The cooccurrence of NPM1, FLT3-ITD, and DNMT3A mutations (i.e., triple mutation) is related to dismal prognosis in patients with acute myeloid leukemia (AML) receiving chemotherapy alone. In this multicenter retrospective cohort study, we aimed to identify whether allogeneic hematopoietic stem cell transplantation (allo-HSCT) could overcome the poor prognosis of DNMT3A^mutNPM1^mutFLT3-ITD^mut AML across four transplant centers in China. Fifty-three patients with triple-mutated AML receiving allo-HSCT in complete remission were enrolled. The 1.5-year probabilities of relapse, leukemia-free survival, and overall survival after allo-HSCT were 11.9%, 80.3%, and 81.8%, respectively. Multivariate analysis revealed that more than one course of induction chemotherapy and allo-HSCT beyond CR1 were associated with poor survival. To our knowledge, this work is the largest study to explore the up-to-date undefined role of allo-HSCT in patients with triple-mutated AML. Our real-world data suggest that allo-HSCT could overcome the poor prognosis of DNMT3A^mutNPM1^mutFLT3-ITD^mut in AML.
Humans ; Nucleophosmin ; Leukemia, Myeloid, Acute/mortality* ; Hematopoietic Stem Cell Transplantation/methods* ; Male ; Female ; DNA Methyltransferase 3A ; Adult ; China ; Retrospective Studies ; DNA (Cytosine-5-)-Methyltransferases/genetics* ; Middle Aged ; Prognosis ; fms-Like Tyrosine Kinase 3/genetics* ; Mutation ; Young Adult ; Transplantation, Homologous ; Nuclear Proteins/genetics* ; Adolescent ; Aged

OBJECTIVE: To observe the clinical significance of translocator proteins (TSPO) gene in the treatment of FLT3-ITD/DNMT3A R882 double-mutated acute myeloid leukemia (AML). METHODS: Seventy-six patients with AML hospitalized in the Department of Hematology of the Affiliated People's Hospital of Ningbo University from June 2018 to June 2020 were selected, including 34 patients with FLT3-ITD mutation, 27 patients with DNMT3A R882 mutation, 15 patients with FLT3-ITD/DNMT3A R882 double mutation, as well as 19 patients with immune thrombocytopenia (ITP) hospitalized during the same period as control group. RNA was routinely extracted from 3 ml bone marrow retained during bone puncture, and TSPO gene expression was detected by transcriptome sequencing (using 2-deltadeltaCt calculation). RESULTS: The expression of TSPO gene in FLT3-ITD group and DNMT3A R882 group at first diagnosis was 2.02±1.04 and 1.85±0.76, respectively, which were both higher than 1.00±0.06 in control group, but the differences were not statistically significant (P=0.671, P=0.821). The expression of TSPO gene in the FLT3-ITD/DNMT3A R882 group was 3.98±1.07, wich was significantly higher than that in the FLT3-ITD group and DNMT3A R882 group, the differences were statistically significant (P=0.032, P=0.021). The expression of TSPO gene in patients who achieved complete response after chemotherapy in the FLT3-ITD/DNMT3A R882 group was 1.19±0.87, which was significantly lower than that at first diagnosis, and the difference was statistically significant (P=0.011). CONCLUSION TSPO gene may be used as an indicator of efficacy in FLT3-ITD /DNMT3A R882 double-mutated AML.
Humans ; DNA (Cytosine-5-)-Methyltransferases/genetics* ; DNA Methyltransferase 3A ; Mutation ; Leukemia, Myeloid, Acute/drug therapy* ; Nucleophosmin ; Prognosis ; fms-Like Tyrosine Kinase 3/genetics* ; Receptors, GABA/therapeutic use*

