AIM: To study the effect of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on heat shock protein 70 (HSP70), human leucocyte antigen-Ⅰ (HLA-Ⅰ) and NY-ESO-1 proteins in exosomes produced by hepatoma cells, HepG2 and Hep3B. METH...AIM: To study the effect of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on heat shock protein 70 (HSP70), human leucocyte antigen-Ⅰ (HLA-Ⅰ) and NY-ESO-1 proteins in exosomes produced by hepatoma cells, HepG2 and Hep3B. METHODS: Exosomes derived from HepG2 and Hep3B cells treated with or without 5-aza-CdR were isolated and purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under electron microscope. Concentration of proteins in exosomes was measured by bicinchoninic acid protein assay. Expression of HSP70, HLA-Ⅰ and NY-ESO-1 proteins in exosomes was detected by Western blotting and immunoelectron microscopy. mRNA expression of p53 gene was detected by reverse transcription polymerase chain reaction.RESULTS: The mRNA expression of p53 gene was increased in both hepatoma cell lines after treatment with 5-Aza-CdR. The number of exosomes and the concentration of total proteins in exosomes were increased signifi cantly after treatment with 5-aza-CdR (P < 0.05). After treatment with 5-Aza-CdR, immunoelectron microscopy and Western blotting showed that the HSP70, HLA-Ⅰ and NY-ESO-1 proteins were increased in exosomes produced by both hepatoma cell lines. CONCLUSION: 5-aza-CdR, an inhibitor of DNA methyltransferase, can increase exosomes produced by hepatoma cells and immune-associated protein component of exosomes, which may be mediated by p53 gene upregulation and 5-Aza-CdR demethylation.展开更多
The non-classical HLA class Ⅰ antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells （DC）. As a result, HLA-G expression in malignant cells may provide them ...The non-classical HLA class Ⅰ antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells （DC）. As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2＇-deoxycytidine （5-AC） and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5＇ regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components （APM） as well as β2 microglobulin （β2-m） expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1A cells.展开更多
Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explor...Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. Methods： Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 （CCK-8） assay. The effect of drug combination was calculated by Jin＇s formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. Results： 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group （P0.05）. Conclusion： Both epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents （5-Aza-Cdr/ TSA）.展开更多
Objective： hMLH1 protein serves to detect the DNA damage caused by cisplatin （DDP） and destroys the cell. The absence of hMLH1 expression has been correlated with acquired resistance of ovarian cancer cells to plat...Objective： hMLH1 protein serves to detect the DNA damage caused by cisplatin （DDP） and destroys the cell. The absence of hMLH1 expression has been correlated with acquired resistance of ovarian cancer cells to platinum. The aim of this study was to determine the possible role of DNA methylation and histone H3 lysine 9 （H3-K9） acetylation on the loss of hMLH1 expression, and to evaluate the reversal effects of 5-Aza-2＇-deoxycytidine （5-Aza-dC） and Trichostatin A （TSA） on DDP-resistance in ovarian cancer cell lines. Methods： Two human ovarian cancer cell lines, COC1 and its DDP-resistant subline, COCI/DDP were cultured. The two cancer cells were treated with 5-Aza-dC or TSA. Using COC1 cells as a control, we used methylation-specific PCR （MSP） to analyze DNA methylation at hMLHI gene promoter, hMLH1 mRNA and protein expressions were analyzed by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blot. Chromatin immunoprecipitation assay （CHIP） was used to test the levels of histone H3-K9 acetylation at hMLH1 gene promoter. Results： In COC1 cells, there was no DNA methylation at hMLH1 gene promoter, while there were hMLH1 mRNA and protein expression. In COC1/DDP cells, there was DNA hypermethylation at hMLH1 gene promoter, while there was no hMLH1 mRNA or protein expression. The treatment with 5-Aza-dC resulted in DNA demethylation at the promoter region, as well as restoration of hMLH1 expression in COC1/DDP cells. The treatment with TSA had no effects on DNA demethylation or restoration of hMLH1 expression in COC1/DDP cells. Conclusion： Hypermethylation of DNA at the promoter is related to the silencing of hMLH1 in COC1/DDP ovarian cancer cells. DNA methylation at hMLH1 promoter could play a significant role in determining the sensitivity of ovarian cancer to DDP. The drug resistance mediated by methylation of hMLH1 could be overcome by 5-Aza-dC.展开更多
Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcino...Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcinoma cell lines SMMC-7721 and HePG2 before and after treatment with 5-Aza-cdR were analyzed via reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistrty Results: The expression levels of p16 mRNA and protein were increased dramatically after treatment with 5-Aza-cdR. Conclusion: Our data show that, 5-Aza-2’ -deoxycytidine can increase the expression of pl6 gene both at transcription and translation. The findings suggested that 5-Aza-cdR may reactivate the pl6 gene by demethylation.展开更多
