Effects of 5-aza-2'-deoxycytidine on proliferation of SPC-Al cells and expression of SFRPl and MGMT methylated proteins in lung cancer / 中国药理学通报

Aim To observe the effect of methylation inhibitors of 5-aza-2'-deoxycytidine (5-Aza-CdR) on SPC-Al - lung cancer cell proliferation, cell scratches and apoptosis, to explore the influence of secretion curl associated protein 1 (SFRPl) and 06-methylguanine-DNA-methyltransferase (MGMT) gene promoter region in which DNA methylation mRNA and protein expression and meaning. Methods The effects of 5-Aza-CdR at different concentrations on the proliferation of human lung cancer SPC-A 1 cells were determined by CCK-8 assay. The effect of 5-Aza-CdR on the migration ability of SPC-A 1 cells was determined by scratch assay. The apoptosis of lung cancer SPC-A 1 cells was detected by Hoechst 33258 staining after treatment with 5-Aza-CdR for 24 h. mRNA and protein expressions of SFRPl and MGMT in SPC-Al cells were detected by RT-PCR and Western blot. Results 5-Aza-CdR could reduce the proliferation of SPC-A 1 cells by concentration gradient,and IC50 was 21.2 jjimol • L

Development of precision medicine approaches based on inter-individual variability of BCRP/

Precision medicine is a rapidly-developing modality of medicine in human healthcare. Based on each patient׳s unique characteristics, more accurate dosages and drug selection can be made to achieve better therapeutic efficacy and less adverse reactions in precision medicine. A patient׳s individual parameters that affect drug transporter action can be used to develop a precision medicine guidance, due to the fact that therapeutic efficacy and adverse reactions of drugs can both be affected by expression and function of drug transporters on the cell membrane surface. The purpose of this review is to summarize unique characteristics of human breast cancer resistant protein (BCRP) and the genetic variability in the BCRP encoded gene in the development of precision medicine. Inter-individual variability of BCRP/ can impact choices and outcomes of drug treatment for several diseases, including cancer chemotherapy. Several factors have been implicated in expression and function of BCRP, including genetic, epigenetic, physiologic, pathologic, and environmental factors. Understanding the roles of these factors in controlling expression and function of BCRP is critical for the development of precision medicine based on BCRP-mediated drug transport.

Effects of DACT1 methylation status on invasion and metastasis of nasopharyngeal carcinoma

BACKGROUND: The purpose of the present study was to investigate the role of the methylation status of the DACT1 gene on the invasion and metastasis of nasopharyngeal carcinoma cells. METHODS: The levels of methylation and expression of the DACT1 gene in nasopharyngeal carcinoma tissues and CNE2 cells were determined by methylation-specific PCR and RT-PCR, respectively. CNE2 cells were treated with 5-aza-2-deoxycytidine, and the variation in the methylation status of the DACT1 gene was detected, as well as the influence of methylation on invasiveness of nasopharyngeal carcinoma cells. RESULTS: The DACT1 gene was hyper-methylated in 44 of 62 cases of nasopharyngeal carcinoma. The DACT1 gene was hyper-methylated in 32 of 38 cases of nasopharyngeal carcinoma with lymph node metastasis, and the DACT1 gene was hyper-methylated in 7 of 24 cases of nasopharyngeal carcinoma without lymph node metastasis. The DACT1 mRNA level was weakly expressed or not expressed in all nasopharyngeal carcinoma tissues with hyper-methylated DACT1 genes; however, the DACT1 mRNA level was highly expressed in nasopharyngeal carcinoma tissues with low expression of the methylated DACT1 gene. The DACT1 gene was hyper-methylated and not expressed in CNE2 cells that did not have 5-aza-2-deoxycytidine treatment. After 5-aza-2-deoxycytidine treatment, the DACT1 gene was demethylated and the expression of DACT1 was restored. Moreover, the invasion ability was inhibited in CNE2 cells treated with 5-aza-2-deoxycytidine. CONCLUSION: The expression of DACT1 was related to the methylation status. High expression of DACT1 may inhibit the invasion and metastasis of nasopharyngeal carcinoma cells.

