Clinical Value of Translocator Protein Gene in Evaluating the Efficacy of FLT3-ITD/DNMT3A R882 Double-Mutated Acute Myeloid Leukemia / 中国实验血液学杂志

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.

Trdmt1 3'-untranslated region functions as a competing endogenous RNA in leukemia HL-60 cell differentiation

Severe blockage in myeloid differentiation is the hallmark of acute myeloid leukemia (AML). Trdmt1 plays an important role in hematopoiesis. However, little is known about the function of Trdmt1 in AML cell differentiation. In the present study, Trdmt1 was up-regulated and miR-181a was down-regulated significantly during human leukemia HL-60 cell differentiation after TAT-CT3 fusion protein treatment. Accordingly, miR-181a overexpression in HL-60 cells inhibited granulocytic maturation. In addition, our "rescue" assay demonstrated that Trdmt1 3′-untranslated region promoted myeloid differentiation of HL-60 cells by sequestering miR-181a and up-regulating C/EBPα (a critical factor for normal myelopoiesis) via its competing endogenous RNA (ceRNA) activity on miR-181a. These findings revealed an unrecognized role of Trdmt1 as a potential ceRNA for therapeutic targets in AML.

miR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis

Abstract Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. Objective: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and −149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. Methodology: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. Results: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. Conclusion: The polymorphism −149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.

Clinical Characteristics of Peripheral Hemogram and DNMT3A Gene Mutation in Patients with Primary AML Treated by FLT3-ITD-mt and FLT3-ITD-wt / 中国实验血液学杂志

OBJECTIVE@#To explore the efficacy and safety of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of relapsed or refractory peripheral T-cell lymphoma(PTCL).@*METHODS@#The clinical data of 6 patients with relapsed or refractory PTCL undergoing allo-HSCT from Sep. 2014 to Sep. 2018 in the department of hematology, aerospace center hospital were retrospectively analyzed. Complications and disease-free survival after HSCT were observed.@*RESULTS@#All the patients could well tolerate the conditioning regimen and acquired hematopoietic recon-struction. Following up till December 2018, with a median time of 11.5 months (1-51); acute GVHD developed in 2 cases and chronic GVHD developed in 5 cases, Among 6 cases one case died of viral pheumonia and the other 5 patients remained disease-free survival. The longest disease-free survival time has reached 51 months.@*CONCLUSION@#allo-HSCT is a safe and effective method for relapsed or refractory peripheral T-cell lymphoma, which can be chosen as salvage treatment method for patients with primary resistance. Optimization of the conditioning regimen may result in better efficacy of allo-HSCT.

DNMT3A/3B overexpression might be correlated with poor patient survival, hypermethylation and low expression of ESR1/PGR in endometrioid carcinoma: an analysis of The Cancer Genome Atlas / 中华医学杂志(英文版)

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.

Study on mechanism of Cuscutae Semen flavonoids in improving reproductive damage of Tripterygium Glycosides Tablets in rats based on high-throughput transcriptome sequencing / 中国中药杂志

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.

Relationship between DNMT1 and Methylation of SHP-1 Promoter 2 in K562 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the relationship of DNA methyltransferase 1 ( DNMT1 ) with hematopoietic cell phosphatase (SHP-1) gene expression and promoter 2 methylation status in cell line K562.</p><p><b>METHODS</b>The promoter sequence of SHP-1 gene promoter 2 in NCBI database was analyzed, the K562 cells were transfected with the lentiviral plasmids-the specified retroviral vector psiHIV-mU6-shDNMT1 and psiHIV-mU6-mcherryFP-control. The methylation status of SHP-1 gene promoter 2 in K562 cells was detected by methylation-specific polymerase chain reaction (MSP) and bisulfite-modified sequencing (BSP). Western blot was used to detect the protein expression level of SHP-1 and DNMT1, the SYBR Green fluorescence quantitative PCR was used to detect the expression of SHP-1 mRNA.</p><p><b>RESULTS</b>It was found that the promoter 2 of SHP-1 gene located between -577 bp to +300 bp, and 22 CpG sites contained between -353 bp-+182 bp were aberrantly hypermethylated and the SHP-1 could not be detected in K562 cells. In vitro, the detection demonstrated that the expression level of DNMT1 in K562 cells transfected with psiHIV-mU6-shDNMT1 was 0.48±0.06 significantly lower than that of psiHIV-mU6-control group (1.33±0.19)(t= 4.18, P<0.05). The expression of SHP-1 mRNA in K562 cells transfected with psiHIV-mU6-shDNMT1 was significantly higher than that in K562 cells transfected with psiHIV-mU6-shDNMT1 (14.23±3.83 vs 1.031±0.156)(P<0.01). DNMT1 silencing induced demethylation of the 22 CpG sites located in the SHP-1 promoter 2, and SHP-1 gene was re-expression in K562 cells.</p><p><b>CONCLUSION</b>The DNMT1 in K562 cells relates with the hypermethylation and silencing of SHP-1 promoter in K562 cells.</p>

