DNMT1 Protein Research Articles

FOXN3-AS1: A Candidate Prognostic Marker and Epigenetic Target with Immunotherapeutic Implications in Acute Myeloid Leukemia.

We focused on the FOXN3 gene and selected its antisense transcripts (FOXN3-AS1) to investigate its potential involvement in acute myeloid leukemia (AML). Several integrated multi-omics datasets have expanded the horizons of the cancer landscape. With the emergence of new high-throughput technologies, a large number of non-coding RNAs have been confirmed to be involved in the pathogenesis of different types of hematological malignancies. We conducted experimental validation using quantitative polymerase chain reaction (qPCR) with bone marrow specimens from AML patients. Then, Kaplan-Meier (KM) and Receiver Operating Characteristic (ROC) curves were used to substantiate the prognostic association between FOXN3-AS1 and AML patients within the TCGA database. Correlation between FOXN3-AS1 expression and gene mutation, immune, and immune function using Spearman correlation analysis. To explore the physical and functional interaction between FOXN3-AS1 and the DNMT1 protein, we utilized the RPISeq web tool from Iowa State University. Subsequently, we performed qPCR experiments to test the effect of 5AzaC (DNMT1 inhibitor) on FOXN3-AS1 expression AML cell lines (THP1 and OCI-AML3). We leveraged the "OncoPredict" R package in conjunction with the Genomics of Drug Sensitivity (GDSC) database to predict drug response in AML patients expressing FOXN3-AS1. We observed a significant upregulation of FOXN3-AS1 expression in AML patients compared to healthy controls using clinical samples. The TCGA database revealed an association between high FOXN3-AS1 expression and adverse prognosis. In our subsequent analysis, genes with poor prognostic implications in AML patients were exclusively identified in the FOXN3-AS1 high-expression group, further corroborating this relationship. AML patients with higher FOXN3-AS1 expression levels may respond less optimally to immunotherapy than patients with lower levels. Besides, we computationally predicted the interaction of FOXN3- AS1 and DNMT1 protein and experimentally confirmed that DNMT1i (GSK-3484862) affects the expression level of FOXN3-AS1. We also found that the chemotherapy drugs (5-Fluorouralic, Cisplatin, Dactolisib, Sapitinib, Temozolomide, Ulixertinib, Vinorelbine, Ruxolitinib, Osimertinib and Cisplatin) showed favorable responses in AML patients with high FOXN3-AS1 expression levels. Our candidate approach identifies FOXN3-AS1 as a prognostic indicator of survival in AML with a potential immune-related role. The preliminary observations we made on FOXN3-AS1/DNMT1 crosstalk warrant more in-depth invested immunotherapeutic approaches in AML.

The scaffolding function of LSD1 controls DNA methylation in mouse ESCs

Lysine-specific histone demethylase 1 (LSD1), which demethylates mono- or di- methylated histone H3 on lysine 4 (H3K4me1/2), is essential for early embryogenesis and development. Here we show that LSD1 is dispensable for mouse embryonic stem cell (ESC) self-renewal but is required for mouse ESC growth and differentiation. Reintroduction of a catalytically-impaired LSD1 (LSD1MUT) recovers the proliferation capability of mouse ESCs, yet the enzymatic activity of LSD1 is essential to ensure proper differentiation. Indeed, increased H3K4me1 in Lsd1 knockout (KO) mouse ESCs does not lead to major changes in global gene expression programs related to stemness. However, ablation of LSD1 but not LSD1MUT results in decreased DNMT1 and UHRF1 proteins coupled to global hypomethylation. We show that both LSD1 and LSD1MUT control protein stability of UHRF1 and DNMT1 through interaction with HDAC1 and the ubiquitin-specific peptidase 7 (USP7), consequently, facilitating the deacetylation and deubiquitination of DNMT1 and UHRF1. Our studies elucidate a mechanism by which LSD1 controls DNA methylation in mouse ESCs, independently of its lysine demethylase activity.

