Effects of DNMT3A on mouse cardiac fibroblast proliferation and migration under high glucose environment / 中国药理学通报

Aim To investigate the effect of DNA methyltransferase 3A (DNMT3A) on the proliferation and migration of cardiac fibroblasts (CFs) in C57 mice under high glucose environment. Methods The hearts of C57 mice were taken from 1 to 3 days. After cutting and digesting, CFs were extracted by differential adherance centrifugattion and observed under microscope. After cell attachment, the cells were cultured under low glucose (5.5 mmol • L

Mechanism of heroin promoting addiction and relapse in progeny of male rats by inhibiting promoter methylation of imprinted gene H19 by down-regulating DNMT3a / 中国药理学通报

Aim To investigate the effect of heroin use in male rats of F0 generation on heroin addiction and relapse in rats of Fl generation and the underlying mechanism. Methods Male rats of F0 generation were treated with different doses of heroin (1, 3, 9 mg • kg

Role of DNMT3a in Hydroquinone-Induced Hematopoietic Stem Cell Toxicity / 中国实验血液学杂志

OBJECTIVE@#To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity.@*METHODS@#Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively.@*RESULTS@#Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001).@*CONCLUSION@#DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.

Aging-elevated inflammation promotes DNMT3A R878H-driven clonal hematopoiesis

Aging-elevated DNMT3A R882H-driven clonal hematopoiesis (CH) is a risk factor for myeloid malignancies remission and overall survival. Although some studies were conducted to investigate this phenomenon, the exact mechanism is still under debate. In this study, we observed that DNMT3A R878H bone marrow cells (human allele: DNMT3A R882H) displayed enhanced reconstitution capacity in aged bone marrow milieu and upon inflammatory insult. DNMT3A R878H protects hematopoietic stem and progenitor cells from the damage induced by chronic inflammation, especially TNFα insults. Mechanistically, we identified that RIPK1-RIPK3-MLKL-mediated necroptosis signaling was compromised in R878H cells in response to proliferation stress and TNFα insults. Briefly, we elucidated the molecular mechanism driving DNMT3A R878H-based clonal hematopoiesis, which raises clinical value for treating DNMT3A R882H-driven clonal hematopoiesis and myeloid malignancies with aging.

Effects of DNMT3A regulating Drp1 on proliferation and migration of activated hepatic stellate cells / 中国药理学通报

Aim To investigate the effects of DNMT3A regulating Drp1 mediated mitochondrial fission on the proliferation and migration of active hepatic stellate cells. Methods HSC-T6 cells were activated by 5 μg·L-1 TGF-β1 for 24 h, and DNMT3A lentivirus infection model was established to silence DNMT3A. The experiment was divided into control group, TGF-β1 group, TGF-β1+LV5-NC group and TGF-β1+ LV5-DNMT3A group. The effects of DNMT3A on related mRNA and protein expression were detected by RT-qPCR and Western blot. The cell proliferation was detected by CCK-8. The effect of DNMT3A on the migration ability of HSCs cells was observed by Wound healing assay and Transwell migration assay. Results Lentivirus infection successfully constructed a DNMT3A silencing model. Compared with the control group, the level of DNMT3A significantly increased, the mRNA and protein levels of the fibrosis markers collagen and α-SMA in the TGF-β1 group significantly increased, and the mRNA and protein levels of the mitochondrial fission marker Drp1 significantly increased; At the same time, the proliferation and migration ability of HSCs cells was significantly improved. Compared with the NC group, the DNMT3A level of the DNMT3A silenced group was significantly reduced, the expressions of collagen I, α-SMA and Drp1 were significantly inhibited, and the proliferation and migration capabilities of HSCs were also significantly inhibited. Conclusions Silencing DNMT3A inhibits the level of Drp1 and inhibits the proliferation and migration of HSCs at the same time. It is suggested suggest that DNMT3A-mediated low level DNA methylation modification may inhibit the occurrence of mitochondrial fission by inhibiting the level of Drp1, thereby inhibiting the activation of HSCs and affecting the occurrence and development of liver fibrosis. ,,,,.