Effects of 5-Aza-2′-deoxycytidine on proliferation of human breast cancer cell line Hs578T and methylation of PRDM10 gene / 天津医药

Objective To investigate effects of 5-Aza-2′-deoxycytidine (5-Aza-CdR) on proliferation of human breast cancer cell line Hs578T,and the methylation status of PRDM10 gene in vitro in this cell line.Methods The human breast cancer cell line Hs578T was cultured with 1,3 and 5 μmol/L DNA methylation inhibitor 5-Aza-CdR respectively, and untreated cells were used as control.Cell proliferation was detected by MTT assay.Methylation-Specific PCR(MSP)was used to detect the methylation status of PRDM10 gene. The mRNA and protein expression levels of PRDM10 gene were detected by RT-PCR and Western blot assay. Results MTT results showed that the higher the concentration of 5-Aza-CdR,and the longer the treatment time,the more significant inhibitory effect on the proliferation of Hs578T cells.Compared with the control group(0 μmol/L),the proliferation of Hs578T was significantly inhibited after the treatment for 72 h in the 1 μmol/L group, and for 48 h in the 3 μmol/L and 5 μmol/L groups (P<0.05). MSP results showed that the higher the concentration of 5-Aza-CdR,the more significant demethylation of PRDM10.Results of RT-PCR and Western blot showed that the higher the concentration of 5-Aza-CdR, the higher the expression levels of mRNA and protein in PRDM10 (P<0.05).Conclusion 5-Aza-CdR could inhibit the cell proliferation of Hs578T,which might be related to the demethylation of PRDM10 gene in the cells.

Negative regulation of microRNA - 9 by ecotropic viral integration site1 involved in the pathogenesis of acute myeloid leukemia / 中华实用儿科临床杂志

Objective To investigate the regulation mechanism of microRNA - 9(miR - 9)by ecotropic viral integration site1(EVI1)its impact on proliferation of AML cells and its role in the pathogenesis of myelogenous leuke-mia. Methods EVI1 was forced to express in Uocm1 cell lines by murine stem cell virus - EVI1(MSCV - EVI1) plasmid infection. EVI1 overexpressed Uocm1 cells were then treated with 0. 1 μmol/ L 5 - aza - 2′ - deoxycytidine (5 - AZA)dissolved in dimethyl sulfoxide (DMSO). The methylation level of miR - 9 promoter was tested by DNA bi-sulfite sequencing technology. The cell cycle was observed by flow cytometry (FCM). The proliferation ability of the cells was detected by the colony forming assay in semi - solid Methylcellulose medium culture. Results EVI1 level was dramatically increased after being infected by MSCV - EVI1 plasmid. Forced expression of EVI1 in Uocm1 signifi-cantly downregulated miR - 9 by inducing hypermethylation of miR - 9 promoter. Relative expression level of miR - 9 was lower in EVI1 overexpressed group(0. 004 ± 0. 000)than that of the control group(0. 006 ± 0. 001)(t = 4. 09,P <0. 05). When EVI1 was overexpressed in Uocm1,the rate of G0 / G1 cells decreased markedly(P < 0. 05),while rates of S phage and G2 phage increased significantly(all P < 0. 05). Seven days after 500 cells plated in semi - solid medium, EVI1 overexpressed Uocm1 cells gave rise to more colony (122. 3 ± 7. 8)than Uocm1 cells infected with vector (45. 7 ± 6. 1)(t = - 13. 44,P < 0. 01). 0. 1 μmol/ L 5 - AZA recovered miR - 9 expression(P < 0. 01)by decreasing EVI1 induced hypermethylation of miR - 9 promoter. G0 / G1 phase cell proportion was(48. 25 ± 2. 19)％ in control group,while (65. 90 ± 2. 90)％ in 5 - AZA group (t = - 6. 85,P < 0. 05). 5 - AZA group formed less colony (51. 00 ± 10. 01)than the control group (123. 40 ± 8. 12)(t = 9. 59,P < 0. 01),which indicated that 5 - AZA inhibi-ted cell proliferation by G0 / G1 cell cycle retardation in EVI1 overexpressed uocm1 cells. Conclusions EVI1 may en-hance proliferation ability of myeloid leukemia cells by downregulating miR - 9 through inducing hypermethylation of miR - 9 promotor,which plays a crucial role in the pathogenesis of AML. 5 - AZA may be an effective hypomethylating agent in the therapy of EVI1 high acute myeloid leukemia.

