| Objective: To study the effects of procaine hydrochloride on methylation state of ID4 gene and cell proliferation of human acute myeloid leukemia(AML) cells HL-60, and to explore the potential target for leukemia gene therapy.Methods : Human AML cells HL-60 were cultivated in vitro, then treated with different concentrations of procaine hydrochloride, and cell inhibitive rate at 24 hours, 48 hours and 72 hours were investigated by Cell Counting Kit(CCK8). The change of methylated degree of ID4 gene in HL-60 cells treated with procaine hydrochloride for 48 hours was detected by Methylation-sensitive high resolution melting(MS-PCR). The expression of ID4 mRNA in HL-60 cells treated with procaine hydrochloride for 48 hours was detected by quantitative real-time polymerase chain reaction(qPCR).The expression of ID4 protein in HL-60 cells treated with procaine hydrochloride for 48 hours was detected Western blot(WB). All the data was undertook statistical analysis with SPSS 17.0 software, the data which had equal variance was compared by Independent sample t-test or one-way ANOVA, the data which had no equal variance was compared by Nonparametric test.Results:(1) CCK8 showed: Cell inhibitive rate of HL-60 cells after treated with different concentrations of procaine hydrochloride for 24 hours were 8.39%, 16.14%, 20.74% respectively; cell inhibitive rate of HL-60 cells after treated with different concentrations of procaine hydrochloride for 48 hours were 24.20%, 41.41%, 50.74% respectively; cell inhibitive rate of HL-60 cells after treated with different concentrations of procaine hydrochloride for 72 hours were 39.03%, 50.48%, 65.27% respectively. Compared with the control group(0mmol/L), there was a significant difference(P <0.05). Under the same time, cell inhibitive rate of HL-60 was continuously increased along with the increase of drug concentration, the difference among experimental groups were statistically significant(P <0.05); Under the same concentration, cell inhibitive rate of HL-60 was increased continuously along with the extension of time, the difference among experimental groups were statistically significant(P <0.05).(2) MS-HRM showed: The CpG island of ID4 gene promoter region in control group presented methylation, after HL-60 cells treated with different concentrations of procaine hydrochloride for 48 hours, the degree of methylation of CpG island in ID4 gene promoter region was reduced, which was dose-dependent in the range of 2-6mmol/L.(3) qPCR showed: As compared with control group, the expression of ID4 mRNA in HL-60 cells after treated with different concentrations of procaine hydrochloride for 48 hours, increased continuously with procaine hydrochloride concentrations increased in the range of 2-6mmol/L, the difference among concentration groups were statistically significant(P <0.05).(4) WB showed: After treated with different concentrations of procaine hydrochloride for 48 hours, the expression of ID4 protein in experiment groups(0.44, 0.56, 1.01) was increased than that in control group(0.34), and it increased continuously with procaine hydrochloride concentrations increased, the difference among concentration groups were statistically significant(P <0.05).Conclusions:(1) The survival of HL-60 cells cultured in vitro was inhibited by procaine hydrochloride with a certain dose and time dependent manners.(2) Procaine hydrochloride can reverse the hypermethylated state of CpG island in ID4 gene promoter region and upregulate the expression of Id4 mRNA and Id4 protein with a certain dose dependent manners.(3) The demethylation may be one of the important molecular mechanism which procaine hydrochloride inhibit leukemia HL-60 cell proliferation. |
