SKM-1细胞地西他滨耐药机制中hENT1功能的研究

Functional study of hENT1 on SKM-1 cell resistance to decitabine

目的 观察干扰1型平衡核苷转运体（human equilibrative nucleoside transporters 1,hENT1）表达后,地西他滨（DAC）对人骨髓增生异常综合征（MDS）细胞系SKM-1细胞的增殖、凋亡和去甲基化的影响.方法 采用慢病毒干扰技术沉默SKM-1细胞中hENT1,采用RT-PCR方法检测靶细胞内hENT1 mRNA表达水平,采用CCK-8法检测不同浓度DAC（0.5、1、5 μmol/L）作用24、48及72 h对hENT1沉默SKM-1细胞的增殖抑制情况,应用流式细胞术及Western blot法观察DAC诱导的凋亡情况,采用甲基化特异性PCR检测p15^INK4B去甲基化水平,分析hENT1沉默后DAC的去甲基化作用.结果 hENT1干扰组hENT1 mRNA表达水平（0.253±0.030）相对于对照组（1.000±0.091）显著降低（P＜0.01）;经0.5、1、5μmol/L DAC作用24、48及72 h后,相对于同时期对照组,hENT1干扰组的增殖抑制率均明显下降（P＜0.05）,其中5 μmol/L DAC作用72 h最为明显,hENT1干扰组和对照组分别为（49.41±4.02）％和（33.03±2.47）％（P=0.007）;hENT1干扰组Caspase-3的剪切体水平及Annexin Ⅴ＋细胞比例减少,并且p15^INK4B去甲基化水平也显著降低.结论 hENT1沉默后SKM-1细胞对DAC诱导的凋亡及去甲基化作用敏感性下降.

Objective To investigate the effect of human equilibrative nucleoside transporters 1 （hENT1） silencing on proliferation,apoptosis and demethylation of human myelodysplastic syndrome （MDS） derived cell line SKM-1 treated with 5-aza-2＆#39;-deoxycytidine （decitabine,DAC）.Methods hENT1 was silenced in SKM-1 cells mediated by lentivirus transfection.The infection efficiency was detected by flow cytometry,and the mRNA expression level of hENT 1 was confirmed by qRT-PCR.The proliferation ratio of SKM-1 cells treated with different concentrations （0.5,1,5 mmol/L） of DAC for 24,48 and 72h was detected by CCK-8 method after hENT1 silencing.The apoptosis of SKM-1 cells was detected by Western blot for cleaved level of caspase-3 and evaluated by flow cytometry after staining with anti-Annexin Ⅴ-PE and 7-AAD.The p15^INK4B DNA methylation status was measured by methylation specific PCR using EZ DNA Methylation-Gold^TM Kit.Results The expression level of hENT1 silenced group （0.253±0.030） was statistically decreased compared with that in control group （1.000±0.091） （P〈0.01）.Compared with control,the proliferation inhibition rate of hENT1 silenced group was significantly decreased by different concentrations of DAC （0.5,1,5 μmol/L） treatment for 24,48,72 h （P〈0.05）,which was （49.41±4.02）% and （33.03±2.47）%,respectively （P=0.007） at 5 μmol/L DAC treatment for 72 h in hENT1 silenced group and the control group.Western blot showed that cleaved caspase3 of hENT1 silenced group was also significantly inhibited.The percentage of Annexin Ⅴ ＋ cells and demethylation status of p15^INK4B were significantly decreased.Conclusion Apoptosis of hENT1 silenced SKM-1 cells induced by DAC was decreased,and the susceptibility of these cells to demethylation treatment was also decreased.