Epigenetic regulation of microRNA-370 by interleukin-6 in malignant human cholangiocytes

Interleukin-6 (IL-6) is overexpressed and contributes to tumor cell growth in cholangiocarcinoma. Enforced IL-6 production can alter the expression of specific microRNAs (miRNAs) involved in tumor growth, and moreover can modulate expression of methylation-dependent genes. Thus, we assessed the methylation-dependent regulation of miRNA expression in human malignant cholangiocytes stably transfected to overexpress IL-6. The expression of the methyltransferases DNA methyltransferase enzyme-1 and HASJ4442 was increased by IL-6 overexpression, but was decreased by the methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR). Expression profiling identified seven miRNAs that were significantly downregulated by IL-6 overexpression (<0.4-fold) and upregulated (>2-fold) by 5-aza-CdR. One of these, miR-370, is embedded in a CpG island. Although 5-aza-CdR increased miR-370 expression by 2.1-fold in malignant cells, the expression in nonmalignant cells was unchanged. The oncogene mitogen-activated protein kinase kinase kinase 8 (MAP3K8) was identified as a target of miR-370, and its expression was decreased by 5-aza-CdR in cholangiocarcinoma cells. Overexpression of IL-6 reduced miR-370 expression and reinstated MAP3K8 expression in vitro as well as in tumor cell xenografts in vivo. Thus, IL-6 may contribute to tumor growth by modulation of expression of selected miRNAs, such as miR-370. These studies define a mechanism by which inflammation-associated cytokines can epigenetically modulate gene expression and directly contribute to tumor biology.     

Methylation-associated silencing of MicroRNA-335 contributes tumor cell invasion and migration by interacting with RASA1 in gastric cancer

MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of target genes, previous studies have shown that miR-335 play an important role in suppressing metastasis and migration in human cancer including gastric cancer (GC). However, the mechanisms which result in aberrant expression of miR-335 in GC are still unknown. Recent studies have shown that the silencing of some miRNAs is associated with DNA hypermethylation. In this study, we find the promoter of miR-335 we embedded in CpG island by accessing to bioinformatics data and the low expression of miR-335 in 5 gastric cell lines can be restored by 5-aza-2’-deoxycytidine (5-Aza-dC) treatment. So we postulated that the miR-335 genes undergo epigenetic inactivation in GC. Subsequently, in GC cells and tissues, we performed quantitative real-time PCR (RTQ-PCR) to assess the expression of miR-335, and methylation-specific PCR (MSP) and bisulfite sequence-PCR (BSP) to evaluate the DNA methylation status in the CpG islands upstream of MiR-335. The result showed that the expression of miR-335 was significantly reduce in gastric cancer cell lines and tumor tissues compared to matched normal gastric tissues, and cell lines, and which is inverse correlation with DNA hypermethylation of miR-335 both in GC cells lines and tissues, but not in normal tissues. In addition, we found that the lower miR-335 expression induced by abnormal methylation may be mainly involved in gastric cell invasion and metastasis in GC tissues. No statistical significance was found about miR-335 expression and methylation level between healthy individuals with and without H. pylori (HP) infection. Finally, we carry out miRNA transfection, RTQ-PCR and western blot assay to find the RAS p21 protein activator (GTPase activating protein) 1 (RASA1) may be the possible target genes which lead to the gastric cell invasion and metastasis, furthermore, the re-expression of endogenous miR-335 by 5-Aza-dC treatment can exert effects similar to exogenous miRNAs transfection. Taken together, our results suggest that miR-335 may be silenced by promoter hypermethylation and play important roles in gastric cell invasion and metastasis through its target genes, such as RASA1. Its methylation level might be a predictive epigenetic marker of GC and remodeling on the expression by demethylation can provided a potential therapeutic strategy.     

