摘要
目的探讨微小RNA-148b-3p(miR-148b-3p)对急性髓系白血病(AML)细胞增殖和自噬的影响及其机制。方法基于基因表达数据集(GEO)和癌症基因组图谱(TCGA),采用生物信息学软件分析miR-148b-3p在AML细胞中的表达及其与患者临床预后的关系;实时荧光定量PCR检测AML细胞中miR-148b-3p表达。用脂质体法将miR-148b-3p模拟物(miR-148b-3p mimic)和miR-148b-3p抑制物(miR-148b-3p inhibitor)分别瞬时转入THP-1和NB4细胞,采用CCK-8法和5-乙炔基-2′脱氧尿嘧啶核苷(EdU)法检测细胞增殖,Western blot法检测B细胞淋巴瘤因子2(Bcl2)、Bcl2相关X蛋白(BAX)、自噬标志物微管相关蛋白1轻链3(LC3)、P62和自噬相关基因14(ATG14)蛋白水平;双荧光素酶报告基因实验检测miR-148b-3p对ATG14的靶向结合,回复实验观察miR-148b-3p/ATG14轴对AML细胞表型的影响。结果AML患者细胞低表达miR-148b-3p,miR-148b-3p低表达的AML患者总体生存率较对照组显著降低;过表达miR-148b-3p可抑制THP-1细胞增殖和促进凋亡、并下调LC3Ⅱ、ATG14及上调P62蛋白水平,而抑制NB4细胞中miR-148b-3p表达则观察到相反的作用;此外,miR-148b-3p能显著降低野生型ATG14表达载体的荧光素酶活性,回复实验结果显示过表达ATG14可逆转miR-148b-3p上调对细胞增殖和自噬的抑制作用,而抑制ATG14表达则能减弱miR-148b-3p下调对细胞表型的促进作用。结论miR-148b-3p通过靶向结合ATG14抑制AML细胞的体外增殖和自噬。
Objective To investigate the effect of miR-148b-3p on the proliferation and autophagy of acute myeloid leukemia(AML)cells and its molecular mechanism.Methods Based on GEO and TCGA databases,the expression of miR-148b-3p in AML cells and its association with clinical prognosis of patients were analyzed with the bioinformatics software.The expression of miR-148b-3p in AML cells was detected by real-time quantitative PCR.The miR-148b-3p mimic and the miR-148b-3p inhibitor were transiently transfected into AML cell lines THP-1 and NB4 by liposome-mediated transfection,respectively.The proliferation of leukemia cells was evaluated by CCK-8 assay and 5-ethynyl-2′-deoxyuridine(EdU)labeling,and the protein levels of Bcl2,Bcl2-associated X protein(BAX),autophagy marker LC3,P62,and autophagy-related gene 14(ATG14)were detected by Western blotting.The targeted binding of miR-148b-3p to ATG14 was measured by dual-luciferase reporter gene assay,and the effect of miR-148b-3p/ATG14 axis on the phenotype of AML cells was observed in the rescue experiments.ResultsA decreased expression of miR-148b-3p was found in leukemia blasts of AML patients,and the overall survival rate of AML patients with low expression of miR-148b-3p was significantly lower than that of the control group.Overexpression of miR-148b-3p inhibited THP-1 cells proliferation,promoted their apoptosis,downregulated the LC3Ⅱand ATG14 protein levels,and upregulated the P62 protein levels,while inhibiting the expression of miR-148b-3p in NB4 cells had the opposite effect.miR-148b-3p significantly reduced the luciferase activity of the wild-type ATG14 expression vector.The results of rescue experiments showed that overexpression of ATG14 reversed the inhibitory effect of miR-148b-3p upregulation on cell proliferation and autophagy,while inhibition of ATG14 expression weakened the promotive effect of miR-148b-3p downregulation on cell phenotype.Conclusion The miR-148b-3p inhibits the in vitro proliferation and autophagy of AML cells by targeting ATG14.
作者
蒋雪坷
敬一佩
雷力
彭美茜
肖巧玲
任俊
陶永红
黄军鹏
张伶
JIANG Xueke;JING Yipei;LEI Li;PENG Meixi;XIAO Qiaoling;REN Jun;TAO Yonghong;HUANG Junpeng;ZHANG Ling(Key Laboratory of Clinical Laboratory Diagnostics Designated by the Ministry of Education,School of Laboratory Medicine,Chongqing Medical University,Chongqing 400016,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2021年第10期881-890,共10页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81873973)
重庆市科学技术局自然科学基金面上项目(cstc2019jcyj-msxmX0229)。
