Metformin selectively inhibits metastatic colorectal cancer with the KRAS mutation by intracellular accumulation through silencing MATE1.

Metastatic colorectal cancer (mCRC) patients have poor overall survival despite using irinotecan- or oxaliplatin-based chemotherapy combined with anti-EGFR (epidermal growth factor receptor) drugs, especially those with the oncogene mutation of KRAS Metformin has been reported as a potentially novel antitumor agent in many experiments, but its therapeutic activity is discrepant and controversial so far. Inspiringly, the median survival time for KRAS-mutation mCRC patients with diabetes on metformin is 37.8 mo longer than those treated with other hypoglycemic drugs in combination with standard systemic therapy. In contrast, metformin could not improve the survival of mCRC patients with wild-type KRAS Interestingly, metformin is preferentially accumulated in KRAS-mutation mCRC cells, but not wild-type ones, in both primary cell cultures and patient-derived xenografts, which is in agreement with its tremendous effect in KRAS-mutation mCRC. Mechanistically, the mutated KRAS oncoprotein hypermethylates and silences the expression of multidrug and toxic compound extrusion 1 (MATE1), a specific pump that expels metformin from the tumor cells by up-regulating DNA methyltransferase 1 (DNMT1). Our findings provide evidence that KRAS-mutation mCRC patients benefit from metformin treatment and targeting MATE1 may provide a strategy to improve the anticancer response of metformin.

Epigenetic Metabolic Reprogramming of Right Ventricular Fibroblasts in Pulmonary Arterial Hypertension: A Pyruvate Dehydrogenase Kinase-Dependent Shift in Mitochondrial Metabolism Promotes Right Ventricular Fibrosis.

RATIONALE: Right ventricular (RV) fibrosis in pulmonary arterial hypertension contributes to RV failure. While RV fibrosis reflects changes in the function of resident RV fibroblasts (RVfib), these cells are understudied. OBJECTIVE: Examine the role of mitochondrial metabolism of RVfib in RV fibrosis in human and experimental pulmonary arterial hypertension. METHODS AND RESULTS: Male Sprague-Dawley rats received monocrotaline (MCT; 60 mg/kg) or saline. Drinking water containing no supplement or the PDK (pyruvate dehydrogenase kinase) inhibitor dichloroacetate was started 7 days post-MCT. At week 4, treadmill testing, echocardiography, and right heart catheterization were performed. The effects of PDK activation on mitochondrial dynamics and metabolism, RVfib proliferation, and collagen production were studied in RVfib in cell culture. Epigenetic mechanisms for persistence of the profibrotic RVfib phenotype in culture were evaluated. PDK expression was also studied in the RVfib of patients with decompensated RV failure (n=11) versus control (n=7). MCT rats developed pulmonary arterial hypertension, RV fibrosis, and RV failure. MCT-RVfib (but not left ventricular fibroblasts) displayed excess mitochondrial fission and had increased expression of PDK isoforms 1 and 3 that persisted for >5 passages in culture. PDK-mediated decreases in pyruvate dehydrogenase activity and oxygen consumption rate were reversed by dichloroacetate (in RVfib and in vivo) or siRNA targeting PDK 1 and 3 (in RVfib). These interventions restored mitochondrial superoxide and hydrogen peroxide production and inactivated HIF (hypoxia-inducible factor)-1α, which was pathologically activated in normoxic MCT-RVfib. Redox-mediated HIF-1α inactivation also decreased the expression of TGF-ß1 (transforming growth factor-beta-1) and CTGF (connective tissue growth factor), reduced fibroblast proliferation, and decreased collagen production. HIF-1α activation in MCT-RVfib reflected increased DNMT (DNA methyltransferase) 1 expression, which was associated with a decrease in its regulatory microRNA, miR-148b-3p. In MCT rats, dichloroacetate, at therapeutic levels in the RV, reduced phospho-pyruvate dehydrogenase expression, RV fibrosis, and hypertrophy and improved RV function. In patients with pulmonary arterial hypertension and RV failure, RVfib had increased PDK1 expression. CONCLUSIONS: MCT-RVfib manifest a DNMT1-HIF-1α-PDK-mediated, chamber-specific, metabolic memory that promotes collagen production and RV fibrosis. This epigenetic mitochondrial-metabolic pathway is a potential antifibrotic therapeutic target.

