutations in the DNMT genes and pathologic DNMT protein expression were found to cause hematologic diseases, like myeloproliferative disease and acute myeloid leukemia, but recently they have been shown to promote cardiovascular diseases, including coronary artery disease and pulmonary hypertension. We reviewed the regulation and functions of DNMTs, with an emphasis on somatic mutations in DNMT3A, a common cause of clonal hematopoiesis of indeterminant potential (CHIP) that may also be involved in the development of pulmonary arterial hypertension (PAH). Accumulation of somatic mutations in DNMT3A and other CHIP genes in hematopoietic cells and cardiovascular tissues creates an inflammatory environment that promotes cardiopulmonary diseases, even in the absence of hematologic disease. This review summarized the current understanding of the roles of DNMTs in maintenance and de novo methylation that contribute to the pathogenesis of cardiovascular diseases, including PAH.

The relationship between DNA methyltransferase 3B (DNMT3B) and miR 124-3pa expressions in bladder cancer tissues.

BACKGROUND: Cancer bladder is the most common malignant tumor affecting the urinary tract. Genetic alterations are tightly associated with the development of cancer bladder. MicroRNAs (miRNA) are small, noncoding single-stranded RNA molecules that have been linked to bladder cancer. miR-124-3pa exhibits altered expression in various types of human malignancies. DNA methyltransferase 3B (DNMT3B) is responsible for de novo DNA methylation which is a fundamental epigenetic process in carcinogenesis. This work was performed to study the expression of DNMT3B and miR 124-3pa in bladder cancer tissues, and investigate their significance in the diagnosis and prognosis of the disease. SUBJECTS & METHODS: This case-control study included one hundred and six tissue samples of patients with primary urothelial bladder cancer. The tissues were separated into two parts. The first part was immediately frozen and kept at - 80 °C for total RNA extraction with subsequent detection of miR 124-3pa and DNMT3B expressions. The other part was preserved in formalin solution for histopathological examination. RESULTS: There was a highly statistically significant difference between the cancerous and the normal tissues as regarding miRNA-124-3pa and DNMT3B expression (P < 0.001) for each. Also, there was a highly statistically significant strong negative correlation between miRNA-124-3pa and DNMT3B expression (r=-0.750, P < 0.001). The combined performance of miR-124-3pa and DNMT3B revealed that the cutoff point of ≥ 3.3 can be used as a predictor of the presence of cancer bladder with sensitivity of 98.1% and specificity of 80%. CONCLUSION: miR-124-3pa and DNMT3B can be used as predictors of the presence of cancer bladder.

Effect of Long-Term Low-Dose Arsenic Exposure on DNA Methylation and Gene Expression in Human Liver Cells.

Millions of people around the world are exposed to elevated levels of arsenic through food or drinking water. Epidemiological studies have linked chronic arsenic exposure to an increased risk of several cancers, cardiovascular disease, central nervous system neuropathies, and genotoxic as well as immunotoxic effects. In addition to the induction of oxidative stress and inhibition of DNA repair processes, epigenetic effects, including altered DNA methylation patterns resulting in aberrant gene expression, may contribute to carcinogenicity. However, the underlying mechanisms by which chronic micromolar concentrations of arsenite affect the methylation status of DNA are not fully understood. In this study, human HepG2 hepatocarcinoma cells were treated with 0.5-10 µM sodium arsenite for 24 h, 10, or 20 days. During these periods, the effects on global DNA methylation, cell cycle phase distribution, and gene expression were investigated. While no impact on DNA methylation was seen after short-term exposure, global hypomethylation was observed at both long-term exposure periods, with concomitant induction of the DNA methyltransferase genes DNMT1 and DNMT3B, while DNMT3A was slightly down-regulated. Pronounced time- and concentration-dependent effects were also seen in the case of genes involved in DNA damage response and repair, inflammation, oxidative stress response, and metal homeostasis. These results suggest that chronic low-dose arsenite exposure can lead to global hypomethylation. As an underlying mechanism, the consistent down-regulation of DNA methyltransferase genes could be excluded; alternatively, interactions at the protein level could play an important role.

Downregulation of DNA methylation enhances differentiation of THP-1 cells and induces M1 polarization of differentiated macrophages.

DNA methylation is an epigenetic modification that regulates gene expression and plays an essential role in hematopoiesis. UHRF1 and DNMT1 are both crucial for regulating genome-wide maintenance of DNA methylation. Specifically, it is well known that hypermethylation is crucial characteristic of acute myeloid leukemia (AML). However, the mechanism underlying how DNA methylation regulates the differentiation of AML cells, including THP-1 is not fully elucidated. In this study, we report that UHRF1 or DNMT1 depletion enhances the phorbol-12-myristate-13-acetate (PMA)-induced differentiation of THP-1 cells. Transcriptome analysis and genome-wide methylation array results showed that depleting UHRF1 or DNMT1 induced changes that made THP-1 cells highly sensitive to PMA. Furthermore, knockdown of UHRF1 or DNMT1 impeded solid tumor formation in xenograft mouse model. These findings suggest that UHRF1 and DNMT1 play a pivotal role in regulating differentiation and proliferation of THP-1 cells and targeting these proteins may improve the efficiency of differentiation therapy in AML patients.