The pyrimidine analog, 5-aza-2’-deoxycytidine (5-aza-dC) is a DNA methyltransferase inhibitor that triggers DNA demethylation leading to the reactivation of epigenetically silenced tumor suppressor genes. To understa...The pyrimidine analog, 5-aza-2’-deoxycytidine (5-aza-dC) is a DNA methyltransferase inhibitor that triggers DNA demethylation leading to the reactivation of epigenetically silenced tumor suppressor genes. To understand the shift in gene expression which mediates the beneficial 5-aza-dC effects in leukemia, we have treated human myeloid derived leukemic cells with 5-aza-dC. Target genes were identified first in MV4-11 cells using a genome-wide gene expression profiling assay to detect differences in treated and untreated cells. From this analysis six genes were identified (HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2) as being significantly different expressed after treatment. To validate microarray data, we performed quantitative PCR on these genes from multiple leukemic cells. The results suggest that these genes are epigenetically regulated indicating that dysregulation of HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2 expression may play an important role in establishing the malignant phenotype in AML.展开更多
The effects of 5-Aza-2＇-deoxycytidine （5-Aza-CdR） on the proliferation of MDA-MB-435S cells and the expression of tumor suppressor gene maspin were investigated. Human breast cancer cell line MDA-MB-435S was treate...The effects of 5-Aza-2＇-deoxycytidine （5-Aza-CdR） on the proliferation of MDA-MB-435S cells and the expression of tumor suppressor gene maspin were investigated. Human breast cancer cell line MDA-MB-435S was treated with 5 μmol/L 5-Aza-CdR, a specific demethylating agent for 0 to 8 days. The growth of MDA-MB-435S cells was observed by MTT assay before and after 5-Aza-CdR treatment, respectively. The expression of maspin mRNA was detected by reverse transcfiption-polymerase chain reaction （RT-PCR）. The cell cycle of MDA-MB-435S cells was analyzed by flow cytometry. The results showed that the growth of MDA-MB-435S cells treated with 5-Aza-CdR for 8 days was significantly suppressed as compared with the control groups, and the inhibition rate increased sharply from 5 day to 8 day （35.42% to 71.29%）. Flow cytometry showed that 5 μmol/L 5-Aza-CdR could induce G2/M cell cycle arrest and decrease the percentage of mitosis cell number in this cell line. Maspin mRNA was expressed in MDA-MB-435S cells after 5-Aza-CdR treatment, but it was weakly detectable before the treatment. It was concluded that Maspin gene might be transcriptional silencing by hypermethylation and the re-expression of maspin gene by 5-Aza-CdR can inhibit the proliferation and induce the G2/M arrest of MDA-MB-435S breast cancer cells.展开更多
[目的]探讨暴露于DNA甲基化抑制剂5-Aza-2’-deoxycytidine(5-Aza-CdR)对新生SD大鼠生长发育和成年精子发生的影响。[方法]新生大鼠随机分为3组,每组24只雄鼠。自出生第3天(postnatal day 3,PND 3)开始经口给予5-Aza-CdR 25、250μg/kg...[目的]探讨暴露于DNA甲基化抑制剂5-Aza-2’-deoxycytidine(5-Aza-CdR)对新生SD大鼠生长发育和成年精子发生的影响。[方法]新生大鼠随机分为3组,每组24只雄鼠。自出生第3天(postnatal day 3,PND 3)开始经口给予5-Aza-CdR 25、250μg/kg,对照组给予等量的溶剂。连续暴露5d,最后一次暴露结束后24h,处死半数雄鼠(幼鼠)。剩余部分继续喂养至12周龄(成鼠),乙醚麻醉。取睾丸组织做病理学检查、精子头计数等,附睾做精子畸形检查。[结果]随着剂量的增加,幼鼠体重出现下降趋势,与对照组比较差异有统计学意义(P<0.05)。组织病理学发现幼鼠睾丸组织中出现空泡变性。检查发现染毒结束后继续喂养至12周的成鼠睾丸组织无明显形态和组织学变化;但每克睾丸组织精子头计数及每日精子生成量随幼年时暴露剂量增加呈现下降趋势(P<0.05),而精子畸形率随剂量增加呈现上升趋势(P<0.05)。[结论]新生大鼠对DNA甲基化抑制制5-Aza-CdR生殖毒性作用敏感,低剂量短时间的暴露即可引起成年期生殖功能的异常。展开更多
Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate t...Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate the role of MBD2 in regulating Th2 cell differentiation.Methods:Splenic mononuclear cells were extracted from C57BL/6 mice,and CD4+T cells were isolated using magnetic beads and confirmed through flow cytometry.Lentivirus was employed to construct MBD2-silenced CD4+T cells.In vitro experiments were performed to treat splenogenic mononuclear cells and CD4+T cells with Ovalbumin(OVA),and Th2 cell ratios and IL-4 levels were assessed using flow cytometry and ELISA.Results:The purity of the isolated CD4+T cells was 95.73%,confirming successful isolation of primary CD4+T cells.Compared to the control group,the Th2 cell ratio exhibited an increase in the Th2-induced group.Treatment with 5-Aza(concentrations,1-100μM)promoted Th2 cell differentiation and increased IL-4 levels.Notably,when combined with Th2 induction and 10μM 5-Aza treatment,silencing MBD2 further amplified Th2 cell ratios and elevated IL-4 levels in cell supernatants.Furthermore,OVA(concentration,200μg/mL)induced the differentiation of CD4+T cells into Th2 cells and increased IL-4 secretion.Interestingly,silencing MBD2 significantly increased the Th2 cell ratio and IL-4 levels in OVA-treated CD4+T cells.Conclusion:In summary,OVA promoted CD4+T cell differentiation into Th2 cells and enhanced IL-4 levels.MBD2 was identified as a mediator of Th2 cell differentiation in splenic-derived CD4+T cells,influenced by OVA or 5-Aza treatment.展开更多