Supplementation of l-arginine in pregnant gilts affects the protein abundance of DNMT1 in 35-day fetuses

Maternal nutrition is one of the main environmental factors regulating gene expression during fetal development through epigenetic modifications. Some nutrients, such as the amino acid l-arginine, are added to maternal diets to modulate gene expression, improve the reproductive performance of females, and enhance conceptus development. This study investigated the hypothesis that supplementation of pregnant gilts with l-arginine regulates gene expression in conceptuses through epigenetic mechanisms. For this, fetal programming phenotypic markers, the expression of key epigenetic genes, and the abundance of DNA methylation proteins (DNMT3A and DNMT1) were evaluated in 25- and 35-day conceptuses from gilts supplemented (ARG) or not (CON) with 1.0 % l-arginine during early gestation. At 25 days, there were no significant differences in phenotypic markers between CON and ARG embryos (P > 0.05). Similarly, no differences were found between CON and ARG fetuses at 35 days (P > 0.05). Maternal supplementation with l-arginine did not influence the expression of the evaluated key epigenetic genes in pig embryos or fetuses, nor DNMT3A protein abundance (P > 0.05); on the other hand, DNMT1 protein abundance was lower in ARG fetuses (P = 0.002). It is concluded that supplementation of l-arginine in pregnant gilts affects epigenetic mechanisms, such as DNA methylation, in 35-day fetuses through regulation of DNMT1 levels. Further studies using transcriptomic and proteomic analysis could reveal additional epigenetic modifications in embryos and fetuses following maternal supplementation with l-arginine.

Long non-coding RNA SIX1-1 promotes proliferation of cervical cancer cells via negative transcriptional regulation of RASD1.

Cervical cancer poses a significant health burden for women globally, and the rapid proliferation of cervical cancer cells greatly worsens patient prognosis. Long non-coding RNAs (lncRNAs) play a crucial role in regulating tumor cell proliferation. However, the involvement of lncRNAs in cervical cancer cell proliferation remains unclear. In this study, we investigated the lncRNA SIX1-1, which was found to be upregulated in cervical cancer tissues and cell lines. Functional assays revealed that knockdown of SIX1-1 inhibited cell proliferation in vitro and reduced tumor growth in vivo. Mechanistically, SIX1-1 was predominantly localized in the nucleus and could bind with DNMT1 protein. The expression of SIX1-1 enhanced the interaction of DNMT1 with RASD1 promoter, leading to the methylation of the promoter and decreased mRNA transcription. Then RASD1 downregulation activated the cAMP/PKA/CREB signaling pathway, promoting cell proliferation. Rescue experiments showed that knockdown of RASD1 restored the inhibited cell proliferation caused by decreased expression of SIX1-1, indicating that RASD1 acted as the functional mediator of SIX1-1. In conclusion, SIX1-1 promoted cervical cancer cell proliferation by modulating RASD1 expression. This suggests that targeting the SIX1-1/RASD1 axis could be a potential antitumor strategy for cervical cancer.

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

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

DNA methyltransferase isoforms regulate endothelial cell exosome proteome composition

Extrinsic and intrinsic pathological stimuli in vascular disorders induce DNA methylation based epigenetic reprogramming in endothelial cells, which leads to perturbed gene expression and subsequently results in endothelial dysfunction (ED). ED is also characterized by release of exosomes with altered proteome leading to paracrine interactions in vasculature and subsequently contributing to manifestation, progression and severity of vascular complications. However, epigenetic regulation of exosome proteome is not known. Hence, our present study aimed to understand influence of DNA methylation on exosome proteome composition and their influence on endothelial cell (EC) function. DNMT isoforms (DNMT1, DNMT3A, and DNMT3B) were overexpressed using lentivirus in ECs. Exosomes were isolated and characterized from ECs overexpressing DNMT isoforms and C57BL/6 mice plasma treated with 5-aza-2′-deoxycytidine. 3D spheroid assay was performed to understand the influence of exosomes derived from cells overexpressing DNMTs on EC functions. Further, the exosomes were subjected to TMT labelled proteomics analysis followed by validation. 3D spheroid assay showed increase in the pro-angiogenic activity in response to exosomes derived from DNMT overexpressing cells which was impeded by inclusion of 5-aza-2′-deoxycytidine. Our results showed that exosome proteome and PTMs were significantly modulated and were associated with dysregulation of vascular homeostasis, metabolism, inflammation and endothelial cell functions. In vitro and in vivo validation showed elevated DNMT1 and TGF-β1 exosome proteins due to DNMT1 and DNMT3A overexpression, but not DNMT3B which was mitigated by 5-aza-2′-deoxycytidine indicating epigenetic regulation. Further, exosomes induced ED as evidenced by reduced expression of phospho-eNOSser1177. Our study unveils epigenetically regulated exosome proteins, aiding management of vascular complications.

