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Objective To investigate the expression of DNA methyltransferase (DNMT) in HL-60 cells induced by tetrandrine (Tet).Methods HL-60 cells were treated with different concentrations of Tet and decitabine (DAC) alone and in combination with both.Methyl thiazolyl tetrazolium (MTT) assay was used to assess cytotoxic effect.Flow cytometry (FCM) was used to determine apoptosis rate.Real-time quantitative polymerase chain reaction (PCR) assay was used to quantify mRNA levels of DNMT.Western blot was used to quantify the expression of DNMT protein.Results Tet inhibited the growth and proliferation of HL-60 cells in a time-and dose-dependent manners (both P ＜0.01).Tet treated HL-60 cells after 48 h at the concentration of 2 μmol/L,and 4 μmol/L,the levels of DNMT gene and protein in the drug administration group decreased compared to the control group.After incubation for 48 h with Tet 2 μmol/L combined with DAC 4 μmol/L,the combination group was significantly depressed.Conclusions Tet could potently inhibit the growth and proliferation of HL-60 cells,reduce the expression levels of DNMT mRNA and protein,and have a more obvious effect in the combination group.
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Objective: To investigate the effect of dihydroartemisinin (DHA) on expression of tumor suppressor gene ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in human prostate cancer cell line PC-3, and explore its regulative mechanism. Methods: PC-3 cells were treated with different concentrations (25, 50, and 100 μmol/L) of DHA for 48 h, while PC-3 cells without DHA treatment were used as the control. Then the apoptosis and cell cycle distribution were detected by flow cytometry. The expressions and cellular locations of DNA methyltransferase 1 (DNMT1) and UCHL1 proteins were detected by immunofluorescence staining. The expression levels of UCHL1, DNMT1, phospho-Akt (p-Akt) and Akt proteins were detected by Western blotting. The 1 μmol/L phosphoinositide-3-kinase (PI3K) inhibitor Wortmanin was used as a positive control to analyze the expression of p-Akt protein in DHA-treated group. Results: DHA significantly induced the apoptosis of PC-3 cells and arrested the cell cycle distribution at phase G2/M as compared with those of the control group (both P 0.05) in the DHA-treated group and the positive control group. The downregulation of p-Akt expression was more obvious in high-concentration of DHA-treated group, much closer to that in the positive control group. Conclusion: DHA can inhibit the expression of DNMT1, restore the function of UCHL1 gene, induce the apoptosis of PC-3 cells, and block the cell cycle progression. These mechanisms may be related to the suppressive activity of PI3K/Akt pathway.
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Objective To investigate the effect of NaAsO2 on the binding levels of methyl CpG binding protein 2(MeCP2),DNA methyltransferase 1 (DNMT1) and histone deacetylase 1(HDAC1) to the hypermethylation promoter region of MGMT gene in HaCaT cells,in order to provide a basis to deepen the interpretation of the role of arsenic poisoning mechanism.Methods HaCaT cells were treated repeatedly and interval with different concentrations of NaAsO2(3.13,6.25,12.50,25.00 μnol/L,respectively) for 72 h.Untreated HaCaT was used as blank control group and human epidermal squamous carcinoma cell line(A431 cells) as positive control group.The binding levels to the two transcription regulatory regions(ChIP1,ChIP2) and to the coding region(ChIP3) of MGMT 8ene were detected by chromatin immuno-precipitation combined with quantitative PCR.Results The differences of binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each group were significant (F=7.387,84.634,78.442 and 19.263,69.649,26.546,all P ＜ 0.05).The binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each NaAsO2 exposed group[3.13 μmol/L NaAsO2 exposed group:(136.00 ±16.97)％,(145.00 ± 2.83)％,(88.50 ± 19.09)％ and (106.50 ± 37.48)％,(112.34 ± 8.73)％,(59.71 ± 8.49)％;6.25 μmol/L NaAsO2 exposed group:(130.00 ± 42.43)％,(154.50 ± 4.95)％,(101.00 ± 1.27)％ and (88.50 ±3.54)％,(134.32 ± 2.82)％,(102.75 ± 19.91)％ ; 12.50 μmol/L NaAsO2 exposed group:(141.50 ± 23.33)％,(161.50 ± 7.78)％,(125.00 ± 11.31)％ and (119.50 ± 24.75)％,(171.59 ± 3.54)％,(167.61 ± 10.61)％; 25.00μmol/L NaAsO2 exposed group:(134.50 ± 43.13)％,(472.50+ 50.20)％,(383.50 ± 30.41)％ and (180.09 ±12.73)％,(348.50 ± 27.58)％,(158.45 ± 12.02)％] were higher than that in the blank control group[(51.50 ±9.19)％,(82.00 ± 12.73)％,(25.03 ± 2.91)％ and (37.02 ± 4.24)％,(91.56 ± 26.16)％,(19.09 ± 2.90)％,all P ＜ 0.05].The differences of binding levels of MeCP2 to ChIP3 in each group were not significant(F =1.670,P ＞0.05),but the differences of binding levels of DNMT1 and HDAC1 to ChIP3 were significant (F =4.404,9.863,all P ＜ 0.05),and only the binding levels in the 25.00 μmol/L NaAsO2 exposed group [(615.85 ± 29.63)％,(306.09 ± 59.40)％] were higher than that in the blank control group[(99.70 ± 12.02)％,(92.45 ± 48.79)％,all P ＜ 0.05].Conclusions MeCP2 can bind to the methylated MGMT gene transcriptional regulatory regions which are induced by arsenic and leads to histone deacetylation by the recruitment of DNMT1 and HDAC1 and,meanwhile,DNMT1 can bind to the coding region of MGMT gene to recruit HDAC1 in a methyl DNA binding protein(MBD) independence manner and media MGMT gene silencing through the chromatin remodeling way,which might be the early molecular events of arsenic poisoning.