Background Aberrant DNA methylation plays a key role in human carcinogenesis. 5-aza-2＇-deoxycytidine inhibits DNA methylation and induces the expression of genes putatively silenced by promoter methylation in vitro. ...Background Aberrant DNA methylation plays a key role in human carcinogenesis. 5-aza-2＇-deoxycytidine inhibits DNA methylation and induces the expression of genes putatively silenced by promoter methylation in vitro. There are few studies of the biological and clinical significance of 5-aza-2＇-deoxycytidine in human hepatocellular carcinoma. This study explored the mechanism of 5-aza-2＇-deoxy.cytidine targeting transcriptional repressor complexes affecting global gene expression in hepatocellular carcinoma cell line. Methods High density oligonucleotide gene expression microarrays were used to examine the effects of 5-aza-2＇-deoxycytidine treatments on human hepatocellular carcinoma cell line SMMC-7721. The 5＇ ends of the genes upregulated or downregulated in this manner were compared with BLAST database to determine whether they might have promoter CpG islands. Flow cytometry was used to detect stages of the cell cycle and apoptosis of SMMC-7721 after being treated with 5-aza-2＇-deoxycytidine. Results Data obtained 3 days after 4 days of treatment with 5-aza-2＇-deoxycytidine showed that more genes were induced in tumorigenic cells including genes that function in cell proliferation, differentiation, regulation of transcription, and cytokine signalling. Approximately 30% of induced genes did not have CpG islands within their 5＇ regions, suggesting that some genes activated by 5-aza-2＇-deoxycytidine may not result from the direct inhibition of promoter methylation. This phenomenon may contribute to a number of upregulated genes involving regulation of transcription in the treated cell. Results showed that 100 lumol/L 5-aza-2＇-deoxycytidine blocked cell cycle at S/G2-M phase increasing rate of apoptosis. Notably, we found differential expression of molecular action in the methylation although DNA methyltransferases did not show significant difference in the treated cell line. Conclusion 5-aza-2＇-deoxycytidine could restore some silenced genes expression independently of DNA rnethylation inhibition and expression of DNA methyltransferases.展开更多
Phthalates are a large family of ubiquitous environmental pollutants suspected of being endocrine disruptors. Epidemiological studies have associated phthalate metabolites with decreased reproductive parameters and li...Phthalates are a large family of ubiquitous environmental pollutants suspected of being endocrine disruptors. Epidemiological studies have associated phthalate metabolites with decreased reproductive parameters and linked phthalate exposure with the level of urinary 5-methyl-2′-deoxycytidine（5mdC, a product of methylated DNA）. In this study, adult male mice were exposed to 450 mg di-isobutyl phthalate（DiBP）/（kg·day） via dietary exposure for 28 days. Mono-isobutyl phthalate（Mi BP, the urinary metabolite） and reproductive function parameters were determined. The levels of 5mdC and 5-hydroxymethyl-2′-deoxycytidine（5hmdC） were measured in urine to evaluate if their contents were also altered by DiBP exposure in this animal model. Results showed that DiBP exposure led to a significant increase in the urinary 5mdC level and significant decreases in sperm concentration and motility in the epididymis, accompanied with reduced testosterone levels and downregulation of the P450 cholesterol side-chain cleavage enzyme（P450scc） gene in the mice testes. Our findings indicated that exposure to DiBP increased the urinary 5mdC levels,which supported our recent epidemiological study about the associations of urinary 5mdC with phthalate exposure in the male human population. In addition, DiBP exposure impaired male reproductive function, possibly by disturbing testosterone levels; P450scc might be a major steroidogenic enzyme targeted by DiBP or other phthalates.展开更多
Background: Telomere length dysregulation plays a major role in cancer development and aging. Telomeres are maintained by a group of specialized genes known as shelterin and shelterin-associated proteins. In breast ca...Background: Telomere length dysregulation plays a major role in cancer development and aging. Telomeres are maintained by a group of specialized genes known as shelterin and shelterin-associated proteins. In breast cancer lines it has been shown that shelterin proteins are dysregulated thereby affecting the telomere stability and contributing to the neoplastic conversion of the mammary epithelial cells. Interestingly, the regulation of some of the shelterin genes is thought to be controlled epigenetically. Methods and Results: In this study, we set out to measure the effect of increased shelterin gene expression on telomere length in breast cancer cell line 21NT treated with 5-aza-2-deoxycytidine (5-aza-CdR) using known telomere length assays. We measured telomere lengths using: Telomere Restriction Fragment length (TRF), absolute quantitative-PCR and cytogenetic Interphase Quantitative Fluorescent in situ Hybridization (iQ-FISH). We found that non-cytotoxic levels of 5-aza-CdR affect telomere lengths by causing a significant and stable increase in telomere lengths of the breast cancer cell line. The increase in telomere lengths was consistently observed when various telomere length methods were used. Conclusions: Further investigation is required to understand the underlying mechanism involved, and the significance of telomere length elongation in relation to clinical outcome when epigenetic modifying drugs are utilized.展开更多
