[Role of DNMT3a in Hydroquinone-Induced Hematopoietic Stem Cell Toxicity].

OBJECTIVE: To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity. METHODS: Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively. RESULTS: Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001). CONCLUSION: DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.

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

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/ß- 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 ß- 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/ß- catenin signaling pathway.

[Effects of WNK1 on Human Chronic Myeloid Leukemia K562 Cells via MAPK7 Phosphorylation and Its Relative Mechanism].

OBJECTIVE: To investigate the biological effects and mechanism of WNK1 on K562 cells through regulating MAPK7. METHODS: Cells were routinely cultured in vitro, the expression of WNK1 and MAPK7 in different blood tumor cell lines was analyzed by RT-qPCR and Western blot analysis.K562 cells were transfected with siRNA-WNK1 lentivirus.The effect of WNK1 on K562 cell proliferation was analyzed by using CCK-8 reagent.And K562 cell apoptosis was analyzed by using flow cytometry. The expression level of phosphorylated MAPK7 protein and total MAPK7 protein in K562 cells was analyzed by Western blot. RESULTS: The mRNA and protein of WNK1 were highly expressed in HL60, THP-1, U266 and K562 cells, however, the expressions were the highest in K562 cells (P<0.05), while the changes of mRNA and protein expressions of MAPK7 were not significantly in HL60, THP-1, U266 and K562 cells (P>0.05), but the phosphorylated MAPK7 expression was the highest in K562 cells (P<0.05). Proliferation of K562 cells transfected by WNK siRNA was significantly suppressed, and apoptosis was obviously increased (P<0.05). And the pMAPK7 protein expression in K562 cells transfected by WNK1 siRNA significantly decreased (P<0.05), however, the total MAPK7 protein expression in K562 cells showed no obvious change (P>0.05). CONCLUSION: WNK1 is highly expressed in K562 cells, which can promote the proliferation of K562 cells and reduce apoptosis probably by promoting phosphorylation of its downstream MAPK7.