BACKGROUND: DNA methylation is involved in numerous biologic events and associates with transcriptional gene silencing, playing an important role in the pathogenesis of endometrial cancer. ESR1/PGR frequently undergoes de novo methylation and loss expression in a wide variety of tumors, including breast, colon, lung, and brain tumors. However, the mechanisms underlying estrogen and progesterone receptors (ER/PR) loss in endometrial cancer have not been studied extensively. The aims of this study were to determine the expression of DNA (cytosine-5)-methyltransferase 3A/3B (DNMT3A/3B) in endometrial cancer to investigate whether the methylation catalyzed by DNMT3A/3B contributes to low ER/PR expression. METHODS: The clinicopathologic information and RNA-Seq expression data of DNMT3A/3B of 544 endometrial cancers were derived from The Cancer Genome Atlas (TCGA) uterine cancer cohort in May 2018. RNA-Seq level of DNMT3A/3B was compared between these clinicopathologic factors with t-test or one-way analysis of variance. RESULTS: DNMT3A/3B was overexpressed in endometrioid carcinoma (EEC) and was even higher in non-endometrioid carcinoma (NEEC) (DNMT3A, EEC vs. NEEC: 37.6% vs. 69.9%, t = -7.440, P < 0.001; DNMT3B, EEC vs. NEEC: 42.4% vs. 72.8%, t = -6.897, P < 0.001). In EEC, DNMT3A overexpression was significantly correlated with the hypermethylation and low expression of the ESR1 and PGR (P < 0.05). The same trend was observed in the DNMT3B overexpression subgroup. In the ESR1/PGR low-expression subgroups, as much as 83.1% of ESR1 and 59.5% of PGR were hypermethylated, which was significantly greater than the ESR1/PGR high-expression subgroups (31.3% and 11.9%, respectively). However, the above phenomena were absent in NEEC, while DNMT3A/3B overexpression, ESR1/PGR hypermethylation, and low ER/PR expression occurred much more often. In univariate analysis, DNMT3A/3B overexpressions were significantly correlated with worse prognosis. In multivariate analysis, only DNMT3A was an independent predictor of disease-free survival (P < 0.05). CONCLUSIONS DNMT3A/3B expression increases progressively from EEC to NEEC and is correlated with poor survival. The mechanisms underlying low ER/PR expression might be distinct in EEC vs. NEEC. In EEC, methylation related to DNMT3A/3B overexpression might play a major role in ER/PR downregulation.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Endometrioid ; genetics ; metabolism ; pathology ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; metabolism ; DNA Methylation ; genetics ; Endometrial Neoplasms ; genetics ; metabolism ; pathology ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Prognosis

Tripterygium Glycosides Tablets has good anti-inflammatory and immunomodulatory activities,but its reproductive damage is significant. Previous studies of the research group have found that Cuscutae Semen flavonoids can improve spermatogenic cell damage caused by Tripterygium Glycosides Tablets by regulating spermatogenic cell cycle,apoptosis and related protein expression,but the mechanism of action at the gene level is still unclear. In this study,Illumina high-throughput sequencing platform was applied in transcriptional sequencing of spermatogenic cells of rats after the intervention of Cuscutae Semen flavonoids and Tripterygium Glycosides Tablets. Differentially expressed genes were screened out and the GO enrichment and KEGG pathway analysis of differentially expressed genes were conducted to explore the mechanism of Cuscutae Semen flavonoids in improving reproductive injury caused by Tripterygium Glycosides Tablets. The results showed that 794 up-regulated genes and 491 down-regulated genes were screened in Tripterygium Glycosides Tablets group compared with the blank group. Compared with Tripterygium Glycosides Tablets,440 up-regulated genes and 784 down-regulated genes were screened in the Cuscutae Semen flavonoids+Tripterygium Glycosides Tablets group. Among them,the gene closely related to reproductive function is DNMT3 L. Analysis of GO function and KEGG signaling pathway enrichment showed that the above differentially expressed genes were mainly enriched in cell,cell process,catalytic activity,binding,ovarian steroid synthesis,thyroid hormone and other functions and pathways. The thyroid hormone signaling pathway was the common enrichment pathway of the two control groups. In a word,Cuscutae Semen flavonoids has a good treatment effect on male reproductive damage caused by Tripterygium Glycosides Tablets. The mechanism may be closely related to up-regulation of DNMT3 L genes and intervention of thyroid hormone signaling pathway. At the same time,the discovery of many different genes provides valuable information for study on the mechanism of Cuscutae Semen flavonoids and Tripterygium Glycosides Tablets compatibility decreasing toxicity and increasing efficiency.
Animals ; Cuscuta ; chemistry ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; Female ; Flavonoids ; pharmacology ; Genitalia ; drug effects ; pathology ; Glycosides ; toxicity ; High-Throughput Nucleotide Sequencing ; Male ; Rats ; Seeds ; chemistry ; Signal Transduction ; Tablets ; Thyroid Hormones ; genetics ; Transcriptome ; Tripterygium ; toxicity

