青蒿琥酯对高危MDS细胞株SKM-1中DNMT1表达的影响

Effects of Artesunate on DNMT1 expression in high-risk MDS cell strain of SKM-1

目的以骨髓增生异常综合征(myelodysplastic syndromes,MDS)细胞系SKM-1细胞为研究对象,在体外观察青蒿琥酯(artesunate,ART)对SKM-1细胞脱氧核糖核酸甲基转移酶(DNA methyltransferase,DNMT1)基因表达的影响,以期为临床应用ART治疗中高危MDS患者提供体外实验的理论依据。方法 ART干预体外细胞培养MDS细胞SKM-1细胞系,采用逆转录聚合酶链反应(revers transcription-polymerase chain reaction,RTPCR)法观察DNMT1的mRNA改变,采用蛋白印迹法观察DNMT1蛋白表达改变。采用实时定量RT-PCR法观察DNMT1靶基因p15INK4b表达的改变,采用甲基化PCR法观察p15INK4b启动子甲基化的改变。结果 1、5、10μmol/L的ART处理SKM-1细胞后DNMT1基因表达低于对照组,差异有统计学意义(P<0.05)。1、5、10μmol/L各剂量组间差异无统计学意义(P>0.05)。DNMT1蛋白表达1、10μmol/L低于对照组,差异有统计学意义(P<0.05);1、10μmol/L组低于5μmol/L组,差异有统计学意义(P<0.05);ART处理SKM-1细胞后DNMT1基因3、6、12h处理组低于对照组,差异有统计学意义(P<0.05);3h、6h与12h差异有统计学意义(P<0.05)。DNMT1蛋白3、6、12h处理组低于对照组,差异有统计学意义(P<0.05)。ART处理后p15INK4b的mRNA水平5、10μmol/L组、地西他宾组高于对照组,差异有统计学意义(P<0.05)。p15INK4b的mRNA水平在10μmol/L组是这5个处理组中最高的,并且1μmol/L、5μmol/L、地西他宾组与10μmol/L组比较,差异有统计学意义(P<0.05);ART处理SKM-1细胞后DNMT1蛋白靶基因p15INK4b启动子甲基化程度下降,各组差异无统计学意义;ART组非甲基化启动子较其他2组上升,差异有统计学意义(P<0.05)。结论ART通过抑制DNMT1的转录和蛋白表达来恢复SKM-1细胞中的p15INK4b的表达,进而发挥其抑癌作用。

Objective To observe the effects of artesunate（ART）on gene expression levels of DNMT1 by using SKM-1cells as the research object,and provide laboratory evidence in vitro for the usage of ART in MDS treatment.Methods After being treated with ART（1,5and 10μmol/L）,DNMT1 mRNA changes in SKM-1cells were observed with RT-PCR,and the protein expression changes were observed with West Blot.Then,the expression changes of p15INK4 b,the DNMT1 targeting gene were observed by using real-time quantitative RT-PCR,and the changes of p15INK4 bpromoter methylation state were observed by using methylation PCR.Results After being treated with ART（1,5and 10μmol/L）,the difference was statistically significant for DNMT1 gene expression in SKM-1cells compared with control group（P〈0.05）,but there was no significant difference in the three dose groups（1,5and 10μmol/L）（P〈0.05）.As to DNMT1 protein expression,compared with 5μmol/L group,there was significantly lower in the other two groups（1and 10μmol/L）（P〈0.05）,and also significantly lower than that in control group（P〈0.05）.However there was no significant difference among the other groups（P〈0.05）.After SKM-1cells being treated with ART for 3h,6hand 12 h,compared with control group,the difference of DNMT1 gene and protein expressions in the three groups（3h,6h and 12h）was significant（P〈0.05）.Besides,the difference of gene expression between the two groups（3h,6h）and the 12 hgroup was also significant（P〈0.05）.But there was no significant different of protein expression in other treated groups（P〉0.05）.After SKM-1cells being treated with ART,p15INK4 B mRNA level of SKM-1cells in the three groups（5and 10μmol/L groups,and decitabine group）was statistically higher than that in control group（P〈0.05）,with the level in 10μmol/L group the highest in the five groups,and the significant difference was only found between 10μmol/L group and the four groups（control group,1μmol group,5μmol group and decitabine group）（P〈0.05）.After being treated with ART,p15INK4 bpromoter methylation level was decreased in SKM-1cells,but the difference was not significant（P〈0.05）.On the contrary,the level of promoter unmethylation state in ART group was increased compared with two other groups（control group and the decitabine group）（P〈0.05）.Conclusion ART can restore the expression of p15INK4 b by inhibiting DNMT1 transcription and protein expression in SKM-1cells,and thus to suppress tumor.