Characteristics of DNMT3A mutations in acute myeloid leukemia

BACKGROUND: DNMT3A mutations occur in approximately 20% of AML cases and are associated with changes in DNA methylation. CDKN2B plays an important role in the regulation of hematopoietic progenitor cells and DNMT3A mutation is associated with CDKN2B promoter methylation. We analyzed the characteristics of DNMT3A mutations including their clinical significance in AML and their influence on promoter methylation and CDKN2B expression.METHODS: A total of 142 adults, recently diagnosed with de novo AML, were enrolled in the study. Mutations in DNMT3A, CEBPA, and NPM1 were analyzed by bidirectional Sanger sequencing. We evaluated CDKN2B promoter methylation and expression using pyrosequencing and RT-qPCR.RESULTS: We identified DNMT3A mutations in 19.7% (N=28) of enrolled patients with AML, which increased to 29.5% when analysis was restricted to cytogenetically normal-AML. Mutations were located on exons from 8–23, and the majority, including R882, were found to be present on exon 23. We also identified a novel frameshift mutation, c.1590delC, in AML with biallelic mutation of CEBPA. There was no significant difference in CDKN2B promoter methylation according to the presence or type of DNMT3A mutations. CDKN2B expression inversely correlated with CDKN2B promoter methylation and was significantly higher in AML with R882H mutation in DNMT3A. We demonstrated that DNMT3A mutation was associated with poor AML outcomes, especially in cytogenetically normal-AML. The DNMT3A mutation remained as the independent unfavorable prognostic factor after multivariate analysis.CONCLUSION: We characterized DNMT3A mutations in AML and revealed the association between the DNMT3A mutation and CDKN2B expression and clinical outcome.

First identified Korean family with Tatton-Brown-Rahman Syndrome caused by the novel DNMT3A variant c.118G>C p.(Glu40Gln)

Tatton-Brown-Rahman Syndrome (TBRS), an overgrowth syndrome caused by heterozygous mutation of DNMT3A, first was described in 2014. Approximately 60 DNMT3A variants, including 32 missense variants, have been reported, with most missense mutations located on the DNMT3A functional domains. Autosomal dominant inheritance by germ-line mutation of DNMT3A has been reported, but vertical transmission within a family is extremely rare. Herein, we report the first Korean family with maternally inherited TBRS due to the novel heterozygous DNMT3A variant c.118G>C p.(Glu40Gln), located outside the main functional domain and identified by multigene panel sequencing. The patient and her mother had typical clinical features, including tall stature during childhood, macrocephaly, intellectual disability, and characteristic facial appearance. TBRS shows milder dysmorphic features than other overgrowth syndromes, potentially leading to underdiagnosis and underestimated prevalence; thus, targeted multigene panel sequencing including DNMT3A will be a useful tool in cases of overgrowth and unexplained mild intellectual disability for early diagnosis and genetic counseling.

Impact of DNMT3A Gene Mutation on Response of Acute Myeloid Leukemia Patients to Induction Therapy

Aim: To evaluate the frequency and prognostic impact of DNA methyltransferase 3A (DNMT3A) gene mutation on response to induction therapy in newly diagnosed acute myeloid leukemia patients. Study Design: Cross-sectional descriptive study. Place and Duration: Hematology units of Suez Canal and Ain Shams schools of Medicine, Egypt. Between September 2016 and July 2017. Methodology: The study enrolled forty patients (male: female ratio was 1; mean age was 52.4 ± 19.4 years) with newly diagnosed de novo AML before starting induction therapy. DNMT3A mutations were detected using dye terminator sequencing technique for the second part of DNMT3A, encompassing the PHD and methyltransferase domains and representing exons 11 till the last exon 23. Hematological, cytogenetic studies and DNMT3A mutation results were compared to the patients’ hematological response to induction therapy. Results: Fourteen patients (35%) of the study participants had DNMT3A mutations while 65% had the wild type. Approximately 49.5% of mutations occurred in exon 23, the most common mutations were (R882C and R882H mutations; 28.5% and 21%, respectively). Out of 14 patients with DNMT3A mutation, 9 patients had incomplete remission and only 5 achieved complete remission with no statistically significant association. Odds ratio of the response to induction therapy according to DNMT3A mutation status was 1.32 times higher to show incomplete remission than in wild-DNMT3A patients. Conclusion: DNMT3A mutation has high prevalence in AML Egyptian patients with non-statistically significant difference between mutated DNMT3A and wild type when related to the impact on remission rates after induction therapy.