Effect of FLT3-ITD with DNMT3A R882 double-mutation on the prognosis of acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

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.

Effects of bufalin on up-regulating methylation of Wilm's tumor 1 gene in human erythroid leukemic cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>Bufalin 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.</p>

Genetic genes associated with oligospermia, asthenospermia and teratospermia: Advances in studies / 中华男科学杂志

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.

Role of DNA methyltransferases in the pathogenesis of neuropathic pain in rats / 中南大学学报(医学版)

OBJECTIVE@#To explore the role of DNA methyltransferases (DNMTs) in the pathogenesis of neuropathic pain (NPP) in rats following sciatic nerve chronic constriction injury (CCI).
@*METHODS@#A total of 27 adult male Sprague-Dawley rats with successful implantation of lumbar intrathecal catheter were randomly divided into 3 groups: a sham + normal saline group (sham+NS group), a CCI+NS group, and a CCI+5-azacytidine group (CCI+5-AZA group) (n=9 in each group). The rats in the Sham+NS group and the CCI+NS group received NS, while the rats in the CCI+5-AZA group received 10 μmol/L of 5-AZA (a DNMTs inhibition) once a day through spinal injection from the 3th day to 14th day after CCI surgery. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of ipsilateral hinds in the 3 groups were measured before or at the 3th, 5th, 7th, 10th or 14th day after CCI surgery. At the end of experiments, all rats were killed under deep anesthesia and their lumbar spinal cords were dissected to examine the DNMT1, DNMT3a and DNMT3b expression by RT-PCR, Western blot and immunohistochemistry, respectively.
@*RESULTS@#Compared with the sham+NS group, the MWT and TWL in the CCI+NS group were obviously reduced from the 3th day to the 14th day after surgery (both P<0.05). Compared with the CCI+NS group, the MWT and TWL in the CCI+5-AZA group were obviously increased from the 5th day to the 14th day after surgery (both P<0.05), but they were still reduced compared with the sham+NS group (both P<0.05). The DNMT1, DNMT3a and DNMT3b were highly expressed in the lumbar spinal dorsal horn in all rats, and the positive signals were mainly located in the nucleus. The DNMT1, DNMT3a and DNMT3b levels in the CCI+NS group were increased significantly compared with that in the sham+NS group on the 14th day after surgery (all P<0.05). The DNMT1, DNMT3a and DNMT3b expressions in the CCI+ 5-AZA group were decreased significantly compared with that in the CCI+NS group (all P<0.05), but they still increased compared with that in the sham+NS group (all P<0.05). 
@*CONCLUSION@#Up-regulation of DNMTs in the lumbar spinal may play an important role in the pathogenesis of NPP in CCI rats. DNMTs inhibitors (5-AZA) could reduce expression of DNMTs and attenuate CCI-induced NPP, which might be a potential therapeutic drug for NPP.