Interleukin-6-mediated epigenetic control of the VEGFR2 gene induces disorganized angiogenesis in human breast tumors.

Disorganized vessels in the tumor vasculature lead to impaired perfusion, resulting in reduced accessibility to immune cells and chemotherapeutic drugs. In the breast tumor-stroma interplay, paracrine factors such as interleukin-6 (IL-6) often facilitate disordered angiogenesis. We show here that epigenetic mechanisms regulate the crosstalk between IL-6 and vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in myoepithelial (CD10+) and endothelial (CD31+, CD105+, CD146+, and CD133-) cells isolated from malignant and nonmalignant tissues of clinically characterized human breast tumors. Tumor endothelial (Endo-T) cells in 3D cultures exhibited higher VEGFR2 expression levels, accelerated migration, invasion, and disorganized sprout formation in response to elevated IL-6 levels secreted by tumor myoepithelial (Epi-T) cells. Constitutively, compared with normal endothelial (Endo-N) cells, Endo-T cells differentially expressed DNA methyltransferase isoforms and had increased levels of IL-6 signaling intermediates such as IL-6R and signal transducer and activator of transcription 3 (STAT3). Upon IL-6 treatment, Endo-N and Endo-T cells displayed altered expression of the DNA methyltransferase 1 (DNMT1) isoform. Mechanistic studies revealed that IL-6 induced proteasomal degradation of DNMT1, but not of DNMT3A and DNMT3B and subsequently led to promoter hypomethylation and expression/activation of VEGFR2. IL-6-induced VEGFR2 up-regulation was inhibited by overexpression of DNMT1. Transfection of a dominant-negative STAT3 mutant, but not of STAT1, abrogated VEGFR2 expression. Our results indicate that in the breast tumor microenvironment, IL-6 secreted from myoepithelial cells influences DNMT1 stability, induces the expression of VEGFR2 in endothelial cells via a promoter methylation-dependent mechanism, and leads to disordered angiogenesis.

Mechanical force inhibited hPDLSCs proliferation with the downregulation of MIR31HG via DNA methylation.

OBJECTIVE: This study aimed to investigate how mechanical force affects the proliferation of human periodontal ligament stem cells (hPDLSCs). METHODS: CCK-8 assays and staining of ki67 were performed to evaluate hPDLSCs proliferation. qRT-PCR, ELISA, or Western blot analysis were used to measure the expression levels of interleukin (IL)-6, miR-31 host gene (MIR31HG), DNA methyltransferase 1 (DNMT1), and DNA methyltransferase 3B (DNMT3B). Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays were conducted to determine whether MIR31HG was targeted by DNMT1 and DNMT3B. MassARRAY mass spectrometry was used to quantify DNA methylation levels of the MIR31HG promoter. RESULTS: Mechanical force inhibited hPDLSCs proliferation with the downregulation of MIR31HG and upregulation of IL-6, DNMT1 and DNMT3B. Knockdown of MIR31HG suppressed hPDLSCs proliferation, and knockdown of DNMT1 or DNMT3B reversed mechanical force-induced downregulation of MIR31HG. Dual-luciferase and ChIP assays revealed DNMT1 and DNMT3B bound MIR31HG promoter in the region 1,015 bp upstream of the transcriptional start site. Treatment with 5'-aca-2'-deoxycytidine downregulated DNA methylation level in MIR31HG gene promoter, while mechanical force promoted the methylation of MIR31HG gene promoter. CONCLUSIONS: These findings elucidated how mechanical force affects proliferation via MIR31HG in hPDLSCs, providing clues for possible MIR31HG-based orthodontic therapeutic approaches.

BAH domains and a histone-like motif in DNA methyltransferase 1 (DNMT1) regulate de novo and maintenance methylation in vivo.