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Objective To investigate the effect of sevoflurane anesthesia on DNA methyltransferase (DNMT) mRNA expression in neonatal rat amygdala. Methods Forty-two 8-day-old SD rats were randomly divided into 2 groups ( n = 21 each): control group and experimental group. 5% sevoflurane was inhaled for 1 min, and then the inhaled concentration of sevoflurane was decreased to 3 % and maintained for 4 h. The rats were sacrificed at the end of sevoflurane inhalation and 24 h after the end of sevoflurane inhalation, and amygdala was removed for determination of DNMT, mRNA, DNMT3, mRNA and DNMT3b mRNA expression by RT-PCR. Blood samples were taken at the end of sevoflurane inhalation for blood gas analysis. Results Compared with control group, the DNMT, mRNA and DNMT3, mRNA expression was down-regulated (P ＜ 0.05). There were no significant differences in DNMT3b mRNA expression and parameters of blood gas analysis between the two groups (P ＞ 0.05). Conclusion Sevoflurane anesthesia can down-regulate DNMT, mRNA and DNMT,, mRNA expression in neonatal rat amygdala, which may result in functional deficits during the development of central nervous system.
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Both DNA methylation and microRNAs(miRNA)play important roles in the development and progression of human cancer. Recently, it has been demonstrated that the promoter of miRNA can be methylated and DNA methylation can be regulated by miRNA through DNA methyltransferase. Research targeting this reciprocal regulatory mechanism may shed some light on the diagnosis and treatment of cancer.
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Overexpression of DNA methyltransferases(DNMTs)is observed in many tumors. Overexpressed DNMTs silence tumor suppressor genes by catalyzing the methylation of CpG islands within the promoter regions, and therefore promote the neoplastic transformation of normal cells. Highly elevated DNMT activities have been observed in cancers including gastrointestinal cancers, breast cancer, and lung cancer and have been associated with cancer development and prognosis. Drugs and targeted therapies that inhibit DNMT activities can reactivate methylated tumor suppressor genes, promote apoptosis, and thus inhibit tumor growth.
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Objective To observe the effect of DNA methyltransferase 1 (DNMT1 ) gene silencing by RNA interfering technology on the proliferation and apoptosis of HeLa cells. Methods Recombinant plasmid pshRNA-DNMT1-A, B and C were respectively transfected into HeLa cells by lipofectamine 2000, while cells transfected plasmid vector pSilencer3.1-HI and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and western blotting was used to detected the mRNA and protein expression of DNMT1 in HeLa cells transfected for 24, 48 and 72 hours. Cell counting kit-8 (CCK-8 ) assay was used to investigate the proliferation of the HeLa cells after transfection, while apoptosis was detected by flowcytometry(FCM ) method. Results Three DNMT1-targeted short hairpin RNA (shRNA) A,B and C were successfully inserted into the plasmid vector PShRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragments. The results indicated that both recombinant plasmid pshRNA-DNMT1-A and B could effectively knock down the expression of DNMT1 gene in human cervical cancer cells, of which pshRNA-DNMTI-B was the better choice. While no effect of pshRNA-DNMTI-C was seen. BT-PCR results showed that the relative mRNA expression of DNMT1 gene in Helm cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were 0.406±0.057,0.191±0.036 and 0.104±0.015, which were significantly lower than that in Helm cells transfected by empty vector and non-transfected cells (0.520±0.020, 0.537±0.041, respectively, P ＜ 0.05 ). The western blotting analysis manifested that the relative expression of DNMT1 protein of Helm cells transfected by pshRNA-DNMT1 for 24, 48 and 72 hours were 0.197±0.024, 0.075±0.015, 0.040± 0.013, which were significantly lower than that in transfected cells by empty vector and non-transfected cells (0.273±0.010, 0.283±0.016, respectively, P ＜0.05). The CCK-8 results showed that the cell survival rates of HeLa cells transfected by pshRNA-DNMT1 for 24, 48, 72, 96 and 120 hours were 70.8%, 64.8%, 51.6%, 45.3% and 38.0%, there were statistically different compared with cells transfected by empty vector and non-transfected cells at different time-points (P ＜ 0.01 ). The results of FCM indicated that the apoptesis rate of HeLa cells trandected with pshRNA-DNMTI for 24, 48 and 72 hours were (17.7± 1.3 ) %, (35.3±1.3 ) %, (47.6±1.6 ) %, which were significantly higher than empty vector transfected cells and non-transfected cells [(4.9±0.5 ) %, (5.1±0.7 ) %, respectively, P ＜ 0.05]. Conclusions DNMT1 can be successfully silenced by RNA interfering in cervical Helm cells. Downregulation of DNMT1 can inhibit cervical cancer cells proliferation and induce cell apoptosis.