Effect of Pinggan Qianyang Recipe Containing Serum on Angiotensin II Induced Vascular Smooth Muscle Cell Proliferation and Migration and DNA Methylation / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the effect of Pinggan Qianyang Recipe (PQR) on inhibiting angiotensin II (Ang II) induced proliferation and migration of vascular smooth muscle cells (VSMCs) and changes of DNA methylation.</p><p><b>METHODS</b>VSMCs were cultured using tissue explant method, and PQR containing serum was prepared. Primarily cultured VSMCs were divided into four groups, the normal group, the model group, the folate group (folic acid intervention) , and the PQR group. The proliferation and migration of VSMCs was duplicated by Ang II. After 24-h Ang II induced culture, 40 microg/mL folic acid was added to the folate group for 48 h, while 5% PQR containing serum was added to the PQR group for 48 h. The cell growth curve of VSMCs was drawn by using Cell Counting Kit (CCK-8). The proliferative activity of VSMC was determined by MTT assay. The migration of VSMCs was measured by Millicell chamber. The general level of cytosine methylation in cell nucleus was detected via 5-mC antibodies immunofluorescence, and mRNA expression levels of DNA methyltransferase 1 (DNMT1) were measured by Real-time q-polymerase chain reaction (q-PCR).</p><p><b>RESULTS</b>VSMCs were promoted by Ang II at 10(-6) mol/L for 24 h. Compared with the normal group, the proliferative activity and migration quantity of VSMCs obviously increased, and DNA methylation level obviously decreased (P < 0.05, P < 0.01). Compared with the model group, the cell growth, proliferative activity and migration quantity of VSMCs obviously decreased and the general DNA methylation level increased in the folate group and the PQR group (P < 0.05, P < 0.01). Compared with the normal group, the mRNA expression of DNMT1 decreased in the model group (P < 0.01). Compared with the model group, mRNA expression of DNMT1 in Ang II induced VSMCs was obviously enhanced in the folate group and the PQR group (P < 0.01).</p><p><b>CONCLUSIONS</b>PQR could inhibit Ang II induced proliferation and migration of VSMCs, and cause high genomic DNA methylation level. Changes of DNA methylation might be associated with DNMT1 expression.</p>

Effect of phenelzine on the proliferation, apoptosis and histone methylation and acetylation of Molt-4 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of monoamine oxidase inhibitor phenelzine on proliferation, apoptosis and histone modulation in acute lymphoblastic leukemia cell line Molt-4 cells.</p><p><b>METHODS</b>The effect of Phenelzine on cell proliferation was detected by MTT. Apoptotic rate was measured by flow cytometry. The variation of apoptosis associated proteins Caspase-3, Bcl-2 and Bax, cyclin-dependent kinase inhibitor p21, tumor suppressor protein p15, and the expression level of histone methylation of H3K4, H3K9 and histone acetylation of H3, DNMT1 were detected by Western Blot.</p><p><b>RESULTS</b>① Molt-4 cell proliferation rates were (87.68±3.54)%, (67.84±3.24)%, (51.48±3.37)%, (28.72±2.56)% respectively after exposured to phenelzine at 5, 10, 15, 20 μmol/L for 24 h, P<0.05. ② After 10 μmol/L of phenelzine exposure for 24, 48, 72 h, cell proliferation rates were (67.84±3.24)%, (50.24±2.01)%, (40.31±2.25)%, P<0.05. ③ The apoptotic rates were (13.64±2.58)%, (31.24±3.42)%, (56.37±4.26)% after phenelzine treatment at 5, 10, 20 μmol/L for 24 h, which was concentration dependent. ④ Phenelzine could upregulate the expression of Bax, caspase-3, p21, and downregulate Bcl-2 expression. Phenelzine upregulated the methylation level of histone H3K4me1, H3K4me2 and histone acetylated H3, while it didn't change the level of histone H3K4me3, H3K9me1, H3K9me2. ⑤ Phenelzine inhibited DNMT1 expression and promoted p15 expression.</p><p><b>CONCLUSIONS</b>Phenelzine increased the methylation of histone H3K4me1, H3K4me2, acetylation of histone H3 and p21, and decreased the expression of DNMT1 and p15, and ultimately inhibited the proliferation and apoptosis of Molt-4 cells.</p>

Expression of DNA Methyltransferase Genes and Its Significance in AML Patients / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect the expression of DNA methyltransferases (DNMT) mRNA in the patients with acute myelogenous leukemia (AML) and to analyze the retationship between the mRNA expression of DNMT and cellular and moleculogenetic risk stratifieation in AML patients, and to evaluate the role of the DNMT mRNA expression in AML prognosis and clinical treatment.</p><p><b>METHODS</b>The mRNA expression of DNMT was detected by real-time PCR in 123 AML patients and 20 healthy people.</p><p><b>RESULTS</b>the mRNA expression levels of DNMT were lower in the healthy people and higher in AML patients; the mRNA expression levels of DNMT in the patients after the consolidation therapy were lower than that in the patients of initial diagnosis and replapse; The mRNA expression levels of DNMT did not correlate with age, sex and the clinical characteristics at initial diagnosis, such as white blood cell count, FAB classification and chromosomal karyotype in AML patients. In CR patients after standard treatment, the initial mRNA expression level of DNMT3b was higher. Based on cellular and moleculogenetic risk stratificantion, the DNMT expression level in the intermediate risk AML patients was higher.</p><p><b>CONCLUSION</b>The mRNA expression of DNMT may play an important role in AML pathogenesis and can serve as an index for evaluating AML prognosis and for instructing clinical treatment.</p>