Nanocomposite magnetic hydrogel with dual anisotropic properties induces osteogenesis through the NOTCH-dependent pathways

Physical factors in the cellular microenvironment have critical effects on stem cell differentiation. The utilization of physical factors to promote the osteogenic differentiation of stem cells has been established as a new strategy for developing bone tissue engineering scaffolds. In this context, scaffolds with multiscale anisotropy are considered to possess biomimetic properties, which are advantageous for their biological performance. In the present study, a novel magnetic anisotropic hydrogel (MAH) with magnetic and topographic anisotropy was designed by combining static magnetic field-induced magnetic nanomaterials and a hydrogel. In in vitro studies, the MAH exhibited excellent biocompatibility and osteogenic bioactivity. The alkaline phosphatase activity and the expression of osteogenic-related genes and proteins induced by the MAH were greater than those induced by the pure PEGDA–GelMA hydrogel (PGH) and the magnetic isotropic hydrogel (MIH). In addition, the present study revealed that the dual anisotropic properties of the MAH activated the NOTCH1/2 pathway by upregulating SNHG5 and downstream SIRT6, which modulates the level of NOTCH1/2 by antagonizing DNMT1 protein stability, ultimately inducing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Furthermore, the MAH, MIH, and PGH were tested for in vivo bone regeneration in rabbits with femur defects, and the results demonstrated that the MAH effectively stimulated bone regeneration. Taken together, these findings suggest that this magnetically and topographically anisotropic biomimetic hydrogel might be a promising candidate for application in the field of bone tissue regeneration.

Annonaceous acetogenins as promising DNA methylation inhibitors to prevent and treat leukemogenesis – an in silico approach

Leukemia is a haematological malignancy affecting blood and bone marrow, ranking 10th among the other common cancers. DNA methylation is an epigenetic dysregulation that plays a critical role in leukemogenesis. DNA methyltransferases (DNMTs) such as DNMT1, DNMT3A and DNMT3B are the key enzymes catalysing DNA methylation. Inhibition of DNMT1 with secondary metabolites from medicinal plants helps reverse DNA methylation. The present study focuses on inhibiting DNMT1 protein (PDB ID: 3PTA) with annonaceous acetogenins through in-silico studies. The docking and molecular dynamic (MD) simulation study was carried out using Schrödinger Maestro and Desmond, respectively. These compounds’ drug likeliness, ADMET properties and bioactivity scores were analysed. About 76 different acetogenins were chosen for this study, among which 17 showed the highest binding energy in the range of −8.312 to −10.266 kcal/mol. The compounds with the highest negative binding energy were found to be annohexocin (-10.266 kcal/mol), isoannonacinone (-10.209 kcal/mol) and annonacin (-9.839 kcal/mol). MD simulation results reveal that annonacin remains stable throughout the simulation time of 100 ns and also binds to the catalytic domain of DNMT1 protein. From the above results, it can be concluded that annonacin has the potential to inhibit the DNA methylation process and prevent leukemogenesis. Communicated by Ramaswamy H. Sarma

Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation.

