OBJECTIVE: To examine the expression of DNMT1, DNMT3a, and DNMT3b in the eutopic and ectopic endometrium in women with endometriosis. METHODS: RT-PCR and real-time RT-PCR were used to examine the expression of DNMT1, DNMT3a, and DNMT3b in the eutopic and ectopic endometrium in 20 women with endometriosis and the endometrium in 20 women without endometriosis. Immunofluorescene staining was used to detect the expression of DNMT1 in these tissues. RESULTS: The expression levels of DNMT1, DNMT3a, and DNMT3b were significantly lower in the ectopic endometrium and eutopic endometrium than those of the control endometium (P<0.05). The changes in the ectopic endometium compared with the control endometium were 0.44, 0.12, and 0.27 folds for DNMT1, DNMT3a, and DNMT3b, respectively, and these in the eutopic endometrium were 0.27, 0.13, and 0.15 folds for DNMT1,DNMT3a, and DNMT3b, respectively. The expression level of DNMT1, DNMT3a, and DNMT3b between the ectopic endometrium and eutopic endometrium was not significantly different (P>0.05 ). Immunofluorescence staining that DNMT1 protein level significantly decreased in the ectopic endometrium and eutopic endometrium of endometriosis patients. CONCLUSION Decreased expression levels of DNMT1, DNMT3a, and DNMT3b in the ectopic endometrium and eutopic endometrium may play a role in patients with abnormal epigenetics which may lead to endometriosis.
Adult ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; Endometriosis ; enzymology ; genetics ; Endometrium ; enzymology ; Epigenomics ; Female ; Humans

Epigenetic changes, including abnormal DNA methylation, have been identified to play significant roles in tumor initiation and progression. Recently, mutations of DNMT3A were identified in acute myeloid leukemia (AML), which possibly caused changes in DNA methylation, and indicated a poor prognosis. Sequencing analysis showed that most of the mutations were single nucleotide variations, including a hotspot Arg882. DNMT3A mutations were detected in about 20% AML patients, and closely associated with the age over 60, the M(4), M(5) subtypes and intermediate-risk cytogenetics. Others showed that these alterations also present in myelodysplastic syndrome (MDS) and primary myelofibrosis (PMF) prior to development of the obvious leukemia, indicating that these mutations might contribute to leukemogenesis. However, its prognostic value of minimal residual disease and role of therapeutic targets are still unclear, focusing on a large cohort of AML patients will solve these issues. In this review, the achievement in studying DNMT3A gene mutation are summarized, and the latest research progress is briefly discussed.
DNA (Cytosine-5-)-Methyltransferases ; genetics ; DNA Methylation ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Mutation

OBJECTIVETo evaluate the effects of DNMT3A gene mutation on prognosis of patients with acute myeloid leukemia (AML) by a meta-analysis.

METHODSMethods of Cochrane systematic review was followed by 7 databases,including PubMed, Embase, Ovid, CNKI, CBM, WanFang Data and VIP, were searched for peer-reviewed articles related to DNMT3A gene mutations and prognosis of patients with AML.Then manual retrieval was applied into literature references. After the evaluation of quality and extract of clinical trialliterature data, Stata 11.0 was employed to perform meta-analysis.

RESULTSSeven randomized controlled trials involving 1493 cases were included in the meta-analysis. The prognosis of patients with DNMT3A mutations and without DNMT3A mutations was compared. There was no statistically significant difference in complete remission(CR) rate (OR=1.034, 95%CI: 0.596~1.796, P=0.905 between two groups, but the overall survival (OS（HR=1.990, 95%CI: 1.463~2.510, P=0.000 and disease free survival (DFS) (HR= 2.840, 95%CI: 1.063~4.613, P=0.002) of patients without DNMT3A mutations were longer than those with DNMT3A mutation.

CONCLUSIONDNMT3A gene mutation is an independent risk factor of poor prognosis of patients with acute myeloid leukemia.

DNA (Cytosine-5-)-Methyltransferases ; genetics ; Humans ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Mutation ; Prognosis ; Risk Factors

OBJECTIVETo investigate the correlation of genomic DNA methylation level with unexplained early spontaneous abortion and analyze the role of DNMT1, DNMT3A and DNMT3B.

METHODSForty-five villus samples from spontaneous abortion cases (with 33 maternal peripheral blood samples) and 44 villus samples from induced abortion (with 34 maternal peripheral blood samples) were examined with high-pressure liquid chromatography (HPLC) to measure the overall methylation level of the genomic DNA. The expressions of DNMT mRNAs were detected using fluorescence quantitative-PCR in the villus samples from 33 induced abortion cases and 30 spontaneous abortion cases.

RESULTSGenomic DNA methylation level was significantly lower in the villus in spontaneous abortion group than in induced abortion group (P<0.01), but similar in the maternal blood samples between the two groups (P>0.05). The mean mRNA expression levels of DNMT1 and DNMT3A in the villus were significantly lower in spontaneous abortion group than in induced abortion group (P<0.05), but DNMT3B expression showed no significant difference between them (P>0.05).