DNA methyltransferase 1 (DNMT1) is a multidomain protein believed to be involved only in the passive transmission of genomic methylation patterns via maintenance methylation. The mechanisms that regulate DNMT1 activity and targeting are complex and poorly understood. We used embryonic stem (ES) cells to investigate the function of the uncharacterized bromo-adjacent homology (BAH) domains and the glycine-lysine (GK) repeats that join the regulatory and catalytic domains of DNMT1. We removed the BAH domains by means of a CRISPR/Cas9-mediated deletion within the endogenous Dnmt1 locus. The internally deleted protein failed to associate with replication foci during S phase in vivo and lost the ability to mediate maintenance methylation. The data indicate that ablation of the BAH domains causes DNMT1 to be excluded from replication foci even in the presence of the replication focus-targeting sequence (RFTS). The GK repeats resemble the N-terminal tails of histones H2A and H4 and are normally acetylated. Substitution of lysines within the GK repeats with arginines to prevent acetylation did not alter the maintenance activity of DNMT1 but unexpectedly activated de novo methylation of paternal imprinting control regions (ICRs) in mouse ES cells; maternal ICRs remained unmethylated. We propose a model under which DNMT1 deposits paternal imprints in male germ cells in an acetylation-dependent manner. These data reveal that DNMT1 responds to multiple regulatory inputs that control its localization as well as its activity and is not purely a maintenance methyltransferase but can participate in the de novo methylation of a small but essential compartment of the genome.

MicroRNA-377 Inhibits Atherosclerosis by Regulating Triglyceride Metabolism Through the DNA Methyltransferase 1 in Apolipoprotein E-Knockout Mice.

BACKGROUND: Lipoprotein lipase (LPL) plays an important role in triglyceride metabolism. It is translocated across endothelial cells to reach the luminal surface of capillaries by glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), where it hydrolyzes triglycerides in lipoproteins. MicroRNA 377 (miR-377) is highly associated with lipid levels. However, how miR-377 regulates triglyceride metabolism and whether it is involved in the development of atherosclerosis remain largely unexplored. Methods and Results: The clinical examination displayed that miR-377 expression was markedly lower in plasma from patients with hypertriglyceridemia compared with non-hypertriglyceridemic subjects. Bioinformatics analyses and a luciferase reporter assay showed that DNA methyltransferase 1 (DNMT1) was a target gene of miR-377. Moreover, miR-377 increased LPL binding to GPIHBP1 by directly targeting DNMT1 in human umbilical vein endothelial cells (HUVECs) and apolipoprotein E (ApoE)-knockout (KO) mice aorta endothelial cells (MAECs). In vivo, hematoxylin-eosin (H&E), Oil Red O and Masson's trichrome staining showed that ApoE-KO mice treated with miR-377 developed less atherosclerotic plaques, accompanied by reduced plasma triglyceride levels. CONCLUSIONS: It is concluded that miR-377 upregulates GPIHBP1 expression, increases the LPL binding to GPIHBP1, and reduces plasma triglyceride levels, likely through targeting DNMT1, inhibiting atherosclerosis in ApoE-KO mice.

Exosomal DNMT1 mediates cisplatin resistance in ovarian cancer.

Ovarian cancer is the most common malignancy in women. Owing to late syndromic presentation and lack of efficient early detection, most cases are diagnosed at advanced stages. Surgery and platinum-based chemotherapy are still the standard care currently. However, resistance invoked often compromises the clinical value of the latter. Expression of DNA methyltransferase 1 (DNMT1) was analysed by gene array. Protein was determined by immunoblotting. Exosome was isolated with commercial kit. Cell proliferation was measured by CCK8 method. Annexin V-PI double staining was performed for apoptosis evaluation. Xenograft model was established and administrated with exosome. Tumour growth and overall survival were monitored. We demonstrated the upregulation of DNMT1 in both tumour and derived cell line. DNMT1 transcripts were highly enriched in exosomes from conditioned medium of ovarian cells. Co-incubation with exosomes stimulated endogenous expression and rendered host cell the resistance to cytotoxicity of cisplatin. In vivo administration of DNMT1-containing exosomes exacerbated xenograft progression and reduced overall survival significantly. Moreover, treatment with exosome inhibitor GW4869 almost completely restored sensitivity in resistant cells. Our data elucidated an unappreciated mechanism of exosomal DNMT1 in cisplatin resistance in ovarian cancer, also indicating the potential of the combination of exosome inhibitor with cisplatin in resistant patients.