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 (M3 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 M0, 24 cases of M1, 56 cases of M2, 39 cases of M4, 63 cases of M5, 6 cases of M6 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.

DNA methyltransferase 3A regulates the expressions of collagens during the activation of cardiac fibroblasts / 医学研究生学报

Objective The mechanisms of methylation acting on myocardial fibrosis are not yet clear at present. The aim of this study was to investigate the role of DNA methyltransferase 3A (DNMT3A) in regulating the expressions of collagens during the activation of cardiac fibroblasts.Methods Cardiac fibroblasts were obtained from 50 neonatal mice and divided into three groups: blank control, DNMT3A overexpression plasmid (mDNMT3A-pEGFP-C3) and small interference DNMT3A siRNA. The contents of collagens in the cell supernatant were detected by ELISA. The mRNA and protein expressions of type I collagen (Col Ⅰ), type Ⅲ collagen (Col Ⅲ) and DNMT3A in the cardiac fibroblasts were determined by real-time quantitative PCR and Western blot respectively and the proliferative activity of the cardiac fibroblasts measured by CCK8 assay.Results The contents of Col I and Col Ⅲ in the cell supernatant were significantly increased in the DNMT3A overexpression plasmid group but decreased in the DNMT3A siRNA group as compared with those in the blank control (P<0.05). The expressions of Col Ⅰ, Col Ⅲ and DNMT3A were remarkably higher in the DNMT3A overexpression plasmid group but lower in the DNMT3A siRNA group than in the blank control (P<0.05). The cell activity was markedly higher in the DNMT3A overexpression plasmid group than in the empty vector plasmid and control groups (2.087±0.317 vs 1.063±0.223 and 1.082±0.207, P<0.05) but lower in the DNMT3A siRNA group (0.463±0.087) than in the latter two (P<0.05).Conclusion DNMT3A can increase the proliferation and activation of cardiac fibroblasts, upregulate the expressions of collagens and thus promote myocardial fibrosis.

The challenge for allogeneic hematopoietic stem cell transplantation in acute leukemia/ myelodyplastic syndrom with TP53, TET2 or DNMT3A gene mutations / 天津医药

@#Recently, much gene mutations have been detected in patients with acute leukemia or myelodysplastic syndrome (MDS) using next-generation sequencing (NSG) technology. Some of them are proved to be important prognostic markers. It has been showed that TP53, TET2 or DNMT3A gene mutations are associated with poor prognosis in acute leukemia or MDS patients. The prognosis of these patients is poor with short remission and survival. Allogeneic hematopoietic stem cell transplantation is the only way to cure these patients. However, the outcomes after transplantation are inferior to those in patients without these mutations. The hypomethylating agents or immune targeting therapy might improve their prognosis when combined with the present strategies. Here, the impact of TP53, TET2 and DNMT3A gene mutations on the prognosis after chemotherapy or transplantation is reviewed.

The progress of pathogenesis and treatment in angioimmunoblastic T-cell lymphoma / 中国肿瘤临床

Angioimmunoblastic T-cell lymphoma (AITL) is one of the most common subtypes of peripheral T-cell lymphomas. It is a fol-licular T-helper-derived neoplasm, and characterized by the presence of some genetic alterations, such as mutations in TET2, DN-MT3A, IDH2, and RHOA. Anthracycline-containing regimens represent the most widely adopted first-line option; however, new biolog-ic agents should be designed and incorporated to improve treatment response. The elucidation of the specific roles of these genetic al-terations in AITL will facilitate the development of molecularly targeted therapies to treat this disease.

mRNA expression of DNA methyltransferase 2 and 3a in the epidermis of patients with psoriasis vulgaris / 中华皮肤科杂志

