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

Silencing DNMT1 Attenuates the Effect of WIF-1 Gene Promoter Methylation on the Biological Behavior of Chronic Myeloid Leukemia K562 Cells

目的: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。方法: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。结果: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)。结论: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。

Objective: 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. Methods: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. Results: 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). Conclusion: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.