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

Objective: To investigate the impact of FLT3-ITD and DNMT3A R882 double mutations to the prognosis of acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: FLT3-ITD, DNMT3A, C-kit, CEBPA, FLT3-TKD and NPM1 mutations were detected in 206 newly diagnosed AML patients by Sanger sequencing (M(3) and those received FLT3 inhibitor were excluded). Clinical data of AML patients were retrospectively analyzed to compare the prognosis of each gene mutation group. Results: ①Of 206 patients, 104 were male and 102 female with a median age of 38 (3-63) years, including 6 cases of M(0), 24 cases of M(1), 56 cases of M(2), 39 cases of M(4), 63 cases of M(5), 6 cases of M(6) and 12 unclassified cases. ②All 206 patients were divided into four groups according to the mutation gene at the time of diagnosis: FLT3-ITD(+) DNMT3A R882(+) group (group A), FLT3-ITD(+) DNMT3A R882(-) group (group B), FLT3-ITD(-) DNMT3A R882(+) group (group C) and FLT3-ITD(-) DNMT3A R882(-) groups (group D). Gender, leukocyte count at diagnosis, chromosome karyotype, the median age, FAB classification, disease status prior to transplantation, type of donor, conditioning regimen and GVHD were not significantly different between four groups (P>0.05). ③The 2-year cumulative recurrence rate (CIR) of group A was significantly higher than that of other groups [group A (72.2±2.6)%, group B (38.6±0.6)%, group C (36.8±1.6)%, group D (27.8±0.1)%, respectively, P<0.05], while the 2-year overall survival (OS) rate and 2-year leukocyte-free survival (LFS) rate were lower than those of other groups [group A (30.9±13.3)%, (11.3±10.2)%; group B (67.5±7.8)%, (47.9±8.4)%; group C (61.4±12.4)%, (56.8±12.5)%; group D (80.1±3.7)%, (79.7±3.6)%, respectively, P<0.05]. Conclusion: AML patients with FLT3-ITD and DNMT3A R882 double mutations had a very high CIR and low OS, LFS after transplantation.
Adolescent ; Adult ; Child ; Child, Preschool ; DNA (Cytosine-5-)-Methyltransferases/genetics* ; DNA Methyltransferase 3A ; Female ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia, Myeloid, Acute ; Male ; Middle Aged ; Mutation ; Nucleophosmin ; Prognosis ; Retrospective Studies ; Young Adult ; fms-Like Tyrosine Kinase 3/genetics*

OBJECTIVETo explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.

METHODSThe HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.

RESULTSThe bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner.

CONCLUSIONSBufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G/Gphase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.

Apoptosis ; drug effects ; genetics ; Bufanolides ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; drug effects ; genetics ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; Leukemia, Erythroblastic, Acute ; enzymology ; genetics ; pathology ; RNA, Messenger ; genetics ; metabolism ; Up-Regulation ; drug effects ; genetics ; WT1 Proteins ; genetics ; metabolism

Approximately 2,300 genes are found to be associated with spermiogenesis and their expressions play important roles in the regulation of spermiogenesis. In recent years, more and more attention has been focused on the studies of the genes associated with oligospermia, asthenospermia and teratospermia and their molecular mechanisms. Some genes, such as GSTM1, DNMT3L, and CYP1A1, have been shown to be potentially associated with oligospermia; some, such as CATSPER1, CRISP2, SEPT4, TCTE3, TEKT4, and DNAH1, with asthenospermia; and still others, such as DPY19L2 and AURKC, with teratospermia. These findings have provided a molecular basis for the studies of the pathogenesis of oligospermia, asthenospermia and teratospermia, as well as a new approach to the exploration of new diagnostic and therapeutic techniques.
Asthenozoospermia ; genetics ; Aurora Kinase C ; genetics ; Calcium Channels ; genetics ; Cytochrome P-450 CYP1A1 ; genetics ; Cytoplasmic Dyneins ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; Dyneins ; genetics ; Glutathione Transferase ; genetics ; Glycoproteins ; genetics ; Humans ; Male ; Membrane Proteins ; genetics ; Microtubule Proteins ; genetics ; Oligospermia ; genetics ; Spermatogenesis ; genetics ; Teratozoospermia ; genetics

OBJECTIVETo investigate the effect of stable knockdown of DNA methyltransferase 3b (DNMT3b) on the proliferation and apoptosis of bladder cancer cells.