The DNA methyltransferase inhibitors zebularine and decitabine induce mitochondria-mediated apoptosis and DNA damage in p53 mutant leukemic T cells

DNA methyltransferase (DNMT)-inhibiting nucleoside analogs reactivate the expression of tumor suppressor genes and apoptosis-related genes silenced by methylation, thus favoring the induction of apoptosis in tumor cells. Moreover, induction of DNA damage seems to contribute to their antitumor effect. However, the apoptotic signaling pathway induced by these demethylating drugs is not well understood. Here, we have investigated the induction of apoptosis by two nucleoside DNMT inhibitors, decitabine and zebularine, in leukemic T cells. Both inhibitors induced caspase-dependent apoptosis in Jurkat, CEM-6 and MOLT-4 leukemia T cell lines, all with mutant p53, whereas resting and activated normal T lymphocytes were highly resistant to these demethylating agents. Although decitabine and zebularine showed different ability to induce apoptosis and cell cycle arrest among the three cell lines, they similarly activated the intrinsic apoptotic pathway by inducing mitochondrial alterations such as Bak activation, loss of transmembrane potential and generation of reactive oxygen species (ROS). Accordingly, Bcl-2- and Bcl-x(L) -overexpressing Jurkat cells, as well as caspase-9-deficient Jurkat cells, were resistant to apoptosis induced by decitabine and zebularine. Interestingly, ROS production seemed to be necessary for the induction of apoptosis. Apoptotic events, such as Bak and caspase activation, started as soon as 20 hr after treatment with either decitabine or zebularine. In addition, progression of apoptosis triggered by both DNMT inhibitors was paralleled by the induction of DNA damage. Our results suggest that the mitochondrial apoptotic pathway activated by decitabine and zebularine in p53 mutant leukemic T cells depends mainly on the induction of DNA damage.     

Epigenetic regulation of miR-34b and miR-129 expression in gastric cancer

MicroRNAs (miRNAs) are small noncoding RNAs that play fundamental roles in diverse biological and pathological processes by targeting the expression of specific genes. Here, we identified 38 methylation-associated miRNAs, the expression of which could be epigenetically restored by cotreatment with 5-aza-2'-deoxycytidine and trichostatin A. Among these 38 miRNAs, we further analyzed miR-34b, miR-127-3p, miR-129-3p and miR-409 because CpG islands are predicted adjacent to them. The methylation-silenced expression of these miRNAs could be reactivated in gastric cancer cells by treatment with demethylating drugs in a time-dependent manner. Analysis of the methylation status of these miRNAs showed that the upstream CpG-rich regions of mir-34b and mir-129-2 are frequently methylated in gastric cancer tissues compared to adjacent normal tissues, and their methylation status correlated inversely with their expression patterns. The expression of miR-34b and miR-129-3p was downregulated by DNA hypermethylation in primary gastric cancers, and the low expression was associated with poor clinicopathological features. In summary, our study shows that tumor-specific methylation silences miR-34b and miR-129 in gastric cancer cells.     

Investigation the Role of Autophagy in Non-Small Cell Lung Cancer

Objective: Recent studies have shown the role of autophagy in different types of cancer including lung cancer. MicroRNAs are considered as key factors in regulation of autophagy related genes. miR-30d, miR-204-5p and miR-20a are regulatory markers which can suppress the expression of beclin1, LC3, bcl2 and ULK1 as their target genes and they lead to decrement of autophagy in human cancer cells. Moreover, epigenetic modifications DNA methylation has been indicated in regulation of autophagy in different stages of cancer. Methods: In this study, the expression levels of miR-30d, miR-204-5p and miR-20a as well as their target genes were analyzed in 30 non-small cell lung cancers (NSCLCs) patients sample and adjacent normal tissues by real-time qPCR. In addition, DNA methylation of beclin1, LC3, bcl2 and ULK1 genes were assessed by MS-HRM method. Results: MiR-30d (p value= 0.01) and miR-204-5p (P=0.048) significantly down-regulated in tumor samples compared to normal adjacent tissues, while there was no significant change in expression level of miR-20a. On the other hand, target genes expression level was significantly increased in NSCLC tissues, however methylation pattern of the target gene promoters, did not show any significant alteration. Conclusion: These results indicate roles for miR-30d, miR-204-5p as tumor suppressor genes as well as target genes as oncogenes in NSCLC patients. Although these factors may have a significant role in NSCLC progression, further studies are necessary to investigate the implications of these findings for treatment of lung cancer.     

