李海英 周斌 张力 高申孟

[摘要] 目的 研究地西他濱抑制AML1-ETO+白血病细胞增殖和诱导凋亡，并初步探讨其可能的机制。 方法 Kasumi-1细胞常规培养在含10%胎牛血清的RPMI-1640培养基中，CCK8法检测地西他滨抑制Kasumi-1细胞增殖，流式细胞术检测Kasumi-1凋亡，Western Blot检测相关蛋白的表达，RT-PCR检测AML1-ETO和miR-193a的表达。结果 地西他滨可以抑制Kasumi-1细胞增殖，具有浓度效应和时间效应；并能诱导凋亡，对照组、24 h和48 h组细胞凋亡率分别为（5.29±0.88）%、（9.83±1.71）%和（19.47±1.84）%；地西他滨能减少AML1-ETO蛋白的表达，0.1、0.5和1 μmol/L地西他滨组与对照组的比值分别为（0.85±0.21）、（0.28±0.06）和（0.10±0.07），24、48 h组AML1-ETO蛋白与对照组的比值为（0.31±0.21）和（0.24±0.11），但不影响AML1-ETO mRNA表达，24和48 h组与对照组的比值分别为（0.96±0.19）和（0.84±0.11），统计分析无显著性差异（F=1.22，P>0.05）；地西他滨能上调miR-193a，24和48 h分别上升（3.61±0.06）和（6.99±0.74）倍，并减少MDM2和Cyclin D1蛋白的表达，MDM2和Cyclin D1蛋白加药组与对照组的比值分别为（0.51±0.19）和（0.50±0.10）。 结论 地西他滨通过上调miR-193a阻遏AML1-ETO的翻译，并减少MDM2和Cyclin D1蛋白的表达，从而抑制AML1-ETO+白血病细胞增殖和诱导凋亡。

[关键词] 地西他滨；AML1-ETO；白血病；miR-193a

[中图分类号] R733.7 [文献标识码] A [文章编号] 1673-9701（2018）15-0004-05

Mechanism of decitabine in inhibiting cell proliferation and inducing apoptosis of AML1-ETO+ leukemia cells

LI Haiying1 ZHOU Bin1 ZHANG Li2 GAO Shenmeng1

1.Central Laboratory， Wenzhou Medical University First Affiliated Hospital， Wenzhou 325000， China； 2.Department of Radiotherapy and Chemotherapy， Wenzhou Medical University First Affiliated Hospital， Wenzhou 325000， China

[Abstract] Objective To study the decitabine inhibits the proliferation and induces apoptosis of AML1-ETO+ leukemia cells and to explore its possible mechanism. Methods Kasumi-1 cells were commonly cultured in RPMI-1640 medium containing 10% fetal bovine serum. The proliferation of Kasumi-1 cells was determined by CCK8 assay. Flow cytometry was used to detect Kasumi-1 apoptosis. Related protein expression was measured by Western Blot. The expressions of AML1-ETO and miR-193a were measured by RT-PCR. Results Decitabine could inhibit the proliferation of Kasumi-1 cells with concentration effect and time effect， and could induce apoptosis. The apoptosis rates of control group， 24 hours and 48 hours group were （5.29±0.88）% and （9.83±1.71）% and （19.47±1.84）%， respectively. Decitabine could decrease the expression of AML1-ETO protein， and the AML1-ETO protein ratios of 0.1， 0.5 and 1 μmol/L decitabine group to the control group were （0.85±0.21）， （0.28±0.06） and （0.10±0.07）. The ratios of AML1-ETO protein of 24 hours group， 48 hours group to control group were （0.31±0.21） and （0.24±0.11）. But decitabin did not affect the expression of AML1-ETO mRNA， and the ratios of AML1-ETO mRNA of 24 hours group and 48 hours group to control group were （0.96±0.19） and （0.84±0.11）. The difference was not significant（F=1.22， P>0.05）. Decitabine could up-regulate the expression of miR-193a by （3.61±0.06） and （6.99±0.74） fold respectively at 24 and 48 hours， and decreased the expression of MDM2 and Cyclin D1.The protein expression ratios of MDM2 and Cyclin D1 of dosing group and control group were （0.51±0.19） and （0.50±0.10）， respectively. Conclusion Decitabine inhibits the proliferation and induces apoptosis of AML1-ETO+ leukemia cells by up-regulating miR-193a to repress the translation of AML1-ETO and inhibiting the expression of MDM2 and Cyclin D1.

[Key words] Decitabine； AML1-ETO； Leukemia； miR-193a特异性AML1-ETO（eight-twenty one）融合基因是由t（8；21）（q22；q22）染色体易位所导致，一般急性髓系白血病（acute myeloid leukemia，AML）发生的比例约12%～20%，但在M2 型中，该比例显著升高，为40%～80%[1]。世界卫生组织将AML伴有t（8；21）（q22；q22）定义为独立类型[2]。在各类型AML中，AML1-ETO+预后较好，患者经过大剂量阿糖胞苷化疗方案治疗后，疗效显著[3]。但近年来有研究发现，并不是所有患者均能取得较好的治疗效果，部分AML1-ETO+患者存在难缓解、易复发现象，而对于此类患者的治疗，国内外相关研究依然较少。有研究表明，AML1-ETO+细胞对去甲基化药物很敏感，尤其是DNA甲基化转移酶（DNA methyltransferase，DNMT）抑制剂，如地西他滨（decitabine，DAC）和阿扎胞苷[4-8]。本……