While extensive investigations have been devoted to the study of genetic pathways related to fatty liver diseases, much less is known about epigenetic mechanisms underlying these disorders. DNA methylation is an epigenetic link between environmental factors (e.g., diets) and complex diseases (e.g., non-alcoholic fatty liver disease). Here, it is aimed to study the role of DNA methylation in the regulation of hepatic lipid metabolism. A dynamic change in the DNA methylome in the liver of high-fat diet (HFD)-fed mice is discovered, including a marked increase in DNA methylation at the promoter of Beta-klotho (Klb), a co-receptor for the biological functions of fibroblast growth factor (FGF)15/19 and FGF21. DNA methyltransferases (DNMT) 1 and 3A mediate HFD-induced methylation at the Klb promoter. Notably, HFD enhances DNMT1 protein stability via a ubiquitination-mediated mechanism. Liver-specific deletion of Dnmt1 or 3a increases Klb expression and ameliorates HFD-induced hepatic steatosis. Single-nucleus RNA sequencing analysis reveals pathways involved in fatty acid oxidation in Dnmt1-deficient hepatocytes. Targeted demethylation at the Klb promoter increases Klb expression and fatty acid oxidation, resulting in decreased hepatic lipid accumulation. Up-regulation of methyltransferases by HFD may induce hypermethylation of the Klb promoter and subsequent down-regulation of Klb expression, resulting in the development of hepatic steatosis.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Peripheral T-cell lymphomas (PTCL) are a rare and heterogeneous group of malignancies that share a unifying feature of epigenetic dysregulation. Recent evidence suggests that PTCL, unlike other forms of cancer or even lymphoma, exhibits a marked vulnerability to hypomethylating agents (HMAs) alone and especially in combination with HDAC inhibitors. HMAs like azacitidine (AZA) and decitabine (DAC) have been shown to reverse transcriptional repression secondary to hypermethylation. Cladribine (CLAD) is FDA-approved for the treatment of hairy cell leukemia and is postulated to inhibit both DNA and histone methylation. Here, we sought to define the mechanistic differences between different DNA HMAs in order to inform a rationale on the optimal combinations that might exploit the epigenetic vulnerabilities of PTCL. Methods: Cell viability, caspase activity assay, and western blotting (WB) were performed on a panel of T-cell lymphoma cell lines to identify the effects of AZA/DAC/CLAD on cell survival, apoptosis, and DNA methyltransferase (DNMT) levels. Liquid chromatography-based mass spectrometry (LC-MS) was done to quantify 5-methyl cytosine (5-mC) levels. Results: Cell viability analysis confirmed that the dose and time-dependent sensitivity of the six PTCL cell lines differed following exposure to AZA/DAC/CLAD, where CLAD showed the lowest IC50 value (0.1–1 µM) across all cell lines, followed by AZA (IC50: 1–10 µM), and DAC had the least effect on cell viability (IC50: >100 µM, except T-ALL). A differential threshold of caspase activation was observed across the PTCL cell lines where CLAD showed the most superior caspase activity leading to the highest apoptotic potential. However, WB analysis showed that after 24 hours of treatment, DNMT1 and DNMT3A protein levels were significantly depleted at low concentrations of DAC (<0.01 µM) compared to AZA whereas CLAD did not affect the DNMT1 and DNMT3A levels. LC-MS-based quantification of 5-mC showed a similar relationship in methylation, with initial changes observed at doses >0.01 μM of DAC and >0.1 μM of AZA/CLAD. Taking the short half-life of AZA/DAC into account, cell viability assays with daily addition of AZA/DAC showed increased cell cytotoxicity for AZA/DAC treated samples, which was comparable to CLAD. Ongoing analyses of gene expression and metabolism of the universal methyl donor S-adenosylmethionine (SAM) will help to understand the mechanistic difference between these HMAs. Conclusions: These data suggest that the HMAs have a distinct mechanism of action for hypomethylation. While AZA/DAC induces hypomethylation by depletion of DNMTs, CLAD acts in a DNMT depletion-independent pathway. A mechanistic understanding of AZA/DAC/CLAD will inform how best to use them in clinic as well as assist in the strategic development of biological correlates to further support their therapeutic application. The research was funded by: Translational Orphan Blood Cancer Research Initiative Fund and RO1 # FD-R-006814-01. Keywords: Genomics, Epigenomics, and Other -Omics, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Combined inhibition of histone deacetylase and cytidine deaminase improves epigenetic potency of decitabine in colorectal adenocarcinomas

BackgroundTargeting the epigenome of cancerous diseases represents an innovative approach, and the DNA methylation inhibitor decitabine is recommended for the treatment of hematological malignancies. Although epigenetic alterations are also common to solid tumors, the therapeutic efficacy of decitabine in colorectal adenocarcinomas (COAD) is unfavorable. Current research focuses on an identification of combination therapies either with chemotherapeutics or checkpoint inhibitors in modulating the tumor microenvironment. Here we report a series of molecular investigations to evaluate potency of decitabine, the histone deacetylase inhibitor PBA and the cytidine deaminase (CDA) inhibitor tetrahydrouridine (THU) in patient derived functional and p53 null colon cancer cell lines (CCCL). We focused on the inhibition of cell proliferation, the recovery of tumor suppressors and programmed cell death, and established clinical relevance by evaluating drug responsive genes among 270 COAD patients. Furthermore, we evaluated treatment responses based on CpG island density.ResultsDecitabine caused marked repression of the DNMT1 protein. Conversely, PBA treatment of CCCL recovered acetylation of histone 3 lysine residues, and this enabled an open chromatin state. Unlike single decitabine treatment, the combined decitabine/PBA treatment caused > 95% inhibition of cell proliferation, prevented cell cycle progression especially in the S and G2-phase and induced programmed cell death. Decitabine and PBA differed in their ability to facilitate re-expression of genes localized on different chromosomes, and the combined decitabine/PBA treatment was most effective in the re-expression of 40 tumor suppressors and 13 genes typically silenced in cancer-associated genomic regions of COAD patients. Furthermore, this treatment repressed expression of 11 survival (anti-apoptotic) genes and augmented expression of X-chromosome inactivated genes, especially the lncRNA Xist to facilitate p53-mediated apoptosis. Pharmacological inhibition of CDA by THU or its gene knockdown prevented decitabine inactivation. Strikingly, PBA treatment recovered the expression of the decitabine drug-uptake transporter SLC15A1, thus enabling high tumor drug-loads. Finally, for 26 drug responsive genes we demonstrated improved survival in COAD patients.ConclusionThe combined decitabine/PBA/THU drug treatment improved drug potency considerably, and given their existing regulatory approval, our findings merit prospective clinical trials for the triple combination in COAD patients.