miR-152 is a tumor suppressor microRNA that is silenced by DNA hypermethylation in endometrial cancer

The etiology and development of human cancers that remain little understood might be enlightened by defining tumor suppressor microRNAs (TS-miRNA). In this study, we identified TS-miRNAs silenced by aberrant DNA hypermethylation in endometrial cancer. Functional screening of 327 synthetic miRNAs in an endometrial cancer cell proliferation assay identified 103 miRNAs that inhibited cell growth. We then determined the sequence, DNA methylation status, and expression levels of these miRNAs in endometrial cancer cell lines and primary tumors. These determinations led to the identification of miR-152 as a candidate TS-miRNA gene in endometrial cancer. Epigenetic silencing documented in miR-152 was consistent with its location at 17q21.32 in intron 1 of the COPZ2 gene, which is also silenced often in endometrial cancer by DNA hypermethylation, and also with evidence that miR-152 targets the DNA methyltransferase DNMT1. Notably, restoration of miR-152 expression in endometrial cancer cell lines was sufficient to inhibit tumor cell growth in vitro and in vivo. We identified E2F3, MET, and Rictor as novel candidate targets of miR-152, suggesting how its epigenetic silencing can drive endometrial carcinogenesis. Our findings define a central role for miR-152 in endometrial cancer, and they also suggest its use in new therapeutic strategies to treat this cancer.     

DNA methylation regulates MicroRNA expression

MicroRNAs (miRNAs), an important class of small regulatory molecules for gene expression, are transcribed by RNA polymerase II. But little is known about the mechanisms that control miRNA expression. Comparing miRNA expression profiles between colon cancer cell line HCT 116 and its derivative, DNA methyltransferase 1 and 3b (DNMT1 and DNMT3b) double knockout cell line, we found that the expression of about 10% miRNAs was regulated by DNA methylation. In addition, neither 5-aza-2'-deoxycytidine treatment nor deletion of DNMT1 alone recapitulated miRNA expression profile seen in the double knockout cell line, suggesting that miRNA expression was tightly controlled by DNA methylation and partial methylation reduction was not sufficient for miRNA reexpression. We also found that HOXA3 and HOXD10 were putative targets of mir-10a, one of the differentially expressed miRNAs that is located in HOX gene cluster.     

DNA去甲基化对急性B淋巴细胞白血病细胞株中DNA甲基转移酶基因及微RNA作用的研究

目的：探索甲基化转移酶抑制剂5-杂氮-2'-脱氧胞苷（5-Aza-CdR）对急性B淋巴细胞白血病（B-ALL）细胞株NALM-6的作用以及对细胞中微RNA（miRNA）表达水平的影响。方法用不同浓度5-Aza-CdR处理NALM-6细胞,采用四甲基偶氮唑盐（MTT）法检测细胞增殖情况,采用荧光定量反转录聚合酶链反应（RT-PCR）检测5-Aza-CdR处理后细胞DNA甲基转移酶（DNMT）基因mRNA表达水平的变化,采用miScript miRNA PCR Array芯片检测去甲基化后细胞中表达量发生改变的miRNA。结果 NALM-6细胞经不同浓度5-Aza-CdR处理不同时间后,细胞生长受抑,最高抑制率达（74.163±0.381）%。5-Aza-CdR作用浓度与DNMT基因mRNA表达水平呈反比,浓度为1000μmol／L的5-Aza-CdR作用细胞72 h后,DNMT-1的相对表达量降至0.453±0.021,DNMT-3L的相对表达量为0.003±0.001, DNMT-3B的相对表达量为0.395±0.019。miScript miRNA PCR Array筛选出3个miRNA（miR-184、miR-23a-3p、miR-34a-5p）与DNA甲基化相关。结论5-Aza-CdR可下调NALM-6细胞中DNMT基因的表达,并对细胞增殖有抑制作用。miR-184、miR-23a-3p和miR-34a-5p在B-ALL的发生、发展中与DNA甲基化相关。     

DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma

Aberrant DNA methylation plays a relevant role in multiple myeloma (MM) pathogenesis. MicroRNAs (miRNAs) are a class of small non-coding RNAs that recently emerged as master regulator of gene expression by targeting protein-coding mRNAs. However, miRNAs involvement in the regulation of the epigenetic machinery and their potential use as therapeutics in MM remain to be investigated. Here, we provide evidence that the expression of de novo DNA methyltransferases (DNMTs) is deregulated in MM cells. Moreover, we show that miR-29b targets DNMT3A and DNMT3B mRNAs and reduces global DNA methylation in MM cells. In vitro transfection of MM cells with synthetic miR-29b mimics significantly impairs cell cycle progression and also potentiates the growth-inhibitory effects induced by the demethylating agent 5-azacitidine. Most importantly, in vivo intratumor or systemic delivery of synthetic miR-29b mimics, in two clinically relevant murine models of human MM, including the SCID-synth-hu system, induces significant anti-tumor effects. All together, our findings demonstrate that aberrant DNMTs expression is efficiently modulated by tumor suppressive synthetic miR-29b mimics, indicating that methyloma modulation is a novel matter of investigation in miRNA-based therapy of MM.     

DNMT3B regulates proliferation of A549 cells through the microRNA-152-3p/NCAM1 pathway

The purpose of the present study was to examine the epigenetic mechanism by which microRNA (miR)-152-3p regulates proliferation in non-small cell lung cancer A549 cells via neural cell adhesion molecule 1 (NCAM1). Bisulfite sequencing PCR (BSP), the gold standard for methylation detection, uses bisulfite-treated DNA to determine its pattern of methylation. Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. It was conducted and demonstrated a relatively high level of methylation in the miR-152-3p promoter region. Chromatin immunoprecipitation was combined with PCR to detect the binding of DNA methyltransferase 3B (DNMT3B) protein to miR-152-3p, which tends to occur in the core region of the miR-152-3p gene in A549 cells. Luciferase assay identified NCAM1 as the target gene of miR-152-3p. MTT, colony formation and Transwell assays indicated that miR-152-3p could decrease cell proliferation and invasion and in addition to reducing the expression level of NCAM1. Overexpression of NCAM1 could attenuate the effect of miR-152-3p. DNMT3B knockdown decreased the proliferative ability of A549 cells and increased the expression of miR-152-3p, while decreased that of NCAM1. After treatment with miR-152-3p inhibitor, these effects were attenuated and the NCAM1 expression level was upregulated. The results indicated that miR-152-3p may suppress the proliferation of A549 cells by downregulating NCAM1. In addition, DNMT3B negatively regulated the expression of miR-152-3p via modulation of the methylation level in the miR-152-3p core region, thus mediating the proliferation of lung tumor cells.     

The tumor suppressive microRNA miR-218 targets the mTOR component Rictor and inhibits AKT phosphorylation in oral cancer

The incidence of oral squamous cell carcinoma (OSCC) is rising rapidly in developed countries, posing a growing challenge due to the poor management of this type of malignancy at present. In this study, we profiled tumor suppressive microRNAs (miRNAs) that are silenced by DNA hypermethylation in OSCC using a function-based screening approach. This approach employed a cell proliferation assay for 327 synthetic miRNAs in two OSCC cell lines. Among the 110 miRNAs identified in this set that exhibited inhibitory properties, we compared DNA methylation and expression status in a wider panel of OSCC cell lines and primary tumor tissues, resulting in the identification of miR-218 and miR-585 as functionally significant miRNA genes that are frequently silenced in OSCC by DNA hypermethylation. Ectopic expression of miR-218 and miR-585 in OSCC cells lacking endogenous expression reduced cell growth in part through caspase-mediated apoptosis. Notably, miR-218 reduced levels of the rapamycin-insensitive component of mTOR, Rictor, in a manner associated with a suppression of Akt S473 phosphorylation. Together our findings define miR-585 as a tumor suppressive function that is often epigenetically silenced in OSCC, and they identify Rictor as a novel target of miR-218, suggesting that activation of the mTOR-Akt signaling pathway induced by Rictor contributes centrally to oral carcinogenesis.     