Aberrant DNA methyltransferase 1 expression in clear cell renal cell carcinoma development and progression.

OBJECTIVE: To better understand the contribution of dysregulated DNA methyltransferase 1 (DNMT1) expression to the progression and biology of clear cell renal cell carcinoma (ccRCC). METHODS: We examined the differences in the expression of DNMT1 in 89 ccRCC and 22 normal tissue samples by immunohistochemistry. In addition, changes in cell viability, apoptosis, colony formation and invading ability of ccRCC cell lines (786-0 and Caki-1) were assessed after transfection with DNMT1 siRNA. RESULTS: We found DNMT1 protein was significantly higher expressed in ccRCC than that of in no-tumor tissues (56.2% and 27.3%, respectively, P=0.018). The expression of DNMT1 was strongly associated with ccRCC tumor size, tumor pathology stage, histological grading, lymph node metastasis, vascular invasion, recurrence and prognosis. Moreover, knockdown of DNMT1 expression significantly inhibited ccRCC cell viability, induced apoptosis, decreased colony formation and invading ability. CONCLUSIONS: Expression of DNMT1 protein is increased in ccRCC tissues, and DNMT1 expression is associated with poor prognosis of patients. Experiments in vitro further showed DNMT1 played an essential role in proliferation and invasion of renal cancer cells. Moreover, targeting this enzyme could be a promising strategy for treating ccRCC, as evidenced by inhibited cell viability, increased apoptosis, decreased colony formation and invading ability.

Simvastatin prevents BMP-2 driven cell migration and invasion by suppressing oncogenic DNMT1 expression in breast cancer cells.

Both epigenetic and genetic changes in the cancer genome act simultaneously to promote tumor development and metastasis. Aberrant DNA methylation, a prime epigenetic event, is often observed in various cancer types. The elevated DNA methyltransferase 1 (DNMT1) enzyme creates DNA hypermethylation at CpG islands to drive oncogenic potential. This study emphasized to decipher the molecular mechanism of endogenous regulation of DNMT1 expression for finding upstream signaling molecules. Cancer database analyses found an upregulated DNMT1 expression in most cancer types including breast cancer. Overexpression of DNMT1 showed an increased cell migration, invasion, and stemness potential whereas 5-azacytidine (DNMT1 inhibitor) and siRNA mediated knockdown of DNMT1 exhibited inhibition of such cancer activities in breast cancer MDA-MB-231 and MCF-7 cells. Infact, cancer database analyses further found a positive correlation of DNMT1 transcript with both cholesterol pathway regulatory genes and BMP signaling molecules. Experimental observations documented that the cholesterol-lowering drug, simvastatin decreased DNMT1 transcript as well as protein, whereas BMP-2 treatment increased DNMT1 expression in breast cancer cells. In addition, expression of various key cholesterol regulatory genes was found to be upregulated in response to BMP-2 treatment. Moreover, simvastatin inhibited BMP-2 induced DNMT1 expression in breast cancer cells. Thus, this study for the first time reveals that both BMP-2 signaling and cholesterol pathways could regulate endogenous DNMT1 expression in cancer cells.

Protein phosphatase 6 (Pp6) is crucial for regulatory T cell function and stability in autoimmunity.

Regulatory T (Treg) cells constitute a dynamic population that is critical in autoimmunity. Treg cell therapies for autoimmune diseases are mainly focused on enhancing their suppressive activities. However, recent studies demonstrated that certain inflammatory conditions induce Treg cell instability with diminished FoxP3 expression and convert them into pathogenic effector cells. Therefore, the identification of novel targets crucial to both Treg cell function and plasticity is of vital importance to the development of therapeutic approaches in autoimmunity. In this study, we found that conditional Pp6 knockout (cKO) in Treg cells led to spontaneous autoinflammation, immune cell activation, and diminished levels of FoxP3 in CD4+ T cells in mice. Loss of Pp6 in Treg cells exacerbated two classical mouse models of Treg-related autoinflammation. Mechanistically, Pp6 deficiency increased CpG motif methylation of the FoxP3 locus by dephosphorylating Dnmt1 and enhancing Akt phosphorylation at Ser473/Thr308, leading to impaired FoxP3 expression in Treg cells. In summary, our study proposes Pp6 as a critical positive regulator of FoxP3 that acts by decreasing DNA methylation of the FoxP3 gene enhancer and inhibiting Akt signaling, thus maintaining Treg cell stability and preventing autoimmune diseases.