Effects of DNA methylation inhibitor 5-Aza-2'-deoxycytidine on biological behavior of esophageal squamous carcinoma cells / 肿瘤研究与临床

Objective To investigate the effects of methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC) on biological behavior of esophageal squamous carcinoma cell (ESCC) lines KYSE140 and KYSE150. Methods KYSE140 and KYSE150 cell lines were divided into the blank group, the control group and the experimental group. The cells in the blank group didn't do the treatment, and the cells in the control group were added to DMSO 2 μmol/L, while in the experimental group, cells were treated with different concentration (1, 2, 3 and 4 μmol/L) of 5-Aza-dC which affected respectively at different time (24, 48, 72 and 96 h). Cell proliferation was detected by using methyl thiazolyl tetrazolium (MTT) assay and the optimal drug concentration and time point were selected. Transwell assay was performed to detect the change of cell migration and invasion. Flow cytometry was used to observe the effects of drugs on cell apoptosis and cell cycle.The expression of PARP,Caspase-3,CCNB-1,and CCNE-1 were detected by Western blot. Results MTT result showed that the effective function time of 5-Aza-dC on KYSE140 and KYSE150 was 96 h at the concentration of 4 μmol/L. Under this condition, the cell ability of migration and invasion was decreased significantly. The migrated cell number of KYSE140 and KYSE150 respectively in the blank group, the control group and the experimental group was (193.3±8.6), (184.0±10.4), (61.7±7.1) and (112.0±6.4), (101.3± 7.9), (26.3±5.7). The invasive cell number was (47.3±7.3), (38.7±5.1), (8.0±3.9) and (83.3±6.8), (74.7±5.7), (21.0±2.7), respectively. The difference was statistically significant (P <0.05). Flow cytometry revealed that 5-Aza-dC increased the apoptosis of KYSE140 and KYSE150. The apoptosis rate of the blank group, the control group and the experimental group was (2.8±0.3) %, (11.2±0.7) %, (18.6±0.6) % for KYSE140 and (2.7±0.4)%,(9.8±0.4)%,(17.7±0.5)% for KYSE150.Compared with the other two groups,the cell number of G2/M phase in the experimental group was increased remarkably (P < 0.05). PARP and Caspase-3 were sheared evidently and the protein expression of CCNB-1 was up-regulated while the expression of CCNE-1 was down-regulated in the experimental group. Conclusion 5-Aza-dC can inhibit the proliferation and promote apoptosis of ESCC cells.

Epigenetic modification by 5-Aza/TSA treatment induces loss of B-cell phenotype in B-cell derived non-Hodgkin lymphoma / 中国病理生理杂志

AIM:To investigate influence of demethylation/acetylation by 5-Aza-2'-deoxycytidine/trichostatin A(5-Aza/TSA)treatment on B-cell specific phenotype of non-Hodgkin lymphoma cells.METHODS:CD19 promoter-driven reporter with NEO cassette was constructed to realize transfection and stable selection of Hodgkin and non -Hodgkin lymphoma cells.The exogenous CD19 promoter activity in both cell line clusters with and without 5-Aza/TSA treatment was detected and compared.The B-cell specific expression profiling in Eμ-myc transgenic mouse model developed lymphoma was isolated and identified.The effects of 5-Aza/TSA treatment on B-cell specific phenotype were analyzed.RESULTS:Epigenetic modification via 5-Aza/TSA repressed B-cell specific phenotype in B-cell-derived non-Hodgkin lymphoma cells. CONCLUSION:Epigenetic modification of pivotal master repressor genes plays an essential role in B -cell phenotype of both human and murine developed B-cell non-Hodgkin lymphoma cells.

