[Silencing DNMT1 Attenuates the Effect of WIF-1 Gene Promoter Methylation on the Biological Behavior of Chronic Myeloid Leukemia K562 Cells].
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To investigate the effect of silencing DNA methyltransferase 1(DNMT1) to the methylation of the promoter of the tumor suppressor gene wnt-1 (WIF-1) in human chronic myeloid leukemia (CML) cells.DNMT1 siRNAi plasmid was constructed and DNMT1 siRNAi was transfected into CML K562 cells. RT-PCR and Western blot were used to detect the expression of DNMT1 gene and related protein, and methylation PCR was used to detect WIF-1 gene promoter methylation level. The trypan blue exclusion and MTT assay were used to detect the cell proliferation, flow cytometry were used to detect the cell apoptosis rate, colony formation assay was used to detect cell colony formation ability. Expression of Wnt/β- catenin and its downstream signaling pathway proteins were detected by Western blot after DNMT1 gene was silenced.The expression level of DNMT1 mRNA and its related protein in the experimental group were significantly lower than those in the control group and negative control group (P<0.05). After 72 hours of successful transfection, the WIF-1 gene in the control group and negative control group were completely methylated, while in the experimental group, the methylation level significantly decreased. The results of MSP showed that the PCR product amplified by the unmethylated WIF-1 primer in the experimental group increased significantly,while by the methylated WIF-1 primer decreased significantly. After 48 h of transfection, the OD value, viable cell number and colony formation of the cells in experimental group were significantly lower than those in the negative control group and the control group (P<0.05). The apoptosis rate of the cells in experimental group was significantly higher than those in the negative control group and control group (P<0.05). The expression levels of β- actin, myc, cyclin D1 and TCF-1 in K562 cells in the experimental group were significantly lower than those in the negative control group and control group (P<0.05).Silencing DNMT1 gene can inhibit the proliferation and promote the apoptosis of K562 cells. The mechanism may be related to reverse the hypermethylation level of the WIF-1 gene promoter, thereby inhibit the activity of the Wnt/β- catenin signaling pathway.沉默DNMT1减弱WIF-1基因启动子甲基化对慢性髓系白血病K562细胞生物学行为的影响.探讨沉默DNA甲基转移酶（DNMT1）对人慢性髓系白血病细胞内抑癌基因wnt抑制因子-1（WIF-1）基因启动子甲基化的影响。.构建DNMT1 siRNAi质粒，将DNMT1 siRNAi转染至慢性髓系白血病K562细胞中，采用RT-PCR和Western blot法检测DNMT1基因和相关蛋白的表达，并利用甲基化PCR检测WIF-1基因启动子甲基化水平，台盼蓝拒染法和MTT 法检测细胞的增殖能力，流式细胞术检测细胞的凋亡率，集落形成实验检测细胞的集落形成能力，Western blot法检测沉默DNMT1基因后 Wnt/β-catenin及其下游信号通路蛋白的表达情况。.实验组DNMT1 mRNA和相关蛋白表达水平显著低于对照组和阴性对照组（P<0.05）；成功转染72 h后，对照组和阴性对照组WIF-1基因处于完全甲基化状态，而实验组WIF-1基因启动子区域甲基化水平显著降低。MSP检测结果显示，实验组未甲基化的WIF-1引物扩增出的PCR产物含量显著增加，而甲基化的WIF-1引物扩增出的PCR产物含量显著降低。转染成功48 h后，实验组的OD值、活细胞数及集落形成数均显著低于阴性对照组和对照组（P<0.05）。实验组的细胞凋亡率显著高于阴性对照组和对照组（P<0.05）。实验组K562细胞中β-actin、myc、cyclin D1和TCF-1蛋白表达水平均显著低于阴性对照组和对照组（P<0.05。.沉默DNMT1基因能够抑制 K562 细胞增殖，促进其凋亡，其作用机制可能与逆转WIF-1基因启动子高甲基化水平，从而抑制Wnt/β-catenin信号通路的活性有关。.Keywords:
DNMT1
K562 cells
To investigate the cytidylyl phosphate guanosine (CpG) islands methylation status of E-cadherin (E-cad) promoter region in human ovarian carcinoma cell lines (ES-2,3AO,SKOV3), and the effect of 5-azacytidine-2'-deoxycytidines (5-Aza-CdR) on the cell proliferative ability, invasion and the expression of E-cad protein.Methylation specific PCR (MSP) was used to detect CpG islands methylation status of E-cad promoter region in ES-2,3AO and SKOV3 cell lines. After treated with different concentrations of 5-Aza-CdR, morphological changes of cell lines were observed under microscope. The proliferative ability was evaluated by methyl thiazolyl tetrazolium (MTT) assay. E-cad protein expression was detected by western-blot and cellular invasion was investigated by 24-well matrigel invasion chambers.Hypermethylation status of CpG islands of E-cad promoter region was observed in ES-2 and SKOV3 cell lines, but not in 3AO cell lines. After treated with 5-Aza-CdR (0.1, 1.0, 10.0 micromol/L), ES-2 and SKOV3 cell lines displayed morphological evidence of differentiation. 5-Aza-CdR was found to decrease proliferation as evidenced by cell growth curve , to increase the level of E-cad protein expression (P < 0.01), and effectively inhibit the ability of cell invasion (P < 0.01).CpG hypermethylation is an important mechanism of E-cad gene inactivation in ES-2 and SKOV3 cell lines. 5-Aza-CdR be found to inhibit proliferation and invasion, and increase the expression of E-cad probably by the inhibition of hypermethylation.
