MicroRNA-125a regulates proliferation and apoptosis of acute myeloid leukemia through targeting NF-κB pathway.
MicroRNA-125a regulates proliferation and apoptosis of acute myeloid leukemia through targeting NF-κB pathway.
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DOI：
10.26355/eurrev_201905_17781
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发表时间：
2019-05
影响因子：
3.3
通讯作者：
Shen My;Ying Wang;Cui Sy;Wu Xl;Y. Guo;Xu Rr
中科院分区：
文献类型：
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作者：
Shen My;Ying Wang;Cui Sy;Wu Xl;Y. Guo;Xu Rr
目的探讨microRNA-125a对急性髓性白血病（AML）细胞生物学行为的影响。材料与方法构建MicroRNA-125a模拟物（mimic）和阴性对照（negative control， NC），分别转染AML细胞株HL60。用MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四氮唑）法测定转染microRNA-125a mimic或NC的HL60细胞的细胞活力。用分光光度法测定microRNA-125a对HL60细胞b细胞淋巴瘤-2 （Bcl-2）、Bcl-xl、caspase-3、caspase-9酶活性的调控作用。流式细胞术和transwell法分别检测过表达microRNA-125a的HL60细胞凋亡和侵袭的变化。Western blot检测转染microRNA-125a mimic或NC的HL60细胞中细胞周期基因（cyclin B、cd -2、mdm-2）、促凋亡基因p53、抗凋亡基因Bcl-2的蛋白表达水平。最后，采用定量实时聚合酶链式反应（qRT-PCR）检测microRNA-125a过表达的HL60细胞中Bax、caspase-8、核因子-κB （NF-κB）和c-myc的mRNA水平。结果MicroRNA-125a mimic转染HL60细胞后，MicroRNA-125a的表达明显增加，转染效果良好。MTT实验显示，microRNA-125a过表达后细胞活力受到抑制。转染microRNA-125a mimic可显著提高HL60细胞中caspase-3和caspase-9酶活性，降低Bcl-2和Bcl-xl酶活性（p<0.05）。此外，FCM数据显示，microRNA-125a过表达可提高细胞凋亡率。Transwell实验显示过表达microRNA-125a的HL60细胞侵袭率降低。Western blot分析显示，转染microRNA-125a mimic后，HL60细胞中细胞周期基因均下调。过表达microRNA-125a的HL60细胞中p53蛋白水平上调，Bcl-2蛋白水平下调（p<0.05）。microRNA-125a过表达后，促凋亡基因Bax、caspase-8 mRNA水平升高，NF-κB、c-myc mRNA水平降低（p<0.05）。结论MicroRNA-125a通过调控NF-κB通路，抑制AML细胞的增殖和侵袭潜能，阻滞细胞周期在G2/M期。
OBJECTIVE To elucidate the influence of microRNA-125a on the biological behaviors of acute myeloid leukemia (AML) cells. MATERIALS AND METHODS MicroRNA-125a mimic and negative control (NC) were constructed and transfected into AML cell line HL60, respectively. Cell viability of HL60 cells transfected with microRNA-125a mimic or NC was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Regulatory effects of microRNA-125a on enzyme activities of B-cell lymphoma-2 (Bcl-2), Bcl-xl, caspase-3, and caspase-9 in HL60 cells were quantified by a spectrophotometry. Changes in apoptosis and invasion of HL60 cells overexpressing microRNA-125a were detected by flow cytometry and transwell assay, respectively. Protein levels of cell cycle genes (cyclin B, cdc-2, mdm-2), pro-apoptotic gene p53 and anti-apoptotic gene Bcl-2 in HL60 cells transfected with microRNA-125a mimic or NC were assessed by Western blot. Finally, the mRNA levels of Bax, caspase-8, nuclear factor-κB (NF-κB), and c-myc in HL60 cells with microRNA-125a overexpression were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). RESULTS MicroRNA-125a expression remarkably increased by transfection of microRNA-125a mimic into HL60 cells, suggesting its sufficient transfection efficacy. MTT assay revealed an inhibited viability after microRNA-125a overexpression. Transfection of microRNA-125a mimic markedly enhanced enzyme activities of caspase-3 and caspase-9, but reduced activities of Bcl-2 and Bcl-xl in HL60 cells than controls (p<0.05). Moreover, microRNA-125a overexpression elevated apoptotic rate as FCM data indicated. Transwell assay demonstrated a decrease in the invasive rate of HL60 cells overexpressing microRNA-125a. Western blot analyses revealed that cell cycle genes all downregulated by transfection of microRNA-125a mimic in HL60 cells. The protein level of p53 upregulated and Bcl-2 downregulated in HL60 cells overexpressing microRNA-125a (p<0.05). Furthermore, mRNA levels of pro-apoptotic genes Bax and caspase-8 were enhanced after microRNA-125a overexpression, while mRNA levels of NF-κB and c-myc were reduced (p<0.05). CONCLUSIONS MicroRNA-125a inhibits proliferative and invasive potentials, arrests the cell cycle in the G2/M phase of AML cells by regulating the NF-κB pathway.