Novel mechanism underlying anticancer effect of methyltransferase inhibitor 5-aza-2′-deoxycytidine / 肿瘤

Objective: To reveal a novel mechanism by which 5-aza-2′- deoxycytidine (AZA) plays its anticancer role, and whether AZA can inhibit the expressions of cancer-associated genes by activating their antisense RNAs. Methods: Six genes were chosen from those having antisense RNAs, which were previously obtained by sequencing a double-stranded RNA library. The selected genes included phosphatase and tensin homolog deleted on chromosome ten (PTEN ), signal transducers and activators of transcription 1 (STAT 1), Ras-like proto-oncogene B (RALB ), MET, CD 44 and heat-shock protein A4 (HSPA 4). The human hepatocellular carcinoma HepG2 cells were treated with AZA (dissolved in DMSO) or DMSO (as the control). The expressions of sense and antisense RNAs for the selected genes were detected by strand-specific RT-PCR and real-time fluorescent quantitative PCR, respectively. Results: The antisense RNAs of all 6 genes were activated by AZA treatment (all P < 0.01). On the other hand, AZA also induced the expressions of tumor suppressor gene PTEN and innate immunity-related gene HSPA 4 (P < 0.01, P < 0.001). In contrast, the expressions of cancer-related genes, CD 44 and HSPA 4, were obviously inhibited by AZA treatment (both P < 0.01). Conclusion: AZA can activate the antisense RNAs, so as to promote the expressions of tumor suppressor genes and innate immunityrelated genes, but to inhibit the expressions of cancer-associated genes; which may be the novel mechanism underlying anticancer effect of AZA.

Effects of 5-Aza-2'-deoxycytidine on proliferation of human gastric cancer BCG-823 cells and the expression of HOXA5 gene / 中国医师杂志

Objective To explore the effects of 5-Aza-2'-deoxycytidine (5-Aza-CdR) on the proliferation of BCG-823 human gastric cancer cells and the expression of HOXA5.Methods The methylation status of the promoter of HOXA5 was measured by methylmion specific PCR (MSP).The BCG-823 cells were treated with different concentrations of 5-Aza-CdR,and then the changes of expression and methylation status of HOXA5 gene were detected by quantitative real-time polymerase chain reaction (QRT-PCR),Western-blot,and MSP.Cells proliferation was assessed by methyl thiazolyl tetrazolium (MTT) assay.Results (1) Different methylation status of HOXA5 gene promoter was detected in BCG-823 cells.The mathylation rate of HOXA5 gene promoter were reduced after treatment with 5-Aza-CdR,and also were negatively related to the concentration of 5-Aza-CdR (F =438.307,P ＜ 0.01).(2) Compared to the control groups,the expressions of HOXA5 mRNA and protein were increased after treatment with 5-Aza-CdR,with statistical significance (P ＜ 0.05).(3) The proliferation rate of BCG-823 cells was significantly inhibited (P ＜0.05).Conclusions The methylation status of HOXA5 promoter was detected in BCG-823 cells.5-AzaCdR is able to inhibit BCG-823 cells growth in vitro,which might be related to the expression of HOXA5.It may be a new way to treat gastric cancer.