miR-10a-5p基因甲基化对卵巢癌铂类耐药的影响

目的 卵巢癌是死亡率最高的妇科肿瘤,较强的化疗耐药性是其预后差的主要原因之一,为了阐明卵巢癌对铂类药物的耐药机制,本研究探讨miRNA基因的甲基化水平对卵巢癌铂类耐药的影响.方法 将卵巢癌组织分为敏感组和耐药组,每组各3例;采用基因芯片技术,对比分析了两组微RNA(microRNA,miRNA)的表达差异;采用实时荧光定量PCR,分别在6例敏感和3例耐药组织、铂类药物敏感(CoC1)和耐药的卵巢癌细胞系(CoC1/DDP),检测了候选miRNA的表达差异;应用Massarray技术,检测敏感组织(15例)与耐药组织(6例)中miRNA基因启动子的甲基化差异;应用生物信息学分析,鉴定目标miRNA的潜在靶基因.结果 以铂类药物敏感的卵巢癌组织样本为对照,利用基因芯片筛选,鉴定了6条在耐药组织样本中出现表达上调的miRNA(miR-493-3p、miR-10a-5 p、miR-16-2-3p、miR-1248、miR-451a、miR-628-3p)和6条表达下调的miRNA(miR-509-3p、miR-1197、miR-376a-3p、miR-1273a、miR-550a-3p、miR-19b-3p).组织验证发现,miR-509-3p、miR-493-3p、miR-10a-5p、miR-16-2-3p和miR-451a,与芯片结果一致;培养细胞研究发现,4条miRNA的表达调控方式与组织芯片结果一致,miR-10a-5p、miR-16-2-3p、miR-1248和miR-628-3p在耐药细胞系中高表达.进一步研究发现,与敏感肿瘤组织相比,耐药组织中miR-10a-5p基因启动子的甲基化水平出现显著降低,P=0.04.结合生物信息学预测HOXA1和USF2为miR-10a-5p与耐药相关的靶基因.结论 与敏感组相比,耐药组miR-10a-5p基因启动子甲基化水平显著降低,miR-10a-5p表达升高,通过抑制 HOXA1和USF2,抑制细胞凋亡,导致铂类化疗药物耐受.     

miR-32-5p通过靶向Dickkopf相关蛋白3的表达调控乳腺癌MDA-MB-231 细胞的生物学行为

目的：通过生物信息学手段筛选乳腺癌中差异表达的关键miRNA及其靶基因,干预其在乳腺癌细胞中的表达并观察对乳腺癌细胞功能的影响。方法：利用GEO数据库筛选在乳腺癌中差异表达的miRNA,ENCORI数据库验证差异miRNA的表达,以选定最显著的差异表达 miRNA 为研究对象;利用 Starbase、miRDB 和 miRWalk 数据库预测 miR-32-5p 的靶基因,利用DAVID数据库对靶基因进行GO分析和KEGG分析,利用String数据库联合Cytoscape3.6.2软件进行PPI网络分析及核心基因的筛选,从核心基因中选择相互联系紧密“度值”最显著的Dickkopf相关蛋白3（DDK3）基因进行后续实验。qPCR检测miR-32-5p在人正常乳腺细胞 MCF10A和人乳腺癌细胞MCF7、MDA-MB-231、MDA-MB-453细胞中的表达。向MDA-MB-231细胞中转染miR-32-5p mimic、miR-32-5p inhibitor及各自的对照（NC）序列,分别用CCK-8法、流式细胞术和Transwell实验检测过表达或抑制miR-32-5p对细胞增殖、凋亡和侵袭的影响。结果：从GEO数据库中获取的两个数据集共识别出两个差异miRNA,ENCORI数据库验证差异miRNA的表达发现miR-32-5p的表达水平与GEO数据库的结果一致,故选择其进行研究;预测得到198个miR-32-5p 潜在的靶基因并鉴定出 10 个核心基因（DKK3、WNT2B、SFRP5、SFRP2、SFRP1、LRP6、WNT6、KREMEN1、NEDD4L、TRIP12）,其中DKK3的度值最大可能在乳腺癌中较为重要,于是选择miR-32-5p/DKK3轴进行后续研究。miR-32-5p在3种乳腺癌细胞中的表达水平显著高于正常乳腺细胞（均P<0.01）,其中以MDA-MB-231细胞中表达最高。双荧光素酶基因报告实验验证了miR-32-5p与DKK3基因的靶向结合及其对后者表达的负向调控。转染miR-32-5p mimic、miR-32-5p inhibitor后成功提高或抑制了MDA-MB-231细胞中miR-32-5p的表达。与对照组相比,过表达miR-32-5p可抑制MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭（P<0.05或P<0.01）,敲低miR-32-5p则起相反的作用（均P<0.01）。结论：miR-32-5p/DKK3轴可能是影响乳腺癌发生发展的关键通路,过表达miR-32-5p能够抑制乳腺癌MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭。     

Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.     

卵巢浆液性囊腺癌化疗耐药和化疗敏感组织中的miRNAs差异表达谱检测及生物信息学分析

目的:寻找卵巢浆液性囊腺癌化疗耐药及化疗敏感组织中microRNA的差异表达,为研究上皮性卵巢癌化疗耐药产生的分子机制及逆转其化疗耐药提供理论基础和依据.方法:首先,应用microRNA表达谱基因芯片寻找卵巢浆液性囊腺癌化疗耐药及化疗敏感组织中差异表达的microRNAs;其次,选取部分差异表达的microRNAs应用RT-PCR实验检测技术在扩大的组织样本中进行验证;最后,运用生物信息学技术对符合要求的microRNAs进行相关生物信息学分析.结果:在microRNA表达谱基因芯片中筛选出与卵巢浆液性囊腺癌化疗耐药相关的miR-27a-3p、miR-141-3p、miR-200b-3p等17个表达上调的miRNAs和miR-93-3p、 miR-497-5p、miR-675-3p等15个表达下调的miRNAs.上调miRNAs预测的靶基因GO(BP模块)分析结果显示凋亡信号通路、Notch信号通路和凋亡过程,KEGG Pathway分析结果显示Wnt信号通路、PI3K-Akt信号通路和ErbB信号通路可能与化疗耐药存在相关性.下调miRNAs预测的靶基因GO(BP模块)分析结果显示负调控凋亡过程、Wnt信号通路生物学过程,KEGG Pathway分析结果显示PI3K-Akt信号通路、细胞循环可能与化疗耐药存在相关性.结论:MicroRNA表达谱基因芯片及生物信息学技术能为研究上皮性卵巢癌化疗耐药产生机制提供新的思路.miR-27a-3p、miR-141-3p、miR-200b-3p、miR-93-3p、miR--p、miR--p可能分别通过调控靶基因PRKCB、MAPK、PAK、PRKAA、CCNE、IGFR参与卵巢浆液性囊腺癌化疗耐药.     

Methylation of secreted frizzled related protein gene in acute leukemia patients in China

Background: DNA methylation of CpG islands within the promoters of specific genes may play roles in tumor initiation and progression. It has been suggested such events may serve as critical check points. Methods:The present study analyzed the methylation status of CpG islands within the promoters of secreted frizzled-related proteins (SFRPs) in 87 acute leukemia (AL) patients, 20 normal controls, and four AL cell lines. 5-aza-2’-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, was employed to determine its effect on SFRP expression. Result: Methylation of at least one SFRP promoter was observed in 69% of the AL patients analyzed. In addition, methylation of all four SFRP promoters was observed in Molt-4, Jurkat, HL60 and NB4 cells. In Jurkat cells, methylation levels of four SFRP promoters decreased in a dose-dependent manner upon treatment with 5-Aza-CdR, which coincided with increased mRNA expression. With increasing 5-Aza-CdR concentrations, the expression of DNA methyltransferases, DNMT3A and DNMT3B, significantly decreased in a dose-dependent manner. Conclusion: The present study demonstrated that SFRP gene methylation may be involved in AL progression, with a possible epigenetic mechanism influencing Wnt signaling.