Involvement of DNA methyltransferase 1 (DNMT1) and multidrug resistance-associated proteins in 2-methoxyestradiol-induced cytotoxicity in EC109/Taxol cells.

BACKGROUND: Due to acquired drug resistance, paclitaxel-based chemotherapy has limited clinical effects in the treatment of various tumors including esophageal cancer. This study analyzes the hypothesis that paclitaxel resistance is related to changes in the expression of DNA methyltransferase 1 (DNMT1). The thesis also studies multidrug resistance-related proteins and the mechanism underlying 2-methoxyestradiol (2-ME)-induced cytotoxicity in EC109/Taxol cells was examined. METHODS: In this study, the mechanisms of 2-ME-induced cytotoxicity in EC109/Taxol cells was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, DNA ladder assay, DNMT activity assay, and Western blotting. The result of 2-ME-induced cytotoxicity EC109/Taxol cells is compared with that of EC109 parental cells. RESULTS: The results show that low concentrations of 2-ME (0.5-10 µM) inhibited cell growth, with IC50 values of 2.04 and 5.38 µmol/L in EC109/Taxol cells and EC109 parental cells after 72 hours of treatment, respectively. Exposure to 2-ME could increase G2/M cell cycle arrest and could increase apoptosis more effectively in EC109/Taxol cells than that observed in the EC109 parental cells. Furthermore, it is observed that paclitaxel resistance is associated with decreased DNMT activity. This study shows that 2-ME decreases DNMT1-mediated paclitaxel resistance by simultaneously reducing the expression of ATP-binding cassette (ABC) transporters, including phosphoglycoprotein (P-gp), breast cancer resistance protein (BCRP), and multi-drug resistance protein 1 (MRP1), in EC109/Taxol cells. CONCLUSIONS: In this study, the co-treatment of Taxol and 2-ME to EC109 could significantly induce cytotoxic effects, whose mechanism might be associated with DNMT1 and multidrug resistance-associated proteins.

Hypermethylation of Hepatic Mitochondrial ND6 Provokes Systemic Insulin Resistance.

Mitochondrial epigenetics is rising as intriguing notion for its potential involvement in aging and diseases, while the details remain largely unexplored. Here it is shown that among the 13 mitochondrial DNA (mtDNA) encoded genes, NADH-dehydrogenase 6 (ND6) transcript is primarily decreased in obese and type 2 diabetes populations, which negatively correlates with its distinctive hypermethylation. Hepatic mtDNA sequencing in mice unveils that ND6 presents the highest methylation level, which dramatically increases under diabetic condition due to enhanced mitochondrial translocation of DNA methyltransferase 1 (DNMT1) promoted by free fatty acid through adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation. Hepatic knockdown of ND6 or overexpression of Dnmt1 similarly impairs mitochondrial function and induces systemic insulin resistance both in vivo and in vitro. Genetic or chemical targeting hepatic DNMT1 shows significant benefits against insulin resistance associated metabolic disorders. These findings highlight the pivotal role of ND6 epigenetic network in regulating mitochondrial function and onset of insulin resistance, shedding light on potential preventive and therapeutic strategies of insulin resistance and related metabolic disorders from a perspective of mitochondrial epigenetics.

MicorRNA-148b inhibits cell proliferation and facilitates cell apoptosis by regulating DNA Methyltransferase 1 in endometrial cancer.