The effects of 5-aza-2-deoxycytidine on RECK gene expression and invasion of salivary adenoid cystic car-cinoma cells / 实用口腔医学杂志

Objective:To investigate the effects of 5-aza-2′deoxycytidine(5-aza-dC),a DNA methyltransferase (DNMT)inhibitor, on the methylation status of the RECK gene and the invasion of salivary adenoid cystic carcinoma cell lines.Methods:Methylation-specific PCR,Western blot analysis and quantitative real-time PCR were used to investigate the methylation status of RECK gene and the expression of RECK mRNA and protein in SACC cell lines.The invasive ability of SACC cells was examined by transwell assay. Results:Promoter methylation was only found in ACC-Mcell line and not in ACC-2 cell line.Treatment of ACC-Mcells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression level of mRNA and pro-tein of RECK,suppressed ACC-Mcell invasive ability.Conclusion:5-aza-dC can inhibit ACC-Mcell invasion by reversal of hyperm-ethylation status of RECK gene.

Effects of 5-aza-2' -deoxycytidine combined with trichostatin A on cell proliferation and PDX-1 methylation on high glucose-induced toxicity in pancreatic β cells / 实用医学杂志

Objective To investigate the effects of 5-aza-2'-deoxycytidine (5-Aza-dC) alone or combined with trichostatin A(TSA) on cell proliferation, promoter methylation and mRNA expression level of PDX-1 gene in pancreatic β cells induced by high glucose toxicity. Method NIT-1 cells were treated in vitro by high glucose (33.3 mmol/L), then divided into five groups, control group, HG grpup, 5-Aza-dC treatment group, TSA interfere group and 5-Aza-dC + TSA group. Proliferation of NIT-1 cells, insulin secretion, promoter methylation and mRNA expression of PDX-1 gene were detected respectively. Results 5-Aza-dC and TSA alone or in combination could promote cell proliferation and recover insulin secretion in NIT-1 cells , could also reduce PDX-1 gene methylation and enhance expression of PDX-1 mRNA. Compared with single-treatment group , combined group was significantly different (all P < 0.05). Conclusion 5-Aza-dC and TSA could activate the expression of PDX-1 and, then recover insulin secretion in NIT-1 cells induced by high glucose. Combination of them had synergistic effect.

Effects of 5-aza-2′deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma

Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoid cystic carcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza-2′deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation-specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECK gene hypermethylation, which might be a promising chemotherapy approach in SACC treatment.

CpG Island Methylation Regulates BNIP3 Gene Expression in Gastric Cancer Cells / 中国医科大学学报

Objective To detect the methylation status of the promoter of BNIP3 gene in gastric cancer cell lines MKN1,and to explore the mecha?nism of DNA methylation regulating the expression of BNIP3 in gastric cancer cells. Methods The methylation status of BNIP3 promoter was de?tected by bisulfate sequencing PCR. Reverse transcription PCR was used to evaluate BNIP3 mRNA expression. MKN1 cells were treated with 5?Aza?2′?deoxycytidine(5?Aza?CdR),and after the treatment,the methylation status and BNIP3 mRNA expression were observed. Chromatin immuno?precipitation(ChIP)was used to determine the combination of BNIP3 with DNA methyltransferase 1(DNMT1). Results The promoter DNA of BNIP3 in MKN1 cells was in state of hypermethylation. Compared to the control group,methylation status and mRNA expression of BNIP3 in the drug treatment group(the 5?Aza?CdR concentration was 10μmol/L)were reversed,which showed statistical differences(P<0.05). 5?Aza?CdR inhibited the combination of BNIP3 with DNMT1. Conclusion CpG island methylation regulates BNIP3 gene expression in MKN1 cells. DNA methylation is related with the binding between the promoter of BNIP3 and DNMT1.

