目的:探索甲基化转移酶抑制剂5-杂氮-2'-脱氧胞苷(5-Aza-CdR)对急性B淋巴细胞白血病(B-ALL)细胞株NALM-6的作用以及对细胞中微RNA(miRNA)表达水平的影响。方法用不同浓度5-Aza-CdR处理NALM-6细胞,采用四甲基偶氮唑盐(MTT)法检测细胞增殖情况,采用荧光定量反转录聚合酶链反应(RT-PCR)检测5-Aza-CdR处理后细胞DNA甲基转移酶(DNMT)基因mRNA表达水平的变化,采用miScript miRNA PCR Array芯片检测去甲基化后细胞中表达量发生改变的miRNA。结果 NALM-6细胞经不同浓度5-Aza-CdR处理不同时间后,细胞生长受抑,最高抑制率达(74.163±0.381)%。5-Aza-CdR作用浓度与DNMT基因mRNA表达水平呈反比,浓度为1000μmol/L的5-Aza-CdR作用细胞72 h后,DNMT-1的相对表达量降至0.453±0.021,DNMT-3L的相对表达量为0.003±0.001, DNMT-3B的相对表达量为0.395±0.019。miScript miRNA PCR Array筛选出3个miRNA(miR-184、miR-23a-3p、miR-34a-5p)与DNA甲基化相关。结论5-Aza-CdR可下调NALM-6细胞中DNMT基因的表达,并对细胞增殖有抑制作用。miR-184、miR-23a-3p和miR-34a-5p在B-ALL的发生、发展中与DNA甲基化相关。
Objective To explore the function of 5-Aza-CdR in B-cell acute lymphocytic leukemia cell line NALM-6 and its influence on the expression of microRNA (miRNA) in the cells. Methods NALM-6 was treated with different concentrations of 5-Aza-CdR. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) test, and DNA methyltransferase (DNMT) mRNA expression level was detected by reverse transcription PCR (RT-PCR). The expression changes of miRNA were detected by miScript miRNA PCR Array chip in cells after methylation. Results NALM-6 cell growth was inhibited by different concentrations of 5-Aza-CdR processing time, reaching to the maximum inhibitory rate was (74.163 ±0.381) %. 5-Aza-CdR affected concentrations was inversely proportional with expression level of DNMT mRNA. After 1 000 μmol/L of 5-Aza-CdR was dealed with cell 72 h, the relative expression of DNMT-1 was reduced to 0.453 ±0.021, DNMT-3L was 0.003±0.001, DNMT-3B was 0.395±0.019. MiScript miRNA PCR array sieved out 3 miRNA (miR-184, miR-23a-3p, miR-34a-5p) associated with DNA methylation. Conclusions 5-Aza-CdR down regulates the expression of DNMT gene in NALM-6 cells, and inhibits the proliferation of cells. MiR-184, miR-23a-3p and miR-34a-5p are related to DNA methylation in the occurrence and development of B-cell acute lymphocytic leukemia.