Inhibition of IL-6 methylation by Saikosaponin C regulates neuroinflammation to alleviate depression.

Saikosaponin C (SSc) increases the expression of synaptic proteins and has a unexplored role in the prevention of AD and other neurodegeneration in humans. Therefore, we hypothesized that SSc has the potential to relief of depressive symptoms. Here, our study assessed the role of SSc on depression-like behaviors caused by a chronic social defeat stress (CSDS) in mice and explored the underlying mechanisms. Behavioral tests were conducted to verify the efficacy of SSC in treating depression-like behavior in mice. The levels of IL-6, TNF-α and IL-1β in brain tissue and BV2 cells were determined by ELISA. The effect of SSc on dendritic spine density was determined by Golgi staining. The percentage of monocytes in peripheral blood was measured using flow cytometry. The levels of STAT3 and DNMT1 under the influence of SSc were assessed by immunofluorescence. Protein expression of DNMT1, DNMT3a, DNMT3b, p-STAT3 and STAT3 in brain and BV2 cells was studied by Western blot. OE-DNMT1 was induced in the experiment to verify the inhibitory effect of DNMT1 on IL-6 methylation in SSC. Luciferase was used to detect SSC specific fragments affecting IL-6 methylation. SSC treatment significantly alleviated depressive-like behavior, inhibited the levels of inflammatory cytokines including IL-6, IL-1β and TNF-α, increased dendritic spine density and promoted synaptic plasticity in mice. SSC downregulated IL-6, STAT3 and DNMT1 expression in vivo and in vitro. SSC also decreased the percentage of monocytes in peripheral blood and suppressed neuroinflammation in mice. Overexpression of DNMT1 by shRNA abolished the inhibitory effect of SSc on IL-6 methylation. This study showed that SSc reduced IL6 methylation by inhibiting DNMT1 protein, induced a decrease in IL6 expression, promoted synaptic plasticity, and attenuated CSDS-induced depression-like behavior.

DNA methylation inhibitor regulates guanylyl cyclase/natriuretic peptide receptor-A gene expression via 5'-untranslated region in mesangial and smooth muscle cells

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is activated by cardiac hormones atrial and brain natriuretic peptides (ANP, BNP) and produces intracellular second messenger cGMP. The physiological actions of ANP/NPRA/cGMP signaling cascade largely include diuresis, natriuresis, vasorelaxation, and organ protection, which maintains blood pressure and opposed renal, cardiac, and vascular injury and remodeling. However, the mechanisms that contribute to the activation of Npr1 (coding for GC-A/NPRA) expression is not well understood. The present study was aimed at understanding the epigenetic mechanisms of DNA methyltransferase (DNMT) inhibitor, 5-Azacytidine (5-Aza)-mediated Npr1 gene transcription. The studies were carried out in mouse mesangial cells (MMCs) and rat thoracic aortic smooth muscle (RTASM) cells, cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and ITS (insulin, transferrin, and sodium selenite) and treated with 5-Aza for 24 h. Western blot analysis showed that 5-Aza enhanced NPRA protein levels by 5-fold and 7-fold in a dose-dependent manner in MMCs and RTASM cells, respectively, and greatly stimulated intracellular accumulation of cGMP in ANP-treated cells. Treatment with 5-Aza attenuated DNMT activity and repressed DNMT1 protein levels in cells. The MethPrimer search result showed the presence of three cytosine-phosphate-guanine (CpG) islands in the Npr1 full length promoter and 5'-untranslated region (UTR), namely island 1 (-886 to -752, 134 bp), island 2 (-310 to -158, 152 bp), and island 3 (-154 to +310, 464 bp) from the transcription start site. Addition of 5'-UTR encompassing +56 to +359 bp region to the Npr1 promoter constructs, (-1982/+55) and proximal promoter (-356/+55), was inhibitory to Npr1 transcriptional activity, independent of the cell type. Furthermore, treatment with 5-Aza distinctly induced Npr1 promoter activity of the construct -356/5'-UTR by 10-fold in MMCs suggesting that the repressive effect of DNA methylation on Npr1 gene transcription might be via 5'-UTR. The results demonstrate that 5-Aza acts as a potent inducer of Npr1 expression and signaling and provides new insights in the epigenetic control of Npr1, an important player in the control of hypertension and cardiorenal axis. Supported by the NIH grants (HL062147 and DK133833). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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

