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Objective To investigate the expression of DNA methyltransferase 3b (DNMT3B) in hepatocellular carcinoma (HCC) and its effect and mechanism on the proliferation,invasion and migration of HCC cells.Methods The expression of DNMT3B gene was detected by qRT-PCR in 46 cases of HCC tissues and corresponding adjacent tissues;the results and clinical pathological parameters were analyzed.SiRNA targeting DNMT3B was transfected into MHCC97-H cells by RNA interference (RNAi) technique.The mRNA and protein expression levels of related genes were detected by qRT-PCR and Western blot.The cell proliferation was measured by MTT assay,and the invasion and migration abilities were measured by Transwell assay.Results In 46 HCC patients,the expression of DNMT3B (73.91％) was significantly higher in HCC than in adjacent normal tissue.The high expression of DNMT3B gene was associated with histological type and tumor size of HCC (all P＜0.05).Inhibition of DNMT3B gene expression decreased proliferation,invasion and migration of MHCC97-H cells.Interference with DNMT3B gene increased the expressions of tumor suppressor genes RASSFA1,APC and MTSS1 at mRNA and protein levels.Conclusion DNMT3B is associated with the progression of HCC.It may inhibit the proliferation,invasion and migration of HCC cells by regulating the methylation of downstream tumor suppressor gene.
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<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>
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Humanos , Apoptose , Genética , Bufanolídeos , Farmacologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Forma Celular , DNA (Citosina-5-)-Metiltransferases , Metabolismo , Metilação de DNA , Genética , Regulação Leucêmica da Expressão Gênica , Leucemia Eritroblástica Aguda , Genética , Patologia , RNA Mensageiro , Genética , Metabolismo , Regulação para Cima , Genética , Proteínas WT1 , Genética , MetabolismoRESUMO
Objective:To determine the clinicopathological significance of the DNA methyltransferase 3B (DNMT3B)overexpression in endometrial carcinomas and to evaluate its correlation with hormone re-ceptor status.Methods:Immunohistochemistry was performed to assess the expression of DNMT3B and hormone receptors in 104 endometrial carcinomas.Results:DNMT3B overexpression occurred frequently in endometrioid carcinoma (EC,54.8%)more than in nonendometrioid carcinoma (NEC,30.0%) with statistical significance (P =0.028).Furthermore,there was a trend that EC with worse clinico-pathological variables and shorter survival had a higher DNMT3B expression,and the correlation between DNMT3B and tumor grade reached statistical significance (P =0.019).A negative correlation between DNMT3B and estrogen receptor (ER)or progesterone receptor (PR)expression was found in EC. NMT3B overexpression occurred frequently in the ER or PR negative subgroups (78.9%,86.7%)more than in the positive subgroups (47.7%,47.8%)with statistical significance (P =0.016,P =0.006). In addition,the DNMT3B overexpression increased in tumors with both ER and PR negative expression (92.9%,P =0.002).However,no such correlation was found in NEC (P >0.05).Sequence analyses demonstrated multiple ER and PR binding sites in the promoter regions of DNMT3B gene.Conclusion:This study showed that the expression of DNMT3B in EC and NEC was different.DNMT3B overexpres-sion in EC was associated with the worse clinicopathological variables and might have predictive value. The methylation status of EC and NEC maybe different.In addition,in EC,DNMT3B overexpression negatively correlated with ER or PR expression.In NEC,the correlation between DNMT3B and ER or PR status was not present.
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Background and purpose:DNMT3B has nearly 40 known splice variants expressed in a tissue-and disease-speciifc manner, but the roles of these splice variants in the cell are still unclear. The aim of this study was to investigate the effects of overexpression of DNA methyltransferase 3B4 (DNMT3B4) gene on proliferation of human embryo kidney 293A cells. Methods:293A cells were transfected with plasmid pCMV-DNMT3B4 or pCMV-2B and then treated with G418 to get the stable cell line. The stable cell lines were determined for proliferation level by MTT method, and for cell cycle distribution by lfow cytometry. The expression of p21 was detected by real-time PCR and Western blot. The methylation status of p21 gene promoter was detected by methylation-speciifc PCR (MS-PCR). Results:The absorbance value in DNMT3B4-1 and DNMT3B4-2 clone were (58.92±3.47)%and (68.82±5.64)%as compared to 293A-vector cells using MTT method. DNMT3B4 overexpression signiifcantly decreased cell proliferation (P<0.05). S phase fraction of 293A-vector cells was (40.44±0.91)%. While in DNMT3B4-1 and DNMT3B4-2 clone cells, the S phase fraction was (35.88±2.00)%and (37.00±1.79)%respectively. Overexpression of DNMT3B4 could significantly decrease S phase fraction (P<0.05). The expression of p21 in DNMT3B4 overexpressed cells was increased, but the methylation status of p21 gene promoter was unchanged.Conclusion:Overexpression of DNMT3B4 can inhibit 293A cell proliferation and can facilitate p21 expression.
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Objective To observe and analyze the correlation between the DNA methyltransferase-3B (DNMT3B) gene single nucleotide polymorphisms (SNP) rs 1569686 and the susceptibility to colorectal cancer (CRC) in Tianjin Han population.Methods A case-control study was performed.89 CRC patients and 94 normal controls were examined,and Sequenom MassArray system was applied to detect the genotype of rs1569686 in DNMT3B gene polymorphisms.The association results were analyzed of the SNP and CRC.Results The frequencies of genotypes TT,GT and GG at SNP rs1569686 were 74.2％,24.7％ and 1.1％ in CRC patients respectively,which there were no significant differences from that in controls(73.4％,23.4％ and 3.2％; P＞0.05).Conclusion It suggests that the SNP rs1569686 of DNMT3B gene may not be related to susceptibility of CRC in Tianjin Han population.
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Objective To investigate the expression of DNMT3b and its correlation with clinicopathological parameters of pancreatic cancer.Methods The expressions of DNMT3b protein in 12 pancreatic cancer tissues and para-cancerous tissues were detected by Western blot.The expressions of DNMT3b protein in 59 pancreatic cancer tissues were detected by immunohistochemistry.The association of DNMT3b expression and clinicopathological parameters of pancreatic carcinoma were analyzed.Results Western blot results showed the expressions of DNMT3b protein in pancreatic cancer tissues and para-cancerous tissues were 0.69 ±0.13and 0.14 ±0.03,and the protein level of DNMT3b in pancreatic cancer tissues were significantly higher than that in para-cancerous tissues (t =4.464,P ＜0.05 ).Immunohistochemistry examination showed that the positive rates of DNMT3b protein were 59％ in pancreatic cancer tissues and negative in para-cancerous tissues.DNMT3b expression was positively correlated with the TNM stage and lymph node metastasis.Conclusions DNMT3b is highly expressed in pancreatic cancer tissues,and it is related with malignant biological behaviors of pancreatic cancer cells.