摘要
目的探讨miR-let-7c-5p/c-myc信号轴在白血病细胞定向单核/巨噬细胞分化中的调控作用。方法采用PMA+LPS+IFN-γ诱导THP-1白血病细胞向单核/巨噬细胞定向分化,PBS作为对照组。诱导48 h后CCK8法测定细胞增殖水平;流式细胞仪测定细胞CD11b与CD14分化抗原表达水平;RT-qPCR检测白血病细胞分化前后miR-let-7c-5p和c-myc的表达变化;蛋白质印迹法检测c-myc蛋白表达变化;双荧光素酶结合实验检测miR-let-7c-5p与c-myc的3′UTR靶向结合和活性调控关系;转染miR-let-7c-5p mimic观察c-myc表达变化后细胞的增殖分化水平变化;过表达c-myc拯救实验观察miR-let-7c-5p对PMA+LPS+IFN-γ诱导的THP-1细胞增殖分化的影响。THP-1细胞转染miR-let-7c-5p inhibitor,观察c-myc表达变化对THP-1定向分化为M1样巨噬细胞的影响。结果与PBS对照组相比,PMA+LPS+IFN-γ诱导48 h后,THP-1细胞的增殖能力明显降低(0.64±0.01 vs 0.33±0.01,t=45.190,P<0.001)。PMA+LPS+IFN-γ实验组的CD11b和CD14平均阳性表达率升高[(5.51±0.89)%vs(17.72±0.86)%,t=17.050,P<0.001;(6.22±0.70)%vs(16.42±0.14)%,t=24.650,P<0.001]。PMA+LPS+IFN-γ实验组的c-myc蛋白表达水平降低(0.87±0.02 vs 0.64±0.06,t=6.041,P=0.004)。PMA+LPS+IFN-γ实验组的miR-let-7c-5p基因表达量高于对照组,而c-myc基因表达量低于对照组,均P<0.001。Luciferase实验证实,miR-let-7c-5p mimic+c-myc-WT组的荧光素酶活性明显低于mimics NC+c-myc-WT组(0.88±0.09 vs 0.62±0.05,t=4.320,P=0.013)。转染miR-let-7c-5p mimic后,miR-let-7c-5p mimic组的c-myc蛋白表达水平低于miR-let-7c-5p mimic NC组(1.03±0.08 vs 0.39±0.07,t=10.420,P<0.001),伴有细胞增殖水平的降低(0.64±0.01 vs 0.42±0.01,t=30.160,P<0.001),CD11b和CD14阳性表达率升高[(2.18±0.53)%vs(22.10±0.87)%,t=33.530,P<0.001;(3.37±0.73)%vs(22.98±5.43)%,t=6.195,P=0.004]。拯救实验结果表明,miR-let-7c-5p mimics+c-myc vector组的c-myc蛋白表达水平高于miR-let-7c-5p mimics组(0.53±0.02 vs 0.84±0.05,t=9.250,P<0.001)。miR-let-7c-5p mimics组的THP-1细胞增殖D值低于miR-let-7c-5p mimics+c-myc vector组(0.62±0.01 vs 0.42±0.01,t=20.330,P<0.001)。miR-let-7c-5p mimics组的平均CD11b、CD14阳性表达率高于miR-let-7c-5p mimics+c-myc vector组[(6.17±0.76)%vs(19.57±5.96)%,t=3.865,P=0.018;(10.36±1.13)%vs(34.89±3.19)%,t=12.56,P<0.001]。miR-let-7c-5p inhibitor干预PMA+LPS+IFN-γ实验组结果显示,与对照组相比较,PMA+LPS+IFN-γ诱导THP-1细胞分化后,c-myc蛋白的表达水平显著降低(0.97±0.05 vs 0.34±0.19,t=5.377,P=0.006),CXCL9、CXCL10、iNOS基因的相对表达水平明显升高,均P<0.001。与PMA+LPS+IFN-γ实验组相比较,PMA+LPS+IFN-γ+miR-let-7c-5p inhibitor组的c-myc蛋白相对表达水平显著升高(0.34±0.06 vs 0.96±0.07,t=11.310,P<0.001),而CXCL9、CXCL10、iNOS基因的相对表达水平明显降低,均P<0.001。结论miR-let-7c-5p可以靶向结合c-myc 3′UTR区,miR-let-7c-5p/c-myc信号轴是白血病细胞定向分化为单核/巨噬细胞的重要途径之一。
Objective To investigate the role of miR-let-7c-5p/c-myc signal axis in the differentiation of THP1 leukemic cells into monocytes/macrophages.Methods Human leukemic THP-1 cells were induced to differentiate into monocytes/macrophages by PMA+LPS+IFN-γ mixture, and PBS as the control group.After 48 hours of induction, the cell proliferation was measured by CCK8 count assay, and expression of differentiation antigens CD11b and CD14 were measured by Flow cytometry assay.The gene expression of miR-let-7c-5p and c-myc was detected by RT-qPCR,the expression of c-myc protein was detected by Western Blot.Dual-luciferase reporter gene analysis was used to detect the targeted binding of miR-let-7c-5p on 3′UTR of c-myc.The effect of miR-let-7c-5p mimics on proliferation and differentiation of THP-1 cells by regulation of c-myc was detected by CCK8 and Flow cytometry respectively.The effect of miR-let-7c-5p mimics on the proliferation and differentiation of THP-1 cells was rescued by transfection of c-myc vector.By transfecting miR-let-7c-5p inhibitor into THP-1 cells, the effect of c-myc expression on the differentiation of THP-1 into m1-like macrophages was observed.Results Compared with PBS control group, the proliferation ability of THP-1 cells was significantly decreased 48 h after induction by PMA+LPS+IFN-γ(0.64±0.01 vs 0.33±0.01,t=45.190,P<0.001).The positive percent of CD11b and CD14 expression by Flow cytometry in the PMA+LPS+IFN-γ group was significantly higher than