Cerebral ischemia/reperfusion injury impairs learning and memory in patients.Studies have shown that synaptic function is involved in the formation and development of memory,and that DNA methylation plays a key role i...Cerebral ischemia/reperfusion injury impairs learning and memory in patients.Studies have shown that synaptic function is involved in the formation and development of memory,and that DNA methylation plays a key role in the regulation of learning and memory.To investigate the role of DNA hypomethylation in cerebral ischemia/reperfusion injury,in this study,we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery and then treated the rats with intraperitoneal 5-aza-2′-deoxycytidine,an inhibitor of DNA methylation.Our results showed that 5-aza-2′-deoxycytidine markedly improved the neurological function,and cognitive,social and spatial memory abilities,and dose-dependently increased the synaptic density and the expression of SYP and SHANK2 proteins in the hippocampus in a dose-dependent manner in rats with cerebral ischemia/reperfusion injury.The effects of 5-aza-2′-deoxycytidine were closely related to its reduction of genomic DNA methylation and DNA methylation at specific sites of the Syp and Shank2 genes in rats with cerebral ischemia/reperfusion injury.These findings suggest that inhibition of DNA methylation by 5-aza-2′-deoxycytidine promotes the recovery of learning and memory impairment in a rat model of cerebral ischemia/reperfusion injury.These results provide theoretical evidence for stroke treatment using epigenetic methods.展开更多
Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are involved in neuroprotection and mitigating endoplasmic reticulum (ER) stress in the brain and peripheral ...Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are involved in neuroprotection and mitigating endoplasmic reticulum (ER) stress in the brain and peripheral organs. In earlier work, an increase in histone acetylation, following treatment with an epigenetic modulator, valproic acid, was associated with induction of CDNF and MANF in cultured cells and rat brain. These findings prompted an investigation of the effects of DNA methyltransferase (DNMT) inhibitors, which can alter epigenetic function, on the expression of CDNF and MANF. Rat C6 glioma cells were treated with a micromolar range of DNMT inhibitors: 5-aza-2’-deoxycytidine (DAC or decitabine), 5-azacytidine (AZA) or zebularine (ZEB) for 24 h. Subsequently, qPCR analysis was used to examine the mRNA expression of DNMT1, ten-eleven translocation methylcytosine dioxygenase 2 (TET-2), CDNF and MANF. A significant dose-dependent decrease in DNMT1 mRNA levels, together with a significant increase in TET-2 expression, was observed following treatment with AZA or DAC. Importantly, DAC, AZA and ZEB caused a significant dose-dependent increase in CDNF mRNA levels. In contrast, MANF mRNA expression decreased following treatment with AZA, with no significant effects observed with DAC or ZEB. Western analysis revealed no significant changes in CDNF protein levels following treatment with DAC for 24 h. The significant increase in CDNF expression, following treatment with DNMT1 inhibitors, suggests that DNA methylation is involved in the regulation of this neurotrophic factor. Clarification of the epigenetic or other mechanisms underlying the regulation of CDNF may provide novel therapeutic approaches in neurodegenerative and ER stress-related disorders.展开更多
Objective： To evaluate the expression of p16INK4A gene in ovarian cancer and analyze the relation between this alteration and the promoter methylation of p16INK4A DNA. Methods： Seven ovarian cancer cell lines and ei...Objective： To evaluate the expression of p16INK4A gene in ovarian cancer and analyze the relation between this alteration and the promoter methylation of p16INK4A DNA. Methods： Seven ovarian cancer cell lines and eighteen ovarian cancer specimens were selected for the study. Genomic DNA and RNA were extracted from fresh tissues and cell lines, DNA was treated with sodium bisulfite and then analyzed with methylation-specific PCR （MSP） to detect p16INK4A methylation. The expression of p16INK4A mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）. In addition, the proliferation of methylated cell lines before and after treatment of demethylating agent 5-Aza-2＇-deoxycytidine （5-ADC） was examined with 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay in vivo. Results： Compared with the control, the expression of p16INK4A mRNA decreased significantly or absolutely defaulted in 10 of 18 （55.56%） ovarian cancer specimens and 71.4% （5/7） ovarian cancer cell lines （P〈0.05）, and the expression of p16INK4A protein also decreased （P〈0.05）. The decrease of p16INK4A was due, in part, to p16INK4A methylation, which was found in the first exon of three cell lines and six ovarian cancer specimens and the rate was 42.86% and 33.33% in ovarian cancer cell lines and specimens respectively. All the methylated cells and tissues showed expression defect of p16INK4A, but the treatment of 5-ADC reactivated the expression of p16INK4A in methylated cells and decreased the proliferation of tumor cells in vitro and in vivo. Conclusion： The expression defect of p16INK4A gene possibly has an important role in the development of ovarian cancer, and this alteration is due, in part, to the methylation of the first exon in p16INK4A.展开更多