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

High nuclear expression of DNMT1 in correlation with inactivation of TET1 portray worst prognosis among the cervical carcinoma patients: clinical implications.

In this study, we aimed to understand the interplay of the epigenetic modifier genes DNMT1 and TET1 along with HPV infection in the cervical epithelium and how it changes during tumorigenesis. For this purpose, initially the bioinformatical analysis (methylation and expression profile) of DNMT1 and TET1 was analyzed in the TCGA dataset. Next genetic (deletion) and epigenetic profiling (promoter methylation) of DNMT1 and TET1 were done in our sample pool and also validated in CACX cell lines as well. The results were further correlated with different clinicopathological parameters. Our data revealed that HPV infection in basal/parabasal layers of cervical epithelium actually disrupts the epigenetic homeostasis of DNMT1 and TET1 proteins which ultimately leads to the high expression of DNMT1 along with further reduction in TET1 protein during the development of carcinoma. Further, in-depth look into the results revealed that comparatively low methylation frequency of DNMT1 coupled with high promoter methylation and deletion frequency [22-46%] of TET1 were the plausible reasons of their antagonistic expression profile during the progression of the disease. Interestingly, the prevalence of DNMT1 [9.1%] and TET1 promoter methylation [22.7%] found in both the plasma DNA of the respective CACX patients implicated its diagnostic importance in this study. Lastly, molecular alteration of TET1 alone or in combination with DNMT1 showed the worst overall survival among the patients. Hence, it may be concluded that an inverse molecular profile of DNMT1 and TET1 genes seen in the proliferative basal-parabasal layers of the cervical epithelium was aggravated during the development of CACX along with genetic and epigenetic changes due to HPV infection.

High-fat diet induced obesity alters Dnmt1 and Dnmt3a levels and global DNA methylation in mouse ovary and testis.

Obesity impairs reproductive capacity, and the link between imprinting disorders and obesity has been discussed in many studies. Recent studies indicate that a high-fat diet may cause epigenetic changes in maternal and paternal genes, which may be transmitted to offspring and negatively affect their development. On this basis, our study aims to reveal the changes in DNA methylation and DNA methyltransferase enzymes in the ovaries and testes of C57BL/6 mice fed a high-fat diet and created a model of obesity, by comparing them with the control group. For this purpose, we demonstrated the presence and quantitative differences of DNA methyltransferase 1 and DNA methyltransferase 3a enzymes as well as global DNA methylation in ovaries and testis of C57BL/6 mice fed a high-fat diet by using immunohistochemistry and western blot methods. We found that a high-fat diet induces the levels of Dnmt1 and Dnmt3a proteins (p < 0.05). We observed increased global DNA methylation in testes but, interestingly, decreased global DNA methylation in ovaries. We think that our outcomes have significant value to demonstrate the effects of obesity on ovarian follicle development and testicular spermatogenesis and may bring a new perspective to obesity-induced infertility treatments. Additionally, to the best of our knowledge, this is the first study to document dynamic alteration of Dnmt1 and Dnmt3a as well as global DNA methylation patterns during follicle development in healthy mouse ovaries.

