Objective: To explore the effects and possible mechanisms of decitabine on Molt4 in vitro.
Methods: Effects of decitabine on cells proliferation were detected by using CCK-8, the apoptosis by Annexin V-FITC, cell cycles by propidium iodide-FACS. Discrepancy genes were screened by RNA-seq technique. The CpG methylation of lactoferrin (LTF) gene in Molt4 cells were identified by Bisulfite sequencing PCR (BSP). The expression of LTF mRNA in Molt4 by RT-PCR and LTF protein expression were analyzed by Western blot.
Results: Decitabine effectively inhibited proliferation and induced apoptosis for Molt4 cells by an time- and dose-dependent manners. Cell cycles were arrested at the G₀/G₁ phase. The promoter methylation degree of LTF gene in Molt4 cells was 72.3% before decitabine treatment and decreased to 45.0% after treatment with 0.50 μmol/L decitabine for 72 h. After the reduction of methylation, expression of its mRNA and protein increased, meanwhile caspase 3 and caspase 9 protein expression levels increased.
Conclusion: The demethylating drug decitabine can induce apoptosis, detain cell cycle at phase G₀/G₁, inhibit proliferation and up-regulate LTF gene expression in Molt4 cells. LTF may become a new target for acute T lymphoblastic leukemia.
目的: 探讨去甲基化药物地西他滨对急性T淋巴细胞白血病细胞株Molt4细胞的影响及其可能的作用机制。
方法: CCK-8法检测地西他滨对细胞增殖的影响；Annexin Ⅴ/PI双重染色法检测地西他滨对细胞凋亡的影响；PI染色法检测细胞周期变化；转录组高通量测序筛选组间差异基因；亚硫酸氢盐测序法检测地西他滨作用Molt4细胞前后乳铁蛋白（LTF）基因启动子CpG岛的甲基化水平变化；实时定量RT-PCR及Western blot法分别检测LTF mRNA及蛋白的表达变化。
结果: 地西他滨能有效抑制Molt4细胞的增殖，抑制率呈时间和剂量依赖性增加，并能诱导细胞凋亡，使细胞阻滞于G0/G1期。0.50 µmol/L地西他滨处理72 h后，Molt4细胞LTF基因启动子甲基化率较对照组降低（45.0%对72.3%），差异有统计学意义（P<0.05），mRNA和蛋白表达增加（P值均<0.05），同时还检测到caspase 3、caspase 9凋亡蛋白表达增加（P值均<0.05）。
结论: 地西他滨诱导Molt4细胞凋亡，阻滞细胞周期在G0/G1期，抑制细胞增殖。LTF基因是地西他滨作用的重要靶点。