基金Supported by Capital Medical Development Scientif ic Research Fund, No. 2005-3086
文摘AIM: To study the effect of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on heat shock protein 70 (HSP70), human leucocyte antigen-Ⅰ (HLA-Ⅰ) and NY-ESO-1 proteins in exosomes produced by hepatoma cells, HepG2 and Hep3B. METHODS: Exosomes derived from HepG2 and Hep3B cells treated with or without 5-aza-CdR were isolated and purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under electron microscope. Concentration of proteins in exosomes was measured by bicinchoninic acid protein assay. Expression of HSP70, HLA-Ⅰ and NY-ESO-1 proteins in exosomes was detected by Western blotting and immunoelectron microscopy. mRNA expression of p53 gene was detected by reverse transcription polymerase chain reaction.RESULTS: The mRNA expression of p53 gene was increased in both hepatoma cell lines after treatment with 5-Aza-CdR. The number of exosomes and the concentration of total proteins in exosomes were increased signifi cantly after treatment with 5-aza-CdR (P < 0.05). After treatment with 5-Aza-CdR, immunoelectron microscopy and Western blotting showed that the HSP70, HLA-Ⅰ and NY-ESO-1 proteins were increased in exosomes produced by both hepatoma cell lines. CONCLUSION: 5-aza-CdR, an inhibitor of DNA methyltransferase, can increase exosomes produced by hepatoma cells and immune-associated protein component of exosomes, which may be mediated by p53 gene upregulation and 5-Aza-CdR demethylation.
文摘The non-classical HLA class Ⅰ antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells （DC）. As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2＇-deoxycytidine （5-AC） and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5＇ regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components （APM） as well as β2 microglobulin （β2-m） expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1A cells.
基金supported by the National Natural Science Foundation of China (No.30772407)
文摘Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. Methods： Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 （CCK-8） assay. The effect of drug combination was calculated by Jin＇s formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. Results： 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group （P0.05）. Conclusion： Both epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents （5-Aza-Cdr/ TSA）.
文摘Objective： hMLH1 protein serves to detect the DNA damage caused by cisplatin （DDP） and destroys the cell. The absence of hMLH1 expression has been correlated with acquired resistance of ovarian cancer cells to platinum. The aim of this study was to determine the possible role of DNA methylation and histone H3 lysine 9 （H3-K9） acetylation on the loss of hMLH1 expression, and to evaluate the reversal effects of 5-Aza-2＇-deoxycytidine （5-Aza-dC） and Trichostatin A （TSA） on DDP-resistance in ovarian cancer cell lines. Methods： Two human ovarian cancer cell lines, COC1 and its DDP-resistant subline, COCI/DDP were cultured. The two cancer cells were treated with 5-Aza-dC or TSA. Using COC1 cells as a control, we used methylation-specific PCR （MSP） to analyze DNA methylation at hMLHI gene promoter, hMLH1 mRNA and protein expressions were analyzed by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blot. Chromatin immunoprecipitation assay （CHIP） was used to test the levels of histone H3-K9 acetylation at hMLH1 gene promoter. Results： In COC1 cells, there was no DNA methylation at hMLH1 gene promoter, while there were hMLH1 mRNA and protein expression. In COC1/DDP cells, there was DNA hypermethylation at hMLH1 gene promoter, while there was no hMLH1 mRNA or protein expression. The treatment with 5-Aza-dC resulted in DNA demethylation at the promoter region, as well as restoration of hMLH1 expression in COC1/DDP cells. The treatment with TSA had no effects on DNA demethylation or restoration of hMLH1 expression in COC1/DDP cells. Conclusion： Hypermethylation of DNA at the promoter is related to the silencing of hMLH1 in COC1/DDP ovarian cancer cells. DNA methylation at hMLH1 promoter could play a significant role in determining the sensitivity of ovarian cancer to DDP. The drug resistance mediated by methylation of hMLH1 could be overcome by 5-Aza-dC.
文摘Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcinoma cell lines SMMC-7721 and HePG2 before and after treatment with 5-Aza-cdR were analyzed via reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistrty Results: The expression levels of p16 mRNA and protein were increased dramatically after treatment with 5-Aza-cdR. Conclusion: Our data show that, 5-Aza-2’ -deoxycytidine can increase the expression of pl6 gene both at transcription and translation. The findings suggested that 5-Aza-cdR may reactivate the pl6 gene by demethylation.