Mitotic Perturbation Is a Key Mechanism of Action of Decitabine in Myeloid Tumor Treatment

Background: Decitabine (DAC) is one of the first generation of epigenetic drugs used clinically to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Historically, DAC was considered as a cytotoxic agent when it was first developed in the 1960s. Subsequent studies in 1980s showed that DAC can induce DNMT1 degradation and DNA hypomethylation at non-cytotoxic concentration. Since then, DNA demethylation has been considered as the main mechanism of action of DAC. The DAC-induced DNA hypomethylation was shown to induce upregulation of tumor suppressor genes or endogenous retroviruses, which could contribute to the antitumor effect of DAC. Recently, DAC was also shown to be effective even in MDS and AML with TP53 mutations. However, it remains unclear why myeloid tumors, especially those with TP53 mutations, are particularly susceptible to DAC treatment. Methods: Cancer Dependency Map (DepMap) database was used to evaluate in vitro sensitivity of human cancer cell lines to DAC. To identify essential genes that regulate sensitivity of myeloid tumors to DAC, we performed a genome-wide CRISPR-dCas9 activation screen using murine MDS/AML cells expressing ASXL1 and SETBP1 mutants (cSAM cells). After transduction of mouse genome-wide CRISPRa-v2 library, cSAM cells were cultured with DMSO or DAC for 1 week. We extracted genomic DNA from the cells at the start and end of our screen and quantified the relative representation of each sgRNA. To assess the effect of DAC on mitosis, we established the time-lapse system optimized for non-adherent MDS-L-2007 cells (a human secondary AML (sAML) cell line derived from an MDS cell line, MDS-92). Results: We first explored DepMap database and found that myeloid tumors are susceptible to DAC at clinically achievable concentration, while most solid tumor cell lines are not. The CRISPR/dCas9-activation screen revealed that genes involved in chromosome segregation, including Cdk1, Cdc20, Cdca8 and Dsn1, were significantly enriched in DAC-resistant cells, indicating that overexpression of mitotic regulators conferred resistance to DAC in cSAM cells. We also found that DAC strongly induced aneuploidy and polyploidy (>4n) in myeloid tumors, especially in those with TP53 mutations and sAML cells. Furthermore, the time-lapse imaging revealed that DAC treatment prolonged the duration of metaphase and increased multipolar mitosis and abscission failure in MDS-L-2007 cells (Figure). Thus, DAC perturbs mitosis and has cytotoxic effects at low concentrations in myeloid tumors. Next, we assessed the role of DNMT1 in the DAC-induced mitotic defects in myeloid tumors. To our surprise, the DAC-induced aneuploidy and apoptosis were markedly attenuated in DNMT1-depleted myeloid tumors. In contrast, overexpression of Dnmt1 increased sensitivity of myeloid tumors to DAC. These results indicate that DAC perturbs mitosis not through DNMT1 degradation and subsequent DNA demethylation, but through DNMT1 itself in myeloid tumors. In line with this, chromatin-bound DNMT1 protein was retained even with DAC, whereas DAC induced rapid degradation of DNMT1 in the nucleoplasm fraction. Importantly, the Dnmt1-C1229A mutant, which loses the ability to induce DNMT1-DNA covalent bond formation, did not enhance the DAC-driven mitotic defects. In addition, DAC treatment increased frequency of cohesion loss between sister chromatids in MDS-L-2007 cells, as evidenced by the absence of centromere connection of sister chromatids at metaphase. Moreover, the in vitroXenopus cell-free system which reproduces replication-dependent DNA methylation maintenance revealed that addition of 5-aza-dCTP strongly promoted chromatin binding of DNMT1, while it inhibited that of cohesion complex members (SMC3 and SCC1). Taken together, these results strongly indicate that the aberrant DNMT1 retention on chromatin induced by DAC leads to cohesion loss and mitotic defects in myeloid tumors. Conclusion: These findings challenge the current assumption that DAC inhibits leukemogenesis by inducing DNA hypomethylation. Rather, the anti-leukemia effect of DAC is likely mediated mainly by the non-epigenetic mechanism: mitotic perturbation through aberrant DNMT1-DNA covalent bonds and cohesion loss of sister chromatids. Our study also suggests that DAC will be most effective on tumors with impaired mitotic fidelity, such as AML and MDS with TP53 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LPS-pretreatment adipose-derived mesenchymal stromal cells promote wound healing in diabetic rats by improving angiogenesis