CONCLUSIONInsufficient genomic DNA methylation in the villus does exist in human early spontaneous abortion, and this insufficiency is probably associated with down-regulated expressions of DNMT1 and DNMT3A.

Abortion, Spontaneous ; genetics ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; Female ; Genomics ; Humans ; Pregnancy ; RNA, Messenger

In order to study the activity and gene expression of DNA methyltransferase (DNMT) on U266 myeloma cells and to analyze their significance, the activity of DNMT was detected by ELISA, and the expressions of DNMT1, DNMT3a and 3b were analyzed by RT-PCR. U266 cells were treated by phenylhexyl isothiocyanate (PHI), and the change of activity and gene expression of DNMT were determined. The results indicated that the activity and expression of DNMT in U266 myeloma cells were higher, compared with normal control. After being treated by different concentration of PHI, U266 cells were driven into apoptosis and the activity of DNMT decreased obviously and the mRNA level of DNMT declined. It is concluded that the activity and gene expression of DNMT on U266 myeloma cells are higher, and DNMT may be a new therapeutic target of multiple myeloma.
Cell Line, Tumor ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; Humans ; Multiple Myeloma ; metabolism ; RNA, Messenger ; genetics

Asian Continental Ancestry Group ; genetics ; China ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; Humans ; Parkinson Disease ; genetics ; Polymorphism, Single Nucleotide

OBJECTIVETo investigate DNA methylation variation in human cells induces by B(a)P, and to explore the role of PARP1 during this process.

METHODSThe changes of DNA methylation of 16HBE and its PARP1-deficient cells exposed to B(a)P (1.0, 2.0, 5.0, 10.0, 15.0, 30.0 µmol/L) were investigated by immunofluorescence and high performance capillary electrophoresis, and simultaneously, the expression level of PARP 1 and DNMT 1 were monitored dynamically.

RESULTSThe percentage of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP1 cells were separately (4.04 ± 0.08)% and (9.69 ± 0.50)%. After being treated by 5-DAC for 72 hours, mCpG% decreased to (3.15 ± 0.14)% and (6.07 ± 0.54)%. After both being exposed to B(a)P for 72 hours, the mCpG% in 16HBE group (ascending rank) were separately (5.10 ± 0.13), (4.25 ± 0.10), (3.91 ± 0.10), (4.23 ± 0.27), (3.70 ± 0.15), (3.08 ± 0.07); while the figures in 16HBE-shPARP1 group (ascending rank) were respectively (10.63 ± 0.60), (13.08 ± 0.68), (9.75 ± 0.55), (7.32 ± 0.67), (6.90 ± 0.49) and (6.27 ± 0.21). The difference of the results was statistically significant (F values were 61.67 and 60.91, P < 0.01). For 16HBE group, expression of PARP 1 and DNMT 1 were 141.0%, 158.0%, 167.0%, 239.0%, 149.0%, 82.9% and 108.0%, 117.0%, 125.0%, 162.0%, 275.0%, 233.0% comparing with the control group, whose difference also has statistical significance (t values were 11.45, 17.32, 32.24, 33.44, 20.21 and 9.87, P < 0.01). For 16HBE-shPARP1 group, expression of PARP 1 and DNMT 1 were 169.0%, 217.0%, 259.0%, 323.0%, 321.0%, 256.0% and 86.0%, 135.0%, 151.0%, 180.0%, 229.0%, 186.0% comparing with the control group, with statistical significance (t values were 9.06, 15.92, 22.68, 26.23, 37.19 and 21.15, P < 0.01). When the dose of B(a)P reached 5.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE group (ascending rank) were 125.0%, 162.0%, 275.0%, 233.0% times of it in control group, with statistical significance (t values were 12.74, 24.92, 55.11, 59.07, P < 0.01); while the dose of B(a)P reached 2.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE-shPARP1 group were 135.0%, 151.0%, 180.0%, 229.0%, 186.0% of the results in control group, and the differences were statistically significant (t values were 23.82, 40.17, 32.69, 74.85, 46.76, P < 0.01).

CONCLUSIONThe hypomethylation of 16HBE cells induced by B(a)P might be one important molecular phenomenon in its malignant transformation process. It suggests that PARP1 could regulate DNA methylation by inhibiting the enzyme activity of DNMT1, and this effect could be alleviated by PARP1-deficiency.

Benzo(a)pyrene ; adverse effects ; Cell Line ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; metabolism ; DNA Damage ; DNA Methylation ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; genetics ; metabolism

OBJECTIVETo construct DNA methyltransferase 1 (DNMT1) low expression 16HBE cell line and observe the variation of cell cycle and global genomic DNA methylation.

METHODSThe method of Lenti-virus induced RNA interference was applied to introduce four different shRNA fragment into 16HBE cells. Flow cytometry and 5-mC immunofluorescence methods were used to observe the cell cycle and global DNA methylation status of DNMT1 low expression 16HBE cells.