BACKGROUND: MicroRNA (miR)-148b has been shown to be dysregulated in a number of cancers; however, studies on the role of miR-148b in the gynaecologic malignancy endometrial cancer (EC) are rare. The purpose of this study was to explore the role of miR-148b in EC and the underlying molecular mechanism. METHODS: The expression levels of miR-148b and DNA methyltransferase 1 (DNMT1) were determined by quantitative real-time PCR (qRT-PCR) in both EC tissues and cell lines (HEC-1A and HEC-1B). These EC cell lines were then transfected with either an miR-148b inhibitor or miR-148b mimics, and cell proliferation, colony, and apoptosis and the cell cycle were measured by the cell counting kit-8, colony formation assay, and flow cytometry assays, respectively. In addition, the expression levels of p16, cyclin-dependent kinase 4 (CDK4), cyclin D1, caspase-3, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X (Bax) were assessed by western blotting. Dual luciferase reporter and RNA pull-down assays were performed to investigate the target genes of miR-148b and validate their relationship. RESULTS: miR-148b expression was down-regulated in both EC tissues and HEC-1A and HEC-1B cells, whereas DNMT1 was highly expressed. Moreover, transfection of miR-148b mimics inhibited cell proliferation and cell cycle progression, but induced cell apoptosis. Western blotting showed that transfection of miR-148b mimics markedly increased caspase-3 and cyclin D1 expression, whereas transfection of miR-148b inhibitor dramatically decreased the expression of caspase-3 and cyclin D1. Importantly, we determined that DNMT1 is a target gene of miR-148b in EC cells, and silencing of DNMT1 reversed the effects of miR-148b inhibitor on cell proliferation, cell cycle progression, and apoptosis in EC. CONCLUSIONS: miR-148b inhibits cell proliferation and facilitates cell apoptosis in EC by regulating DNMT1.

Epigenetic modulation of macrophage polarization prevents lumbar disc degeneration.

Inflammation plays an essential role in the development of lumbar disc degeneration (LDD), although the exact effects of macrophage subtypes on LDD remain unclear. Based on previous studies, we hypothesized that M2-polarization of local macrophages and simultaneous suppression of their production of fibrotic transforming growth factor beta 1 (TGFß1) could inhibit progression of LDD. Thus, we applied an orthotopic injection of adeno-associated virus (AAV) carrying shRNA for DNA Methyltransferase 1 (DNMT1) and/or shRNA for TGFß1 under a macrophage-specific CD68 promoter to specifically target local macrophages in a mouse model for LDD. We found that shDNMT1 significantly reduced levels of the pro-inflammatory cytokines TNFα, IL-1ß and IL-6, significantly increased levels of the anti-inflammatory cytokines IL-4 and IL-10, significantly increased M2 macrophage polarization, significantly reduced cell apoptosis in the disc degeneration zone and significantly reduced LDD-associated pain. The anti-apoptotic and anti-pain effects were further strengthened by co-application of shTGFß1. Together, these data suggest that M2 polarization of macrophages induced by both epigenetic modulation and suppressed production and release of TGFß1 from polarized M2 macrophages, may have a demonstrable therapeutic effect on LDD.

DNMT1 and p38γ are inversely expressed in reactive non-metastatic lymph nodes burdened with colorectal adenocarcinoma.

Lymph nodes are important front-line defense immune tissues, which also act against inflammatory diseases and cancer. Lymph nodes undergo extensive upheavals within newly formed germinal centers (GCs) when exposed to antigens, the molecular mechanisms of which remain elusive. Recently, p38γ was identified as an important target for multiple cancers, including cutaneous T-cell lymphoma (CTCL). We previously observed that p38γ is overexpressed in CTCL versus normal cells, but it is not clear if p38γ is expressed in B or T lymphocytes of GCs of patients in response to a stress such as cancer. Therefore, in this study, we obtained non-metastatic reactive lymph nodes adjacent to cancer lesions (colorectal adenocarcinoma), then performed multicolor immunohistochemical staining for p38γ and other relevant markers. We observed for the first time that p38γ was expressed in the light zone of activated B cells and T helper cells in GCs, whereas DNA-methyltransferase 1 (DNMT1), a marker for GC B cells, was highly expressed in centrocytes and in the dark zone of GCs. This inverse relationship suggests a novel function for p38γ in T cells that cross-talk to B cells in response to stress.