文摘The pyrimidine analog, 5-aza-2’-deoxycytidine (5-aza-dC) is a DNA methyltransferase inhibitor that triggers DNA demethylation leading to the reactivation of epigenetically silenced tumor suppressor genes. To understand the shift in gene expression which mediates the beneficial 5-aza-dC effects in leukemia, we have treated human myeloid derived leukemic cells with 5-aza-dC. Target genes were identified first in MV4-11 cells using a genome-wide gene expression profiling assay to detect differences in treated and untreated cells. From this analysis six genes were identified (HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2) as being significantly different expressed after treatment. To validate microarray data, we performed quantitative PCR on these genes from multiple leukemic cells. The results suggest that these genes are epigenetically regulated indicating that dysregulation of HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2 expression may play an important role in establishing the malignant phenotype in AML.
基金This project was supported by a grant from Hubei Eleventh-Five Major Scientific Key Program (No 2006AA301A05)
文摘The effects of 5-Aza-2＇-deoxycytidine （5-Aza-CdR） on the proliferation of MDA-MB-435S cells and the expression of tumor suppressor gene maspin were investigated. Human breast cancer cell line MDA-MB-435S was treated with 5 μmol/L 5-Aza-CdR, a specific demethylating agent for 0 to 8 days. The growth of MDA-MB-435S cells was observed by MTT assay before and after 5-Aza-CdR treatment, respectively. The expression of maspin mRNA was detected by reverse transcfiption-polymerase chain reaction （RT-PCR）. The cell cycle of MDA-MB-435S cells was analyzed by flow cytometry. The results showed that the growth of MDA-MB-435S cells treated with 5-Aza-CdR for 8 days was significantly suppressed as compared with the control groups, and the inhibition rate increased sharply from 5 day to 8 day （35.42% to 71.29%）. Flow cytometry showed that 5 μmol/L 5-Aza-CdR could induce G2/M cell cycle arrest and decrease the percentage of mitosis cell number in this cell line. Maspin mRNA was expressed in MDA-MB-435S cells after 5-Aza-CdR treatment, but it was weakly detectable before the treatment. It was concluded that Maspin gene might be transcriptional silencing by hypermethylation and the re-expression of maspin gene by 5-Aza-CdR can inhibit the proliferation and induce the G2/M arrest of MDA-MB-435S breast cancer cells.
文摘[目的]探讨暴露于DNA甲基化抑制剂5-Aza-2’-deoxycytidine(5-Aza-CdR)对新生SD大鼠生长发育和成年精子发生的影响。[方法]新生大鼠随机分为3组,每组24只雄鼠。自出生第3天(postnatal day 3,PND 3)开始经口给予5-Aza-CdR 25、250μg/kg,对照组给予等量的溶剂。连续暴露5d,最后一次暴露结束后24h,处死半数雄鼠(幼鼠)。剩余部分继续喂养至12周龄(成鼠),乙醚麻醉。取睾丸组织做病理学检查、精子头计数等,附睾做精子畸形检查。[结果]随着剂量的增加,幼鼠体重出现下降趋势,与对照组比较差异有统计学意义(P<0.05)。组织病理学发现幼鼠睾丸组织中出现空泡变性。检查发现染毒结束后继续喂养至12周的成鼠睾丸组织无明显形态和组织学变化;但每克睾丸组织精子头计数及每日精子生成量随幼年时暴露剂量增加呈现下降趋势(P<0.05),而精子畸形率随剂量增加呈现上升趋势(P<0.05)。[结论]新生大鼠对DNA甲基化抑制制5-Aza-CdR生殖毒性作用敏感,低剂量短时间的暴露即可引起成年期生殖功能的异常。
基金supported by grants from the National Natural Science Foundation of China(Nos.81760009 and 81560007).
文摘Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate the role of MBD2 in regulating Th2 cell differentiation.Methods:Splenic mononuclear cells were extracted from C57BL/6 mice,and CD4+T cells were isolated using magnetic beads and confirmed through flow cytometry.Lentivirus was employed to construct MBD2-silenced CD4+T cells.In vitro experiments were performed to treat splenogenic mononuclear cells and CD4+T cells with Ovalbumin(OVA),and Th2 cell ratios and IL-4 levels were assessed using flow cytometry and ELISA.Results:The purity of the isolated CD4+T cells was 95.73%,confirming successful isolation of primary CD4+T cells.Compared to the control group,the Th2 cell ratio exhibited an increase in the Th2-induced group.Treatment with 5-Aza(concentrations,1-100μM)promoted Th2 cell differentiation and increased IL-4 levels.Notably,when combined with Th2 induction and 10μM 5-Aza treatment,silencing MBD2 further amplified Th2 cell ratios and elevated IL-4 levels in cell supernatants.Furthermore,OVA(concentration,200μg/mL)induced the differentiation of CD4+T cells into Th2 cells and increased IL-4 secretion.Interestingly,silencing MBD2 significantly increased the Th2 cell ratio and IL-4 levels in OVA-treated CD4+T cells.Conclusion:In summary,OVA promoted CD4+T cell differentiation into Th2 cells and enhanced IL-4 levels.MBD2 was identified as a mediator of Th2 cell differentiation in splenic-derived CD4+T cells,influenced by OVA or 5-Aza treatment.