Objective To investigate the expression of DNA methyltransferase 2 (DNMT2) and 3a (DNMT3a) in the epidermis of patients with psoriasis vulgaris.Methods Between March 2009 and December 2010,46 patients with psoriasis vulgaris were enrolled from the Department of Dermatology,Hospital for Skin Diseases,Chinese Academy of Medical Sciences and Peking Union Medical College,and the Department of Dermatology of Yixing People's Hospital,and 28 healthy controls were enrolled from the Department of Dermatologic Surgery,Hospital for Skin Diseases,Chinese Academy of Medical Sciences and Peking Union Medical College.Real-time quantitative PCR was performed to determine the mRNA expression of DNMT2 and DNMT3a in the epidermis of the lesional and nonlesional skin of patients with psoriasis vulgaris,as well as in the epidermis of normal skin of the healthy controls.Results The mRNA expression of DNMT2 (expressed as 2-△△Ct) in the lesional skin,non-lesional skin of the patients and normal skin of the healthy controls was 0.62 ± 0.02,0.36-± 0.05 and 0.15 ± 0.11,respectively.The mRNA expression of DNMT2 was significantly higher in the lesional skin than in the non-lesional skin of the patients (t =6.23,P ＜ 0.01),and higher in the non-lesional skin of the patients than in the normal skin of the healthy controls (t =7.33,P ＜ 0.01).Additionally,the mRNA expression of DNMT3a was significantly higher in the lesional skin (0.85 ± 0.03) than in the non-lesional skin (0.43 ± 0.04) of the patients (t =5.66,P ＜ 0.01),and higher in the non-lesiona] skin of the patients than in the normal skin of healthy controls (0.18 ± 0.09,t =8.62,P ＜ 0.01).Conclusion Both DNMT2 and DNMT3a mRNA were abnormally expressed in the epidermis of patients with psoriasis vulgaris.

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.

Expression of TET2 and DNMT3A in peripheral T cell lymphoma and their significance / 临床与实验病理学杂志

Purpose To investigate the expression of TET2 and DNMT3A in patients with peripheral T cell lymphoma (PTCL) and the relationship to immunophenotypes of PTCL.Methods Using a panel of immunohistochemical markers (CD3,CD4,CD10,BCL-6,CXCL-13,CD30,ALK),all cases of PTCLs were further divided into four groups,including angioimmunoblastic T cell lymphoma (AITL),peripheral T cell lymphoma,not otherwise specified (PTCL-NOS),anaplastic lymphoma kinase negative anaplastic large cell lymphoma (ALK-ALCL) and anaplastic lymphoma kinase positive anaplastic large cell lymphoma (ALK + ALCL).The expression of TET2 and DNMT3A in 89 cases of PTCL was detected by immunohistochemical analysis.Results 89 cases were divide into four subtypes,AITL (36/89),PTCL-NOS (26/89),ALKALCL (18/89),and ALK + ALCL (9/89).Immunohistochemistry staining revealed higher cytoplasmic expression of TET2 and DNMT3A in AITL than that of in PTCL-NOS and ALCL (P ＜ 0.05).And the nuclear expression of DNMT3A in patients with AITL was higher than that of PTCL-NOS and ALCL (P ＜ 0.05).The cytoplasmic expression of TET2 was positively related with both cytoplasmic and nuclear expression of DNMT3A in patients with AITL (P ＜ 0.05).Conclusion TET2 combined with DNMT3A could be used as markers in AITL diagnosis,which could provide new strategy for AITL diagnosis.

Effect of Ligustri Lucidi Fructus on hepatic COX activity in normal rats and preliminary investigation of its DNA methylation mechanism / 中国中药杂志

The study aims to investigate the effect of Ligustri Lucidi Fructus on cytochrome c oxidase(COX) activity in rat hepatic tissues and explore its possible mechanism of DNA methylation. Male SD rats received aqueous extract of Ligustri Lucidi Fructus (2.0，6.0 g•kg⁻¹) by intragastric administration for 30 d. After the rats were sacrificed, hepatic tissues of rats were taken to detect COX activity, protein concentration of COX4I1, TET1 and DNMT3A protein levels, mRNA expression levels of Cox4i1, Dnmt3a and Tet1, and determine the DNA methylation frequency of Cox4i1.Results showed that both low and high doses of Ligustri Lucidi Fructus could significantly increase COX activity and concentration of COX4I1(P<0.05), with a decreasing tendency of both TET1 protein and DNMT3a protein expression； however, mRNA expression levels of Cox4i1, Dnmt3a and Tet1 were not significantly changed by Ligustri Lucidi Fructus. In addition, DNA methylation frequency of Cox4i1 in high dose group showed a declining tendency as compared with the blank control group, but without significant difference.These results indicated that Ligustri Lucidi Fructus had promotive effect on hepatic COX activity in rats, which may be achieved by increasing protein content of COX4I1. Moreover, a decreased tendency of DNMT3A protein could be one of the reasons for the lower trend of Cox4i1 methylation rate. In addition, Ligustri Lucidi Fructus may regulate the expression levels of DNMT3A and TET1 in the same direction and its mechanism is not clear.