that in the PBS control group, such as CD11b [(5.51±0.89)% vs(17.72±0.86)%,t=17.050,P<0.001] and CD14 [(6.22±0.70)% vs(16.42±0.14)%,t=24.650,P<0.001].The relative expression of c-myc in PMA+LPS+IFN-γ group was significantly lower than that of PBS control group(0.87±0.02 vs 0.64±0.06,t=6.041,P=0.004).The expression level of miR-let-7c-5p gene in the PMA+LPS+IFN-γ group was higher than that in the control group, while the relative expression level of c-myc gene was lower than that in the control group, both P<0.001.The results of double luciferase reporter gene detection confirmed that the luciferase activity of the miR-let-7c-5p mimic+c-myc-WT group was significantly lower than that of the mimics NC+c-myc-WT group, and the relative enzyme activity values were 0.88±0.09 and 0.62±0.05,respectively, with a statistically significant difference(t=4.320,P=0.013).After transfection of THP-1 cells, the relative expression of c-myc in miR-let-7c-5p mimic group was lower than that in miR-let-7c-5p mimic NC group(1.03±0.08 vs 0.39±0.07,t=10.420,P<0.001),accompanied by decreased cell proliferation(0.64±0.01 vs 0.42±0.01,t=30.160,P<0.001),the positive rate of CD11b and CD14 increased [(2.18±0.53)% vs(22.10±0.87)%,t=33.530,P<0.001;(3.37±0.73)% vs(22.98±5.43)%,t=6.195,P=0.004].The rescue experiment of miR-let-7c-5p on c-myc showed that the relative expression of c-myc protein in the miR-let-7c-5p mimics+c-myc vector group was higher than that in the miR-let-7c-5p mimics group(0.53±0.02 vs 0.84±0.05,t=9.250,P<0.001).The D values of THP-1 cells in miR-let-7c-5p mimics group was lower than the miR-let-7c-5p mimics+c-myc vector group(0.62±0.01 vs 0.42±0.01,t=20.330,P<0.001).The mean positive rate of CD11b and CD14 in the miR-let-7c-5p mimics group was higher than the miR-let-7c-5p mimics+c-myc vector group [(6.17±0.76)% vs(19.57±5.96)%,t=3.865,P=0.018;(10.36±1.13)% vs(34.89±3.19)%,t=12.560,P<0.001].The miR-let-7c-5p inhibitor was used to interfere the role of PMA+LPS+IFN-γ in inducing differentiation of THP-1 cells.Compared with the control group, the relative expression of c-myc protein in THP-1 cells induced by PMA+LPS+IFN-γ decreased significantly(0.97±0.05 vs 0.34±0.19,t=5.377,P=0.006),the relative expression level of CXCL9,CXCL10 and iNOS gene expression level were significantly increased, both P<0.001.Compared with PMA+LPS+IFN-γ group, relative expression of c-myc protein of PMA+LPS+IFN-γ+miR-let-7c-5p inhibitor group were significantly elevated(0.34±0.06 vs 0.96±0.07,t=11.310,P<0.001),and relative CXCL9,CXCL10,iNOS gene expression level decreased obviously, both P<0.001.Conclusion miR-let-7c-5p can target to the 3′UTR region of c-myc, and the miR-let-7c-5p/c-myc signaling axis is one of the important pathways for the directional differentiation of leukemic cells into monocytes/macrophages.
作者
孙瑞婧
王玉芳
王朝喆
吴沄桦
都鹏超
毕可红
姜国胜
SUN Rujjing;WANG Yufang;WANG Chaozhe;WU Yunhua;DU Pengchao;BI Kehong;JIANG Guosheng(Department of Immunology,School of Basic medicine,Binzhou Medical University,Yantai 264003,China;Department of Laboratory Medicine,Fushan District People's Hospital,Yantai 265500,China;Department of Hematology,First Af filiated Hospital of Shandong First Medical University(Shandong Academy of Medical Sciences),Jinan 250062,China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2023年第10期577-586,共10页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学基金(81573467)
山东省自然科学基金青年项目(ZR2020QH160)
山东省自然科学基金面上项目(ZR2021MH080)
山东大学临床医学科技创新项目(202134001)。