DNMT1 mediated promoter methylation of GNAO1 in hepatoma carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers worldwide and has recently become the second most common cause of cancer-related deaths in men of developing countries. Guanine nucleotide-binding protein (G protein) has been reported to be associated with the early process of HCC. In our previous study, GNAO1, one of members of G protein, was found to be down-regulated in HCC. Thus, the present study aimed to throw light upon the mechanism of the abnormal expression of GNAO1 in HCC. First, qPCR results from two HCC cell lines (SMMC-7721 and QGY-7703) confirmed the down-expression of GNAO1, followed by the validation of the methylation status of the promoter region by bisulfite sequence PCR (BSP). Moreover, 5-Aza-2'-deoxycytidine (DAC) with Trichostatin A (TSA) treatment made it much clear that GNAO1 transcription was inhibited by promoter hypermethylation, contributing to its low expression. It was further revealed that the silencing effect was regulated by methyltransferase 1 (DNMT1), and was further enhanced by transforming growth factor ß (TGF-ß). In addition, the up-regulation of GNAO1 with the help of recombinant plasmid was also found to accelerate cell apoptosis, confirmed by flow cytometry and western blotting analysis. All these results above indicated that the promoter hypermethylation of GNAO1 might play an important role in HCC, suggesting that it might be used as a promising biomarker for HCC diagnosis and targeted therapy.

miR-126 promotes the growth and proliferation of leukemia stem cells by targeting DNA methyltransferase 1.

Previous studies have showed that the interaction between microRNAs (miRNAs) and leukemia stem cells (LSCs) may be a cause of drug resistance of acute myeloid leukemia (AML). However, whether miR-126 participates in the pathogenesis of AML remains unclear. In our study, we first examined the expression of miR-126 in CD34+ or CD34- cells isolated from blood samples and LSC cell line: KG-1a-LSCs and MOLM13-LSCs by qRT-PCR analysis. Then miR-126 inhibitor and mimics were applied to evaluate the roles of miR-126 in cell proliferation of LSC cell lines using CCK-8 assay and Ki-67 staining. Moreover, flow cytometry analysis was used to assess the apoptosis of LSC cell lines treated with miR-126 inhibitor of mimics. In addition, we analyzed the relationship between miR-126 and DNA methyltransferase 1 (DNMT1) by bioinformatics analysis and dual-luciferase reporter assay. Western blot analysis was applied to examine the protein expression level of DNMT1 in miR-126 mimics treated LSC cells. Results showed that miR-126 expression was significantly higher in CD34+ cells and KG-1a-LSCs and MOLM13-LSCs. Knockdown of miR-126 in KG-1a-LSCs and MOLM13-LSCs inhibited cell proliferation, and promoted apoptosis. miR-126 could regulate DNA methyltransferase 1 (DNMT1) expression by directly binding to it. In conclusion, these findings suggested that miR-126 may promote cell proliferation of LSCs by targeting DNMT1.

Circulating MIR148A associates with sensitivity to adiponectin levels in human metabolic surgery for weight loss

Objective: We sought to discover secreted biomarkers to monitor the recovery of physiological adiponectin levels with metabolic surgery, focusing on epigenetic changes that might predict adiponectin function. Design: We conducted a prospective observational study of patients undergoing metabolic surgery by Roux-en-Y Gastric Bypass (RYGB) for weight loss in a single center (IRB GHS # 1207-27). Methods: All patients (n = 33; 27 females; 6 males) signed informed consent. Metabolites, adiponectin and MIR148A were measured in fasting plasma. We followed MIQE for transcript profiles. Results: Patients lost on average 47 ± 12% excess BMI (%EBMI) after 12 weeks. Adiponectin pre, post or delta (post minus pre) did not correlate with %EBMIL. A decrease in adiponectin following weight loss surgery was observed in a subset of patients, chi-square test of independence rejects the null hypotheses that the liver DNA methyltransferase 1 (DNMT1) and delta adiponectin are independent (chi-square statistics χ2 = 6.9205, P = 0.00852, n = 33), as well as MIR148A and delta adiponectin are independent (chi-square statistics χ2 = 9.6823, P = 0.00186, n = 33). The presence of plasma MIR148A allows identification of patients that appear to be adiponectin insensitive at baseline. Conclusion: We combined the presence of plasma MIR148A, the concentration of total adiponectin and the expression of DNA methyltransferase 1 (DNMT1) in liver biopsy tissue to identify patients with non-physiological adiponectin. Weight loss and physical activity interventions complemented with the new method presented here could serve to monitor the physiological levels of adiponectin, thought to be important for long-term weight loss maintenance.