Effects of 5-Aza-CdR on growth of TPC-1 cell line and KLF4 expression / 中华内分泌外科杂志

Objective Methylation of anti-oncogene can be demethylated by related drugs which can help the inactivated gene to express again .This study aims to study the effects of the demethylating agent 5-Aza-2′-deoxycytidine on the growth of human thyroid papillary cancer cell line TPC-1 and mRNA and protein expres-sion of KLF4.Methods TPC-1 cells were treated with different concentration of 5-Aza-CdR.MTT was used to detect the influence of 5-Aza-CdR on cell proliferation .RT-PCR was used to detect mRNA and protein expression levels of KLF4.Results After being treated with 5-Aza-CdR for 24 hours, 48 hours, and 72 hours, the growth of TPC-1 cells was inhibited and the inhibition was in time and concentration depended manner .After treatment with 5-Aza-CdR, mRNA and protein expression levels of KLF 4 were increased, and the difference had statistical significance(P<0.05).Conclusion 5-Aza-CdR can inhibit the cell viability of TPC-1 cells through upregulat-ing KLF4 expression , which may provide experimental basis for 5-Aza-CdR in treating thyroid cancer .

Effects of 5-Aza-CdR on cell apoptosis in lung cancer A549/DDP cell line and expression of tumor suppressor gene hMLH1 / 肿瘤研究与临床

Objective To investigate the effects of 5-Aza-2'-deoxycytidine (5-Aza-CdR) on the apoptosis of A549/DDP cells and the expression of hMLH1 gene.Methods A549/DDP cells were treated with 5-Aza-CdR at 0.5,5,50 μmol/L.The growth curve of A549/DDP cells was investigated by MTT assay.The methylation status of hMLH1 gene was detected by methylation specific PCR (MSP).The expression of hMLH1 mRNA was evaluated by FQ-PCR.The apoptosis rate of A549/DDP cells was analyzed by flow cytometry.Results A549/DDP cells treated with 5-Aza-CdR showed a slow growth in comparison with the control cells,and the growth rates were decreased with the increasing of 5-Aza-CdR concentration.The apoptosis rate after treatment was higher than that before treatment in A549/DDP cells (P ＜ 0.05),and had a positive correlation with 5-Aza-CdR dose (P ＜ 0.001).hMLH1 mRNA expression level was increased in a 5-Aza-CdR concentration dependent manner (P ＜ 0.05).hMLH1 promoter in A549/DDP cells was methylated and hMLH1 mRNA was negatively expressed before treatment,but the mRNA was positively expressed after treatment with 5-Aza-CdR.Conclusions 5-Aza-2'-CdR can induce apoptosis of A549/DDP cells by inducing demethylation of hMLH1 promoter and thereby enhancing hMLH1 gene expression and its tumor suppressor function.

Efefct of adoptive transfer of 5 AzaD-induced re gulator T cells on outcomes of gestation in abortion-prone murine matings / 中国免疫学杂志

Objective:To investigate the effect of the adoptive transfer of CD4+CD25+Foxp3+regulatory T cells ( iTregs) induced by 5-aza-2′-deoxycytidine (5AzaD) on pregnant outcome of the abortion-prone mice.Methods:Sixty cases of female CBA/J × male DBA/2J abortion-prone matings were taken as study group,the CD4+T cells from spleen of twenty female CBA/J mice were separated by magnetic activated cell sorting (MACS),5AzaD was applied to the conversion of CD4+CD25-T cells to iTregs,the expression of Foxp3 in Tregs was characterized by flow cytometry analysis before and after epigenetic modification.The purified iTregs were injected into abortion-prone mice on day 1 or 4 of pregnancy,respectively,which were used as therapy groups,and then the embryo resorption rate was counted on day 14 of pregnancy.Results:After the treatment of 5AzaD,the percentage of iTregs in CD4+T cells was (41.50±8.03)%.The embryonic absorption rates of the two therapy groups were 10.47%(on day 1 of pregnancy) and 21.69%(on day 4 of pregnancy) ,respectively ( P<0.05 ) .Conclusion: Epigenetic modication of CD4+CD25-T cells may solve the problem of nTregs deficiency,particularly adoptive therapy of 5AzaD-induced iTregs at early stage of pregnancy can maintain normal pregnancy.

Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.