miR-200b suppresses proliferation and induces apoptosis in non-small cell lung cancer cells by targeting DNMT3A / 天津医药

Objective To investigate whether miR-200b suppresses proliferation and induces apoptosis of non-small cell lung cancer cells by targeting DNMT3A. Methods A qRT-PCR was employed for detecting the expression of miR-200b in different non-small cell lung cancer cells and human bronchial epithelial cells. A549 cells were transfected with miR-200b mimics, scramble, DNMT3A-siRNA and control-siRNA, respectively. The scramble and control-siRNA were served the negative control of miR-200b mimics and DNMT3A-siRNA, respectively. Western blot assay was conducted to detect the expression of DNMT3A protein in A549 cells. MTT and Annexin V/propidium iodide staining were employed to detect the proliferation ability and apoptosis rate of A549 cells. The effects of miR-200b mimics and DNMT3A-siRNA on the proliferation and apoptosis rate of A549 cells were compared between groups. Results Results of qRT-PCR showed that the expression of miR-200b was significantly down-regulated in A549, H1299, L78 and H460 cells than that of 16HBE cells. Among them, the most obviously reduction was found in A549 cells (P<0.05). Western blot assay showed that the level of DNMT3A protein was inhibited by restored miR-200b or knock-down DNMT3A in A549 cells. After transfection of miR-200b mimics or knock-down DNMT3A for 48 h, 72 h and 96 h, MTT showed that the OD values, which reflected the optical density of cell proliferation were significantly lower than those in the control group (P<0.05). Annexin V/propidium iodide staining showed that apoptosis rates of A549 cells after transfection of miR-200b mimics or knock-down DNMT3A were (23.33%±0.90%and 20.41%±0.70%), compared with the control group (5.28%± 0.55%and 5.68%±0.47%, P<0.01). Conclusion miR-200b suppresses cell proliferation and induces apoptosis by targeting DNMT3A in non-small cell lung cancer.

miR-101 inhibits growth and invasion of ovarian cancer cells by targeting DNMT3A / 中国癌症杂志

Background and purpose:miR-101 has been reported to be down-regulated in gastric cancer, colorectal cancer, breast cancer as well as prostate cancer acting as a tumor suppressor gene. However, its function in ovarian cancer is still unknown. The aim of this study was to investigate whether miR-101 can suppress cell growth and invasion of ovarian cancer cells by targeting DNMT3A, so as to reveal molecular mechanism to inhibit ovarian cancer. Methods:Quantitative real-time palymerase chain reaction (qRT-PCR) method was employed to detect the expression of miR-101 in ovarian cancer and cancer adjacent normal ovarian tissues. SKOV3 cells were transfected with miR-101 mimics, and DNMT3A siRNA was transfected as a positive control. Then Western blot was used to detect the expres-sion of DNMT3A protein regulated by miR-101 in SKOV3 cells. The growth and invasion ability of SKOV3 cells were evaluated by MTT and Transwell invasion assays.Results:qRT-PCR showed that miR-101 was down-regulated in ovarian cancer tissues. Western blot showed that the level of DNMT3A protein was inhibited by restored miR-101 or knock-down of DNMT3A in SKOV3 cells. Following transfection of miR-101 mimics or knock-down of DNMT3A for 48, 72 and 96 h respectively, MTT assay showed that theD values were signiifcantly lower than the control group, (P<0.05). After transfection of miR-101 mimics or knock-down of DNMT3A for 36 h, Transwell invasion assay showed that the numbers of cells through the basement membrane was (105±7) and (107±13), respectively, which are signiifcantly different from the control group (213±11), indicating invasion of SKOV3 cells signiifcantly slowed down (P<0.05).Conclusion:miR-101 suppresses cell growth and invasion by targeting DNMT3A in ovarian cancer.

