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

Abstract

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.