METHODSLentivirus expressing DNMT3b siRNA or the negative control siRNA was infected in human bladder cancer BIU-87 cells. MTT assay and flow cytometry were used to detect cell proliferation and apoptosis, respectively. The inhibitory effect of DNMT3b knockdown on xenograft tumors in nude mice was observed. Real-time PCR and Western blotting were carried out to investigate the expression level of cell apoptosis related genes. Methylation specific PCR was used to examine the methylation in the promoter region of the cell apoptosis related genes.

RESULTSThe results of real-time PCR and Western blotting showed that DNMT3b mRNA and protein level were stably knocked down in BIU-87 cells. Stable DNMT3b knockdown suppressed BIU-87 cell growth and the tumor formation ability of the cells in nude mice. DNMT3b knockdown promoted the apoptosis of BIU-87 cells, increased the mRNA and protein expression of the cell growth and apoptosis related genes including DAPK, Bax and RASSF1A, and significantly decreased the methylation of these genes.

CONCLUSIONStable DNMT3b knockdown can affect the methylation of the cell growth and apoptosis related genes to regulate their expression, which might be a possible mechanism for suppressed cell growth and enhanced apoptosis of BIU-87 cells.

Animals ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; Gene Knockdown Techniques ; Humans ; Mice ; Mice, Nude ; Neoplasm Transplantation ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Urinary Bladder Neoplasms ; genetics ; pathology

OBJECTIVETo determine the interaction between miR-21 and DNA methylation in different breast cancer cells.

METHODSFluorescence tagged miR-21 inhibitor and its negative control (NC) were transient transfected into MCF-7 and MDA-MB-231 cell, the transfection efficiency was observed using fluorescence microscopy, and the miR-21 expression level and genome DNA methylation status before and after transfection were assessed by real-time PCR and bisulfite-qMSP respectively. To investigate the regulation effect of DNA methylation on miR-21, cells were treated with 5-AZA (2.5 µmol/L) for 72 h, with dimethyl sulfoxide (DMSO) treatment as its negative control (NC), and the expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and AKT(also known as Protein Kinase B), two downstream genes of miR-21 were detected by Western blot.

RESULTSThe expression of miR-21 in MCF-7 cell was significantly knocked down (P < 0.01) by miR-21 inhibitor, with the genome DNA methylation level (P < 0.05) and all the three Dnmts: Dnmt1, Dnmt3a, and Dnmt3b unregulated. In contrast, the miR-21 expression in MDA-MB-231 cell was elevated ( P < 0.01) by miR-21 inhibitor, meanwhile, down- regulated of genome DNA methylation (P < 0.05) and Dnmt3b expression, upregulation of Dnmt3a were also observed. In addition, treated with 5-AZA resulted in significant increases of miR-21 expression in both MCF-7 and MDA-MB-231 cells (P < 0.01), with the protein level of PTEN increased in MCF-7 cell, which was further involved in the downregulation of AKT.

CONCLUSIONThe regulation effects of DNA methylation by transient transfection of miR-21 in MCF-7 and MDA-MB-231 cells are almost opposite, whilst the expression of miR-21 in two cell lines were all upregulated by decreased DNA methylation level and our results may provide some experimental evidences for the future development of rational therapy for different breast cancer.

Azacitidine ; Breast Neoplasms ; genetics ; Cell Line, Tumor ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Humans ; MCF-7 Cells ; MicroRNAs ; genetics ; PTEN Phosphohydrolase ; metabolism ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-akt ; metabolism ; Real-Time Polymerase Chain Reaction ; Up-Regulation