The association of DNMT3a, DNMT3b expression with the state of promoter methylation of ERαgene and ERαexpression in sporadic breast cancer / 天津医药

Objective To investigate the correlationship between DNMT3a, DNMT3b protein expressions and the state of promoter methylation of ERα gene and ERα protein expression in the development of sporadic breast cancer. Methods A total of 180 patients with sporadic breast cancer and 30 patients with breast fibroadenoma were included in this study. The expressions of DNMT3a and DNMT3b protein were detected by immunohistochemical method. The state of promoter methylation of ERα gene was detected by methylation specific PCR in 97 patients with sporadic breast cancer. Results There were no significant differences in positive expression rates of DNMT3a and DNMT3b protein between breast fibroadenoma and breast cancer. There were higher expression levels of DNMT3a and DNMT3b in breast cancer patient of Ⅲ～Ⅳstages than those of Ⅰ～Ⅱstages. The expression of DNMT3a was significantly higher in patients with lymph node metastasis than that of patients without lymph node metastasis (P＜0.05). Of 97 cases of breast cancer patients, ERα gene promoter methylation occurred in 39 cases (40.2%). The positive expression of DNMT3a protein was positively correlated with the ERα gene methylation (rS=0.250). The DNMT3a protein expression showed a significant influence to the overall survival (OS) in patients of breast cancer (P=0.035), no significant influence to the disease-free survival (DFS) (P=0.064). DNMT3b protein expression showed no significant influence to OS and DFS of patients with breast cancer (P=0.914 and 0.961). Conclusion The positive expressions of DNMT3a and DNMT3b are correlated with the invasion, metastasis and poor prognosis of sporadic breast cancer. DNMT3a was positively correlated with the state of ERα gene promoter methylation. The inhibition of DNMT3a and DNMT3b may have advantages in the prevention and treatment of sporadic breast cancer.

Methylation status of tumor suppressor gene ppENK in the pathogenesis of pancreatic carcinoma / 中华胰腺病杂志

Objective To detect the methylation status of ppENK and its role in the pathogenesis of pancreatic carcinoma.Methods The ppENK methylation status in human tissues of pancreatic cancer,pancreatic carcinoma cell lines and normal pancreas was detected by methylation-specific RT-PCR(MSP).The association of methylation status of ppENK gene with clinicopathological parameters was analyzed. The expression of ppENK mRNA was detected by RT-PCR.Two pancreatic carcinoma cell lines (PANC1,AsPC1 ) were treated with demethylating agent (5-aza-dC).The cell growth was measured by MTT.Apoptosis and cell cycle was analyzed by flow cytometry.The expression of DNMT3a was measured by Western blot.Results ppENK mRNA was expressed in normal pancreas.And methylation of ppENK was not detected in normal pancreas.Methylation of ppENK was detected in 90.3％ (28/31) of pancreatic carcinoma tissue,and there were no correlation between methylated ppENK with clinicopathological features of pancreatic carcinoma.There was no ppENK mRNA expression in SW1990,PANC1,PC3,AsPC1,PuPan-1,and ppENK was methylated.Methylated ppENK was associated with no ppENK mRNA expression.After 5-Aza-dC treatment,PANC1,AsPC1 was demethylated and ppENK mRNA expression was reversed.The proliferation of PANC1 and AsPC1 was inhibited in a dose dependent manner.The apoptotic rates of PANC1 and AsPC1 were increased [ (31.57 ± 6.76)％ts (3.21 ±1.43)％,P =0.002,(16.6 ±8.22)％ vs (3.82 ±1.71)％,P=0.058];the expression of DNMT3a protein was decreased; the PANC1 cells of G1 phase significantly increased [ (67.87 ± 2.72 ) ％ vs (54.57 ± 7.18) ％,P =0.040 ],but PANC1 cells of S phase significantly decreased [ ( 22.37 ± 4.31 )％ vs (33.73 ± 4.63)％,P =0.036 ].But the percentage of G1,S phase in AsPC1 cell line was not significantly changed ( P =0.236,0.075 ).ConclusionsppENK demethylation is an important molecular event in inducing ppENK expression inhibition,which can inhibit pancreatic cancer proliferation,promote apoptosis,arrest cell cycle at G1 and decrease the expression of DNMT3a protein.