摘要
目的:比较miR-199a-5p在急性髓系白血病(AML)耐药细胞株K562/ADM以及敏感细胞株K562中的表达,研究其对AML耐药的调控效应并探索其机制。方法:采用MTT法检测阿柔比星(ADM)对K562/ADM和K562细胞的生长抑制率并计算IC50。采用实时荧光定量RT-PCR的方法检测2种细胞株(K562/ADM和K562)以及患者骨髓标本(复发难治AML患者和化疗后完全缓解AML患者)中miR-199a-5p的表达。通过细胞转染的方法在K562/ADM细胞中转入miR-199a-5p mimic使其表达上调,在K562细胞中转入miR-199a-5p inhibitor使其表达下调,采用CCK-8法检测ADM对2种细胞的增殖抑制率,实时荧光定量PCR和Western blot法分别检测转染后2种细胞中DRAM1基因和蛋白的表达。双荧光素酶报告基因实验检测miR-199a-5p与DRAM13′UTR是否存在直接结合位点。采用siRNA的方法下调K562/ADM细胞中DRAM1表达,CCK-8法检测ADM对细胞增殖抑制率的变化。结果:ADM对K562/ADM和K562细胞的IC50分别为146.14±0.079和3.08±0.056μg/ml。miR-199a-5p在复发难治AML患者骨髓中的表达明显低于完全缓解患者,在K562/ADM细胞中的表达明显低于K562细胞(P<0.05)。当K562/ADM细胞中miR-199a-5p表达上调时,ADM对细胞的增殖抑制率升高,DRAM1基因和蛋白表达明显下降。当K562细胞中miR-199a-5p表达下调时,ADM对细胞的增殖抑制率明显下降,DRAM1基因和蛋白表达明显升高(P<0.05)。双荧光素酶报告基因实验显示,miR-199a-5p与DRAM1的3′UTR区存在直接结合位点。K562/ADM细胞中DRAM1在基因和蛋白水平表达均明显高于K562细胞(P<0.05)。当K562/ADM细胞中DRAM1基因表达下调后,细胞对ADM的敏感性显著升高(P<0.05)。结论:miR-199a-5p在耐药白血病细胞中呈低表达。miR-199a-5p表达能够调控AML细胞对ADM的敏感性。DRAM1是miR-199a-5p调控AML耐药的功能性靶基因。
Objective:To compare the expression of miR-199a-5p between ADM-resistant AML cell(K562/ADM)and ADM-sensitive AML cell(K562),and to investigate the effect of miR-199a-5p on regulating AML drug resistance as well as its molecular mechanism.Methods:MTT method was used to detect the proliferation inhibition effect of ADM on K562 and K562/ADM cells,the IC50 was calculated.miR-199a-5p expression in cell lines(K562 and K562/ADM)and bone marrow sample(refractory/relapsed AML patients and complete remission AML patients)was detected by RT-qPCR.K562/ADM and K562 cells were transfected by miR-199a-5p mimic and miR-199a-5p inhibitor respectively to ensure that miR-199a-5p expression in K562/ADM cells was increased and that in K562 cells was decreased.Then proliferation inhibition effect of ADM on both cells was detected by CCK-8 and mRNA and protein DRAM1 expression in both cells was measured by real time RT-PCR and Western blot respectively.Dual luciferase reporter assay was used to detect wether there were direct binding sites between miR-199a-5p and DRAM13′UTR.CCK-8 was used to measure the proliferation inhibition effect of ADM on K562/ADM cells when DRAM1 was downregulated by siRNA.Results:The IC50 of ADM for K562/ADM and K562 cells was 146.14±0.079 and 3.08±0.056μg/ml respectively.As compared with patients in complete remission group,MiR-199a-5p expression in refractory/relapsed AML patients significantly decreased,and the MiR-199a-5p expression in K562/ADM cells was also dramatically downregulated,compared with K562 cells(P<0.05).When the expression of miR-199a-5p was upregulated in K562/ADM cells,the proliferation inhibition effect of ADM on cells elevated and both DRAM1 mRNA and protein expressions decreased.Conversely,when miR-199a-5p expression was downregulated in K562 cells,the proliferation inhibition effect of ADM on cells obviously reduced and both DRAM1 mRNA and protein expression increased(P<0.05).Dual luciferase reporter Assay showed a direct interaction between miR-199a-5p and its binding site within DRAM1 mRNA.Both DRAM1 mRNA and protein expression in K562/ADM were markedly higher than those in K562 cells(P<0.05).The ADM chemosensitivity of K562/ADM cells was improved significantly when DRAM1 expression was downregulated(P<0.05).Conclusion:miR-199a-5p is downregulated in chemoresistant AML cells.miR-199a-5p expression plays an important role in regulating the sensitivity of AML cells to ADM treatment.DRAM1 is a functional target gene for miR-199a-5p modulating AML chemoresistance.
作者
李旸
孙颖
苗苗
石雪
杨威
刘卓刚
LI Yang;SUN Ying;MIAO Miao;SHI Xue;YANG Wei;LIU Zhuo-Gang(Department of Hematology,Shengjing Hospital of China Medical University,Shenyang 110022,Liaoning Province,China)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2020年第4期1096-1104,共9页
Journal of Experimental Hematology