基金National Natural Science Foundation of China(No. 30470950)Chinese Postdoctoral Science Foundation (No. 2003-2004).
文摘Background Aberrant DNA methylation plays a key role in human carcinogenesis. 5-aza-2＇-deoxycytidine inhibits DNA methylation and induces the expression of genes putatively silenced by promoter methylation in vitro. There are few studies of the biological and clinical significance of 5-aza-2＇-deoxycytidine in human hepatocellular carcinoma. This study explored the mechanism of 5-aza-2＇-deoxy.cytidine targeting transcriptional repressor complexes affecting global gene expression in hepatocellular carcinoma cell line. Methods High density oligonucleotide gene expression microarrays were used to examine the effects of 5-aza-2＇-deoxycytidine treatments on human hepatocellular carcinoma cell line SMMC-7721. The 5＇ ends of the genes upregulated or downregulated in this manner were compared with BLAST database to determine whether they might have promoter CpG islands. Flow cytometry was used to detect stages of the cell cycle and apoptosis of SMMC-7721 after being treated with 5-aza-2＇-deoxycytidine. Results Data obtained 3 days after 4 days of treatment with 5-aza-2＇-deoxycytidine showed that more genes were induced in tumorigenic cells including genes that function in cell proliferation, differentiation, regulation of transcription, and cytokine signalling. Approximately 30% of induced genes did not have CpG islands within their 5＇ regions, suggesting that some genes activated by 5-aza-2＇-deoxycytidine may not result from the direct inhibition of promoter methylation. This phenomenon may contribute to a number of upregulated genes involving regulation of transcription in the treated cell. Results showed that 100 lumol/L 5-aza-2＇-deoxycytidine blocked cell cycle at S/G2-M phase increasing rate of apoptosis. Notably, we found differential expression of molecular action in the methylation although DNA methyltransferases did not show significant difference in the treated cell line. Conclusion 5-aza-2＇-deoxycytidine could restore some silenced genes expression independently of DNA rnethylation inhibition and expression of DNA methyltransferases.
基金supported by the National Basic Research Program of China (973No.2013CB945004)the National Natural Science Foundation of China (No.21477127)
文摘Phthalates are a large family of ubiquitous environmental pollutants suspected of being endocrine disruptors. Epidemiological studies have associated phthalate metabolites with decreased reproductive parameters and linked phthalate exposure with the level of urinary 5-methyl-2′-deoxycytidine（5mdC, a product of methylated DNA）. In this study, adult male mice were exposed to 450 mg di-isobutyl phthalate（DiBP）/（kg·day） via dietary exposure for 28 days. Mono-isobutyl phthalate（Mi BP, the urinary metabolite） and reproductive function parameters were determined. The levels of 5mdC and 5-hydroxymethyl-2′-deoxycytidine（5hmdC） were measured in urine to evaluate if their contents were also altered by DiBP exposure in this animal model. Results showed that DiBP exposure led to a significant increase in the urinary 5mdC level and significant decreases in sperm concentration and motility in the epididymis, accompanied with reduced testosterone levels and downregulation of the P450 cholesterol side-chain cleavage enzyme（P450scc） gene in the mice testes. Our findings indicated that exposure to DiBP increased the urinary 5mdC levels,which supported our recent epidemiological study about the associations of urinary 5mdC with phthalate exposure in the male human population. In addition, DiBP exposure impaired male reproductive function, possibly by disturbing testosterone levels; P450scc might be a major steroidogenic enzyme targeted by DiBP or other phthalates.
文摘Background: Telomere length dysregulation plays a major role in cancer development and aging. Telomeres are maintained by a group of specialized genes known as shelterin and shelterin-associated proteins. In breast cancer lines it has been shown that shelterin proteins are dysregulated thereby affecting the telomere stability and contributing to the neoplastic conversion of the mammary epithelial cells. Interestingly, the regulation of some of the shelterin genes is thought to be controlled epigenetically. Methods and Results: In this study, we set out to measure the effect of increased shelterin gene expression on telomere length in breast cancer cell line 21NT treated with 5-aza-2-deoxycytidine (5-aza-CdR) using known telomere length assays. We measured telomere lengths using: Telomere Restriction Fragment length (TRF), absolute quantitative-PCR and cytogenetic Interphase Quantitative Fluorescent in situ Hybridization (iQ-FISH). We found that non-cytotoxic levels of 5-aza-CdR affect telomere lengths by causing a significant and stable increase in telomere lengths of the breast cancer cell line. The increase in telomere lengths was consistently observed when various telomere length methods were used. Conclusions: Further investigation is required to understand the underlying mechanism involved, and the significance of telomere length elongation in relation to clinical outcome when epigenetic modifying drugs are utilized.
