Relationship between DNMT1 and Methylation of SHP-1 Promoter 2 in K562 Cells / 中国实验血液学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the relationship of DNA methyltransferase 1 ( DNMT1 ) with hematopoietic cell phosphatase (SHP-1) gene expression and promoter 2 methylation status in cell line K562.</p><p><b>METHODS</b>The promoter sequence of SHP-1 gene promoter 2 in NCBI database was analyzed, the K562 cells were transfected with the lentiviral plasmids-the specified retroviral vector psiHIV-mU6-shDNMT1 and psiHIV-mU6-mcherryFP-control. The methylation status of SHP-1 gene promoter 2 in K562 cells was detected by methylation-specific polymerase chain reaction (MSP) and bisulfite-modified sequencing (BSP). Western blot was used to detect the protein expression level of SHP-1 and DNMT1, the SYBR Green fluorescence quantitative PCR was used to detect the expression of SHP-1 mRNA.</p><p><b>RESULTS</b>It was found that the promoter 2 of SHP-1 gene located between -577 bp to +300 bp, and 22 CpG sites contained between -353 bp-+182 bp were aberrantly hypermethylated and the SHP-1 could not be detected in K562 cells. In vitro, the detection demonstrated that the expression level of DNMT1 in K562 cells transfected with psiHIV-mU6-shDNMT1 was 0.48±0.06 significantly lower than that of psiHIV-mU6-control group (1.33±0.19)(t= 4.18, P<0.05). The expression of SHP-1 mRNA in K562 cells transfected with psiHIV-mU6-shDNMT1 was significantly higher than that in K562 cells transfected with psiHIV-mU6-shDNMT1 (14.23±3.83 vs 1.031±0.156)(P<0.01). DNMT1 silencing induced demethylation of the 22 CpG sites located in the SHP-1 promoter 2, and SHP-1 gene was re-expression in K562 cells.</p><p><b>CONCLUSION</b>The DNMT1 in K562 cells relates with the hypermethylation and silencing of SHP-1 promoter in K562 cells.</p>