RESULTSThe DNMT1 protein relative expression level of 16HBE-shDNMT1-4 cell line was down regulated about 44% (P < 0.05) compared with the control. No obvious differences of cell cycle and global genome DNA methylation status were observed between the 16HBE and 16HBE-shDNMT1.

CONCLUSIONThe DNMT1 gene low expression cell is successfully constructed, and there are no obvious changes happened on the cell cycle and global genomic DNA methylation.

Cell Cycle ; Cell Line ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; metabolism ; DNA Methylation ; Down-Regulation ; Epithelial Cells ; metabolism ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVETo reveal the role of poly-ADP-ribosylation and DNA methylation in carcinogenic process induced induced by Cr (VI), and to discuss the relations between them.

METHODSThe pre-established Poly (ADP-ribose) glycohydrolase (PARG) deficient cells and 16HBE cells were treated with different concentrations of Cr (VI), and the changes of total genomic DNA methylation level in different groups were detected by methylation immunofluorescent detection, as well as the changes of the activity of methyltransferases. Moreover, RT-PCR and western blotting method were applied to analyze the changes of expression of DNMT1, DNMT3a, DNMT3b and MBD2, upon the protein level.

RESULTSAfter treated by Cr(VI) for 24 h, the healthy 16HBE cells showed a significant lower level of genomic DNA methylation; however, there was no significant changes (P > 0.05) found in PARG deficient cells by immunofluorescence assay. When the dose of Cr (VI) reached 5.0 µmol/L, the activity of methyltransferases in 16HBE cells and PARG deficient cells (49.33 ± 2.65, 80.05 ± 2.05) decreased by 20% and 50% comparing with contrast group (99.27 ± 1.10, 99.30 ± 0.60) . After treated by Cr (VI) for 24 h, the expression of mRNA and protein level among DNMT1, DNMT3a, DNMT3b and MBD2 decreased significantly in healthy 16HBE cells; and the expression of DNMT1 and DNMT3a decreased in PARG deficiency cells. The relevant expression levels of mRNA of DNMT1 were separately (0.99 ± 0.09), (0.79 ± 0.10), (0.59 ± 0.13) and (0.39 ± 0.02) (F = 247.17, P < 0.01), the expression levels of protein were separately (1.00 ± 0.03), (0.69 ± 0.15), (0.65 ± 0.10) and (0.55 ± 0.13) (F = 214.12, P < 0.01), the expression levels of DNMT3a mRNA were separately (1.00 ± 0.04) , (0.93 ± 0.11) , (0.79 ± 0.07) , (0.59 ± 0.05) (F = 498.16, P < 0.01) , and the expression levels of protein were separately (1.00 ± 0.14) , (0.97 ± 0.11) , (0.79 ± 0.17) , (0.57 ± 0.15) (F = 390.11, P < 0.01) when the dose of Cr (VI) at 0, 0.3, 1.2 and 5.0 µmol/L. However, there were no significant changes of expression found in DNMT3b and MBD2.

CONCLUSIONPoly-ADP-ribosylation could regulate the activity of DNMT3b and MBD2, protect cells against the DNA methylation alteration induced by Cr(VI) and maintain the global genomic DNA methylation level.

Cell Line ; Chromium ; toxicity ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; drug effects ; DNA-Binding Proteins ; metabolism ; Epithelial Cells ; metabolism ; Genome ; Humans ; Poly Adenosine Diphosphate Ribose ; metabolism ; RNA, Messenger ; genetics

The purpose of this study was to explore the effect of epigallocatechin-3-galate (EGCG) on acute monocytic leukemia cell line U937 and its relevant mechanism. The viability of U937 cells were assayed by SRB method. The cell cycle of U937 cells was analyzed by flow cytometry. The mRNA and protein expression of p16 gene were detected by RT-PCR and Western blot, respectively. Methylation level of U937 cells was analyzed by n-MSP. The mRNA expression of DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B genes were analyzed by RT-PCR. The results showed that EGCG could inhibit the growth of U937 cells significantly in dose-and time-dependent manners (r=0.71), and induce the G0/G1 arrest of U937 cells in dose-dependent manner. EGCG could up-regulate the mRNA and protein expression of P16 gene in U937 cells in dose-dependent manner. EGCG could down-regulate the methylation level of p16 gene in U937 cells in dose-dependent manner. EGCG could down-regulate the mRNA expression of DNMT3A, DNMT3B genes, while did not influence the mRNA expression of DNMT1 gene. It is concluded that EGCG can up-regulate the mRNA and protein expression of p16 gene by demethylation or/and by inhibiting DNMT3A and DNMT3B genes, leading, in turn, to G0/G1 arrest and growth inhibition of U937 cells.
Catechin ; analogs & derivatives ; pharmacology ; Cell Proliferation ; drug effects ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; Gene Expression Regulation, Leukemic ; Genes, p16 ; Humans ; Leukemia, Monocytic, Acute ; genetics ; U937 Cells