基金supported by the National Natural Science Foundation of China,No.82101567Doctoral Start-up Foundation of Liaoning Province,No.2021-BS-111345 Talent Project of Shengjing Hospital of China Medical University,No.M0673(all to XYF)。
文摘Cerebral ischemia/reperfusion injury impairs learning and memory in patients.Studies have shown that synaptic function is involved in the formation and development of memory,and that DNA methylation plays a key role in the regulation of learning and memory.To investigate the role of DNA hypomethylation in cerebral ischemia/reperfusion injury,in this study,we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery and then treated the rats with intraperitoneal 5-aza-2′-deoxycytidine,an inhibitor of DNA methylation.Our results showed that 5-aza-2′-deoxycytidine markedly improved the neurological function,and cognitive,social and spatial memory abilities,and dose-dependently increased the synaptic density and the expression of SYP and SHANK2 proteins in the hippocampus in a dose-dependent manner in rats with cerebral ischemia/reperfusion injury.The effects of 5-aza-2′-deoxycytidine were closely related to its reduction of genomic DNA methylation and DNA methylation at specific sites of the Syp and Shank2 genes in rats with cerebral ischemia/reperfusion injury.These findings suggest that inhibition of DNA methylation by 5-aza-2′-deoxycytidine promotes the recovery of learning and memory impairment in a rat model of cerebral ischemia/reperfusion injury.These results provide theoretical evidence for stroke treatment using epigenetic methods.
文摘Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are involved in neuroprotection and mitigating endoplasmic reticulum (ER) stress in the brain and peripheral organs. In earlier work, an increase in histone acetylation, following treatment with an epigenetic modulator, valproic acid, was associated with induction of CDNF and MANF in cultured cells and rat brain. These findings prompted an investigation of the effects of DNA methyltransferase (DNMT) inhibitors, which can alter epigenetic function, on the expression of CDNF and MANF. Rat C6 glioma cells were treated with a micromolar range of DNMT inhibitors: 5-aza-2’-deoxycytidine (DAC or decitabine), 5-azacytidine (AZA) or zebularine (ZEB) for 24 h. Subsequently, qPCR analysis was used to examine the mRNA expression of DNMT1, ten-eleven translocation methylcytosine dioxygenase 2 (TET-2), CDNF and MANF. A significant dose-dependent decrease in DNMT1 mRNA levels, together with a significant increase in TET-2 expression, was observed following treatment with AZA or DAC. Importantly, DAC, AZA and ZEB caused a significant dose-dependent increase in CDNF mRNA levels. In contrast, MANF mRNA expression decreased following treatment with AZA, with no significant effects observed with DAC or ZEB. Western analysis revealed no significant changes in CDNF protein levels following treatment with DAC for 24 h. The significant increase in CDNF expression, following treatment with DNMT1 inhibitors, suggests that DNA methylation is involved in the regulation of this neurotrophic factor. Clarification of the epigenetic or other mechanisms underlying the regulation of CDNF may provide novel therapeutic approaches in neurodegenerative and ER stress-related disorders.
基金This work was supported by grant from the National Natural Science Foundation of China (No. 30070786)as well as Scientific and Technological Development Plan of Hubei Province of China
文摘Objective： To evaluate the expression of p16INK4A gene in ovarian cancer and analyze the relation between this alteration and the promoter methylation of p16INK4A DNA. Methods： Seven ovarian cancer cell lines and eighteen ovarian cancer specimens were selected for the study. Genomic DNA and RNA were extracted from fresh tissues and cell lines, DNA was treated with sodium bisulfite and then analyzed with methylation-specific PCR （MSP） to detect p16INK4A methylation. The expression of p16INK4A mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）. In addition, the proliferation of methylated cell lines before and after treatment of demethylating agent 5-Aza-2＇-deoxycytidine （5-ADC） was examined with 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay in vivo. Results： Compared with the control, the expression of p16INK4A mRNA decreased significantly or absolutely defaulted in 10 of 18 （55.56%） ovarian cancer specimens and 71.4% （5/7） ovarian cancer cell lines （P〈0.05）, and the expression of p16INK4A protein also decreased （P〈0.05）. The decrease of p16INK4A was due, in part, to p16INK4A methylation, which was found in the first exon of three cell lines and six ovarian cancer specimens and the rate was 42.86% and 33.33% in ovarian cancer cell lines and specimens respectively. All the methylated cells and tissues showed expression defect of p16INK4A, but the treatment of 5-ADC reactivated the expression of p16INK4A in methylated cells and decreased the proliferation of tumor cells in vitro and in vivo. Conclusion： The expression defect of p16INK4A gene possibly has an important role in the development of ovarian cancer, and this alteration is due, in part, to the methylation of the first exon in p16INK4A.