Insulin-like growth factor II mRNA-binding protein 3 (IMP3) is a marker that predicts presence of invasion in papillary biliary tumors.

Biliary tumors showing intraductal papillary growth (Pap-BTs) include intraductal papillary neoplasm of the bile duct (IPNB) and papillary cholangiocarcinoma (CC). A differential diagnosis between IPNB and papillary CC currently remains challenging. The aim of the present study is to identify histological features and immunohistochemical markers of malignant potential such as tumor invasion in Pap-BTs. Subjects comprised 37 patients with Pap-BT (intrahepatic and perihilar [proximal], 27: 17 noninvasive and 10 invasive; distal, 10: all invasive). We examined histological features and the expression of p53, enhancer of zeste homolog 2, insulin-like growth factor II mRNA-binding protein 3 (IMP3), and DNA methyltransferase-1 in the intraductal area in Pap-BTs. Noninvasive Pap-BT was characterized by the presence of a low-grade dysplastic area, edematous stroma, and the absence of necrosis. The expression of p53, enhancer of zeste homolog 2, IMP3, and DNA methyltransferase-1 was significantly weaker in noninvasive Pap-BTs than in invasive Pap-BTs (P<.01). Diffuse cytoplasmic IMP3 expression was absent in noninvasive Pap-BTs. IMP3 showed the greatest specificity to predict a presence of invasion. A heatmap demonstrated that proximal noninvasive Pap-BTs and distal Pap-BTs may be completely different. In bile duct biopsies, the expression of IMP3 was the most precise predictor of invasion in Pap-BTs. In conclusion, Pap-BTs may be separated into 3 subgroups: (1) proximal noninvasive Pap-BT, corresponding to IPNB; (2) distal invasive Pap-BT, corresponding to papillary CC; and (3) the remaining Pap-BT including IPNB with associated adenocarcinomas, based on histological and immunohistochemical features. IMP3 may be a useful marker for predicting invasion in Pap-BT.

SOCS1 hypermethylation mediated by DNMT1 is associated with lipopolysaccharide-induced inflammatory cytokines in macrophages.

Macrophages activation which releases the pro-inflammatory cytokines is an essential event in the process of inflammation. SOCS1 has been shown to act as a negative regulator of cytokine signals and plays a key role in the suppression of tissue injury and inflammatory diseases. DNA methylation mediated by specific DNA methyltransferases1 (DNMT1) which contributes to the epigenetic silencing of multiple genes. SOCS1 promoter hypermethylation is by far the best categorized epigenetic change in tumors. Our study with a view to investigate whether the loss of SOCS1 due to SOCS1 promoter methylation was involved in the course of inflammatory cytokines released from lipopolysaccharide (LPS)-stimulated macrophages. Here, we found that treatment of LPS-induced RAW264.7 macrophage cells with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-azadC) reduced aberrant promoter hypermethylation of SOCS1 and prevented the loss of the expression of SOCS1 in macrophages which secret inflammatory cytokines. Knockdown of DNMT1 gene not only attenuated the SOCS1 gene promoter methylation but also up-regulated the expression of SOCS1 in activated RAW264.7 cells. Furthermore, silencing of DNMT1 prevented the activation of JAK2/STAT3 pathway in LPS-induced RAW264.7 cells. These studies demonstrated that DNMT1-mediated SOCS1 hypermethylation caused the loss of SOCS1 expression results in negative regulation of activation of the JAK2/STAT3 pathway, and enhanced the release of LPS-induced pro-inflammatory cytokines such as TNF-α and IL-6 in macrophages.