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genomic DNA
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HpaII
CpG site
Illumina Methylation Assay
Bisulfite sequencing
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Abstract The low level of DNA methylation in tumors compared to the level of DNA methylation in their normal-tissue counterparts or global DNA hypomethylation was one of the first epigenetic alterations to be found in human cancer^1,2^. While the contribution of genome hypomethylation in cancer development and progression is explained by several mechanisms: chromosomal instability, loss of imprinting, and reactivation of transposable elements^3, 4^, the molecular causes of genome hypomethylation remain unclear. Indeed, despite the central roles of the DNA methyltransferases (Dnmts) in the establishment and maintenance of the DNA methylation, no clear consensus appears between the reduction of the Dnmts expression and the genome hypomethylation in human cancers^5^. Nevertheless, the cancer-associated genome hypomethylation could be explained by the disruption of interactions existing between Dnmts and the DNA replication and DNA repair proteins because these interactions play a crucial role in the DNA methylation in mammalian cells^6-8^. We here demonstrate that the disruption of the Dnmt1/PCNA and Dnmt1/UHRF1 interactions induce the genome hypomethylation and act as oncogenic factors promoting the tumorigenesis. We also identify the Akt- and/or PKC-mediated phosphorylations of Dnmt1 as both initiators of these disruptions and as a hallmark conferring poor prognosis in glioma patients.
DNMT1
Cancer Epigenetics
Epigenomics
RNA-Directed DNA Methylation
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Secreted frizzled-related protein 2, (SFRP2) is a Wnt inhibitor whose promoter CpGs were recently found to be methylated at high frequency in colorectal cancers (CRCs). We hypothesized that the pattern of SFRP2 methylation may differ throughout the promoter during progressive tumorigenesis. Using combined bisulfite restriction analysis (COBRA), two methylation-sensitive regions (Regions A and B) of the SFRP2 promoter were investigated in 569 specimens of colorectal tissue:222 CRCs, 103 adenomatous polyps (APs), 208 normal colonic mucosa from CRC patients (N-Cs), and 36 normal colonic mucosa from subjects with no evidence of colorectal neoplasia at colonoscopy (N-Ns). Extensive (including both Regions A and B) and partial (either Region A or B) SFRP2 methylation levels were found in 61.7% and 24.8% of CRCs, 8.7% and 37.9% of APs, 3.9% and 39.9% of N-Cs, and 0% and 30.6% of N-Ns, respectively. Extensive methylation of the SFRP2 promoter was present primarily in CRCs, while partial methylation was common in APs. Whereas APs with the KRAS mutant showed no correlation to any pattern of SFRP2 methylation, extensive methylation of the SFRP2 promoter was significantly associated with KRAS mutant CRCs (p＜.0001), suggesting that genetic alteration in the RAS-RAF pathway might precede the spread of CpG methylation through the SFRP2 promoter, which is observed in over 60% of advanced colorectal tumors.
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Bisulfite sequencing
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Nuclear DNA
Bisulfite
Bisulfite sequencing
Epigenomics
Pyrosequencing
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