Effect of SET deficiency on the trichloroethylene-induced alteration of DNA methylation in human hepatic L-02 cells / 中华预防医学杂志

<p><b>OBJECTIVE</b>To compare the DNA methylation-related alteration induced by trichloroethylene (TCE) in human hepatic L-02 cells (L-02 cells) and SET deficient cells, and reveal the role of SET on the mechanisms in TCE-induced epigenetic pathway.</p><p><b>METHODS</b>The L-02 cells and pre-established SET deficient cells were treated with different TCE concentrations, and the changes of total cell viability, DNA methylation level and DNA methyltransferases (DNMTs) activity were measured, respectively. In addition, the TCE-induced alteration in the protein expression of DNMT1, DNMT3a and DNMT3b were analyzed by Western blotting.</p><p><b>RESULTS</b>After treatment with TCE for 24 h, the cell proliferation level was significantly decreased in both cell lines. When concentrations of TCE were 0, 1.0, 2.0, 4.0 and 8.0 mmol/L, the proliferation levels of L-02 cells were 100.00±2.70, 83.34±2.38, 75.56±4.51, 71.67±2.77 and 66.67±1.63, respectively (F = 58.29, P < 0.001); the cell proliferation levels of SET deficient cells were 101.12±1.67, 85.01±2.33, 79.44±1.67, 78.337±3.89 and 76.11±3.33, respectively (F = 42.41, P < 0.001). When concentration of TCE reached 4.0 mmol/L, the difference of cell proliferation level between two groups was statistically significant (t = -3.51; P = 0.013). After treated by TCE for 24 h, the global DNA methylation significantly decreased in both cell lines (F value was 212.87 and 79.32, respectively, P < 0.001). The difference between two groups was not statistically significant. After treated by TCE for 24 h, the methyltransferases activities were significantly decreased in both cell cells (F values were 77.92 and 113.80, respectively, P-0.001). The SET deficiency could inhibit the decrease of methyltransferases activity under TCE treatment. When the concentration of TCE reached 8.0 mmol/L, the enzymatic activity of L-02 cells and SET deficient cells decreased to 67.61%±2.85% and 72.97%± 1.94%, respectively. The difference between two groups was statistically significant (t = -3.94, P = 0.008). After treated with TCE for 24 h, concentrations of TCE were 0, 1.0, 2.0, 4.0 and 8.0 mmol/L, and the relative protein levels of DNMT1 in normal L-02 cells increased significantly to 1.00±0.03, 1.28±0.04, 1.20±0.04, 1.62±0.05, 1.43±0.04 (F = 103.00, P < 0.001); In SET deficient cells, the expressions of DNMT1 were 1.00±0.04, 0.96±0.02, 1.19±0.05, 0.85±0.03, 0.83±0.03, which was significantly down-regulated under TCE treatment (F = 44.18, P < 0.001).</p><p><b>CONCLUSION</b>SET deficiency can significantly attenuate the TCE-induced decreases of cell viability and DNMTs activity, as well as alteration of protein expression of DNMT1 in L-02 cells, which indicated that SET was involved in the mechanism of TCE-induced cytotoxicity and epigenetic pathway in L-02 cells.</p>

Outcomes of refractory or relapsed DNMT3A + cytogenetically normal acute myeloid leukemia patients followed the therapy including decitabine combined with CAG or CAG-like regimen / 中华血液学杂志