Mesenchymal stem cells (MSCs) play a key role in wound healing, and the advantages of pretreated MSCs in wound healing have previously been reported. In the present study, we investigated the impact of LPS pretreated human adipose-derived MSCs on skin wound healing in diabetic rats. We found that some improvements occurred through improving angiogenesis. Then, we scrutinized the impact of lipopolysaccharide (LPS) treatment on human adipose-derived MSCs in a high-glucose (HG) medium, as an in vitro diabetic model. In vivo findings revealed significant improvements in epithelialization and angiogenesis of diabetic wounds which received LPS pre-MSCs. Particularly, LPS pre-MSCs-treated diabetic wounds reached considerably higher percentages of wound closure. Also, the granulation tissue of these wounds had higher pronounced epithelialization and more vascularization compared with PBS-treated and MSCs-treated diabetic ones by CD31, VEGF, CD90, collagen 1, and collagen 3 immunostaining. Western-blots analyses indicated that LPS pre-MSCs led to the upregulation of vascular endothelial growth factor (VEGF) and DNMT1. In addition, significantly higher cell viability (proliferation/colonie), and elevated VEGF and DNMT1 protein expression were observed when MSCs were treated with LPS (10 ng/ml, 6 h) in HG culture media. Based on these findings, it is suggested that LPS pre-MSCs could promote wound repair and skin regeneration, in some major processes, via the improvement of cellular behaviors of MSCs in the diabetic microenvironment. The beneficial advantages of LPS treated with mesenchymal stem cells on wound healing may lead to establishing a novel approach as an alternative therapeutic procedure to cure chronic wounds in diabetic conditions.

Hydrogen Sulfide Improves Angiogenesis by Regulating the Transcription of pri-miR-126 in Diabetic Endothelial Cells.

Introduction: Diabetes mellitus results in high rates of cardiovascular disease, such as microcirculation disorder of the lower limbs, with angiogenesis impairment being the main factor. The endothelium functions as a barrier between blood and the vessel wall. Vascular endothelial cell dysfunction caused by hyperglycemia is the main factor leading to angiogenesis impairment. Hydrogen sulfide (H2S) and miR-126-3p are known for their pro-angiogenesis effects; however, little is known about how H2S regulates miR-126-3p to promote angiogenesis under high-glucose conditions. Objectives: The main objective of this research was to explore how H2S regulates the miR-126-3p levels under high-glucose conditions. Methods: We evaluated the pro-angiogenesis effects of H2S in the diabetic hindlimb of an ischemia mice model and in vivo Matrigel plugs. Two microRNA datasets were used to screen microRNAs regulated by both diabetes and H2S. The mRNA and protein levels were detected through real-time PCR and Western blot, respectively. Immunofluorescent staining was also used to assess the capillary density and to evaluate the protein levels in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were used in in vitro experiments. A scratch wound-healing assay was applied to detect the migration ability of endothelial cells. Methylated DNA immunoprecipitation combined with real-time PCR was chosen to identify the DNA methylation level in the HUVECs. Results: Exogenous H2S improved angiogenesis in diabetic mice. miR-126-3p was regulated by both diabetes and H2S. Exogenous H2S up-regulated the miR-126-3p level and recovered the migration rate of endothelial cells via down-regulating the DNMT1 protein level, which was increased by high glucose. Furthermore, DNMT1 upregulation in the HUVECs increased the methylation levels of the gene sequences upstream of miR-126-3p and then inhibited the transcription of primary-miR-126, thus decreasing the miR-126-3p level. CSE overexpression in the HUVECs rescued the miR-126-3p level, by decreasing the methylation level to improve migration. Conclusion: H2S increases the miR-126-3p level through down-regulating the methylation level, by decreasing the DNMT1 protein level induced by high glucose, thus improving the angiogenesis originally impaired by high glucose.

Expression and Clinical Significance of HER2 Gene and DNMT1 in Non-Small-Cell Lung Cancer.

Objective To explore the expression and clinical significance of HER2 and DNMT1 in non-small-cell lung cancer. Methods The patients with non-small-cell lung cancer treated in the First Affiliated Hospital of Jiamusi University between 2018 and 2020 were enrolled in this study. The serum DNMT1 concentration and the expression of HER2 protein in lung cancer and adjacent tissues of the two groups were analyzed. Results The DNMT1 protein concentration was significantly correlated with gender, age, and smoking history of patients. HER2-positive expression was significantly related to tumor type, tumor size, tumor differentiation degree, and lymph node metastasis. However, HER2 levels were not related to the gender and smoking history of patients. Conclusion High expression of DNMT1 protein in serum may increase the risk of non-small-cell lung cancer and may play an important role in the early development of lung cancer. HER2-positive expression may promote the development of advanced and metastatic non-small-cell lung cancer.