Effects of 5-Aza-dC on KISS1 gene expression and the abilities of invasion and migration of human colorectal cancer cell line HCT116 / 肿瘤

Objective: To investigate the effects of methylation of KISS1 gene promoter and KISS1 expression on biological characteristics of colorectal cancer HCT116 cells induced by methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC). Methods: The promoter methylation status of KISS1 gene and its mRNA and protein expressions in HCT116 cells were detected by methylation-specific PCR, real-time fluorogenic quantitative-PCR and Western blotting, respectively; after treatment with 5-Aza-dC in vitro. The abilities of invasion and migration of HCT116 cells were detected by Transwell assay, and the cell proliferation was detected by Cell Counting Kit-8 (CCK-8). Results: The demethylation of KISS1 promoter was induced after treatment with 5-Aza-dC. The expressions of KISS1 mRNA and protein in HCT116 cells were significantly increased in a dose-dependent manner after treatment with different doses of 5-Aza-dC for 5 days as compared with those without treatment with 5-Aza-dC (P 0.05). The abilities of invasion and migration of HCT116 cells after 5-Aza-dC treatment were also reduced (P < 0.05). Conclusion: The DNA methyltransferase inhibitor 5-Aza-dC can reverse the methylation of KISS1 promoter, and reduce the abilities of invasion and migration of HCT116 cells by up-regulating the expression of KISS1. Copyright© 2014 by TUMOR.

Application of Cell Counting Kit-8 in detecting the growth inhibiting effect of 5-Aza-2 ’-deoxycytidine on chronic myeloid leukemia cells / 解剖学报

Objective To study the application of Cell Counting Kit-8(CCK-8) in detecting the growth inhibiting effect of 5-Aza-2’-deoxycytidine on chronic myeloid leukemia cell .Methods The proliferation of K562 cells was detected by CCK-8 with different concentrations of 5-Aza-2 ’-deoxycytidine and the cell cycle and apoptosis of K 562 cells were detected after K562 treated by 50% inhibitory concentration of 5-Aza-2 ’-deoxycytidine .Results The 50% inhibitory concentration of 5-Aza-2’-deoxycytidine was 15.55nmol/L, after treated with this concentration , K562 cells showed that G2 phase arrest occurred , proliferation inhibited and apoptosis peaks appeared .Conclusion Inhibition of proliferation of K562 cells with different concentrations of 5-Aza-2’-deoxycytidine varied in a dose-dependent relationship , and 5-Aza-2’-deoxycytidine could promote apoptosis of K 562 cells.CCK-8 can be used in detecting the effect of 5-Aza-2’-deoxycytidine on chronic myeloid leukemia cells .

Expression of p53 in breast cancer and regulatory effects of 5-aza-2′-deoxycytidine on p53 / 医学研究生学报

Objective Tumor suppressor gene p53 can inhibit tumor cell growth, arrest cell cycle, and promote apoptosis.Howev-er, the effects of p53 on the pathogenesis of breast cancer have not been fully elucidated.The aim of this study was to explore the expression of p53 protein and the correlation with clinical pathologic features in breast cancer.Furthermore, the regulatory effects of 5-aza-2′-deoxycyti-dine on p53 in breast cancer cell line were also studied. Methods The expression of p53 protein in 80 cases of breast cancer and normal and adjacent tissue were determined by the immunohistochemical staining .The expressions of p53 mRNA and p53 protein in breast cancer cell line were determined by RT-PCR and Western blotting. Results The positive rate of p53 in breast cancer (41.25%) was higher than that in the normal and adjacent tissue (22.5%) (P0.05).The low expression of p53 both in mRNA and in protein levels were found in breast cancer cell line of MCF-7.The expres-sion of p53 increased after 5-aza-2′-deoxycytidine administration . Conclusion p53 is highly expressed in breast cancer , which may play an im-portant role in the development and progression of breast cancer. 5-aza-2′-deoxycytidine, up-regulating the p53 expression in breast cancer cell line, which provides the evidents for the development of therapeutic drugs for the patients with low expression of p53 breast cancer.