<p><b>OBJECTIVE</b>To study clinical characteristics of refractory or relapsed DNMT3A⁺ cytogenetically normal acute myeloid leukemia（CN-AML）patients, and to explore the overall response rate（ORR）and side effects of these patients followed the therapy including decitabine with CAG or CAGlike regimen.</p><p><b>METHODS</b>In this study we retrospectively analyzed 53 refractory or relapsed CN- AML patients receiving the therapy including decitabine combined with CAG and CAG- like regimen in our center from April 2011 to October 2014. The clinical characteristics and ORR were further analyzed. Based on gene mutations, these patients could be divided into 2 groups: DNMT3A⁺ AML patients（n=24）and DNMT3A- AML patients（n=29）.</p><p><b>RESULTS</b>The median age of DNMT3A⁺AML patients was 46 years old, higher white blood cells and bone marrow blasts were observed in DNMT3A+ AML group. The ORR and complete response（CR）rate of DNMT3A+ group were 62.50% and 54.17%, respectively. No differences were observed in ORR and CR rates（P>0.05）between these two groups. DNMT3A⁺/FLT3-ITD⁺ CN-AML patients（n=14）had higher ORR and CR rates than DNMT3A-/FLT3-ITD⁺CN- AML patients（n=15）（P= 0.040 and 0.042, respectively）. The one- year overall survival （OS） of DNMT3A⁺ AML group and DNMT3A- AML group were 59.58% , 54.09% , no differences were observed （P=0.438）. 25 patients received further therapy of allo-HSCT, the one-year OS of DNMT3A⁺ CN-AML was 87.50% and one-year disease free survival（DFS）was 72.73%, while the one- year OS was 61.54% and one- year DFS was 58.02% in DNMT3A⁻ group. No differences were observed between 2 groups （P=0.456, 0.217）.</p><p><b>CONCLUSION</b>Decitabine combined with CAG or CAG-like regimen was an effective and safe treatment for refractory or relapsed CN- AML patients. Compared to DNMT3A⁻/FLT3- ITD⁺ CN- AML patients, DNMT3A⁺/ FLT3-ITD⁺ CN-AML patients had higher ORR and CR rates. Decitabine bridged hematopoietic stem cells transplant could likely improve the survival of refractory or relapsed CN-AML patients.</p>

DNMT1 mediates chemosensitivity by reducing methylation of miRNA-20a promoter in glioma cells

Although methyltransferase has been recognized as a major element that governs the epigenetic regulation of the genome during temozolomide (TMZ) chemotherapy in glioblastoma multiforme (GBM) patients, its regulatory effect on glioblastoma chemoresistance has not been well defined. This study investigated whether DNA methyltransferase (DNMT) expression was associated with TMZ sensitivity in glioma cells and elucidated the underlying mechanism. DNMT expression was analyzed by western blotting. miR-20a promoter methylation was evaluated by methylation-specific PCR. Cell viability and apoptosis were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and TdT-mediated dUTP-biotin nick end labeling assays, respectively. The results showed that compared with parental U251 cells, DNMT1 expression was downregulated, miR-20a promoter methylation was attenuated and miR-20a levels were elevated in TMZ-resistant U251 cells. Methyltransferase inhibition by 5-aza-2\'-deoxycytidine treatment reduced TMZ sensitivity in U251 cells. In U251/TM cells, DNMT1 expression was negatively correlated with miR-20a expression and positively correlated with TMZ sensitivity and leucine-rich repeats and immunoglobulin-like domains 1 expression; these effects were reversed by changes in miR-20a expression. DNMT1 overexpression induced an increase in U251/TM cell apoptosis that was inhibited by the miR-20a mimic, whereas DNMT1 silencing attenuated U251/TM cell apoptosis in a manner that was abrogated by miR-20a inhibitor treatment. Tumor growth of the U251/TM xenograft was inhibited by pcDNA-DNMT1 pretreatment and boosted by DNMT1-small hairpin RNA pretreatment. In summary, DNMT1 mediated chemosensitivity by reducing methylation of the microRNA-20a promoter in glioma cells.

Effect of stable DNA methyltransferase 3bknockdown on proliferation and apoptosis in bladder cancer cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of stable knockdown of DNA methyltransferase 3b (DNMT3b) on the proliferation and apoptosis of bladder cancer cells.</p><p><b>METHODS</b>Lentivirus 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Stable 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.</p>

Interaction between miR-21 and DNA methylation in different breast cancer cells / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To determine the interaction between miR-21 and DNA methylation in different breast cancer cells.</p><p><b>METHODS</b>Fluorescence 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>

Effects of DNMT3A gene mutations on prognosis of patients with acute myeloid leukemia: a meta-analysis / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To evaluate the effects of DNMT3A gene mutation on prognosis of patients with acute myeloid leukemia (AML) by a meta-analysis.</p><p><b>METHODS</b>Methods 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.</p><p><b>RESULTS</b>Seven 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.</p><p><b>CONCLUSION</b>DNMT3A gene mutation is an independent risk factor of poor prognosis of patients with acute myeloid leukemia.</p>