let-7b-5p通过FTO/m^6 A/MYC信号通路抑制人类白血病THP-1细胞增殖  

Hsa-let-7b-5p Inhibits Proliferation of Human Leukemia THP-1 Cells via FTO/m^6A/MYC Signaling Pathway

作　　者：吴淡森[1] 郑彩罚 曾泳萍 林晶[1] 陈佳龙 石松菁[1] WU Dan-Sen;ZHENG Cai-Fa;ZENG Yong-Ping;LIN Jing;CHEN Jia-Long;SHI Song-Jing(Fujian Provincial Hospital,Shengli Clinical College of Fujian Medical University,Fuzhou 350001,Fujian Province,China)

机构地区：[1]福建省立医院,福建医科大学附属省立临床学院,福建福州350001

出　　处：《中国实验血液学杂志》2020年第6期1873-1879,共7页Journal of Experimental Hematology

摘　　要：目的:探讨let-7b-5p下调FTO并通过m^6A/MYC信号通路抑制急性髓系白血病细胞系THP-1增殖的作用。方法:选取急性髓系白血病细胞系THP-1及正常人外周血单个核细胞(PBMNC)为研究对象,采用RT-PCR检测细胞中let-7b-5p及FTO mRNA表达;Western blot检测细胞中FTO蛋白表达;采用荧光素酶报告基因实验验证let-7b-5p对FTO的靶向作用。分别采用let-7b-5p mimic转染THP-1细胞和let-7b-5p inhibitor转染PBMNC,采用m^6A斑点印迹分析转染后的c-MYC mRNA m^6A富集水平变化,RT-PCR分析转染细胞let-7b-5p、FTO与c-MYC mRNA表达水平变化,Western blot进一步验证FTO、c-MYC表达,验证let-7b-5p对FTO表达的影响;采用MTT法检测转染后细胞的增殖水平。结果:与PBMNC比较,THP-1的let-7b-5p表达显著降低,而FTO表达明显增高,差异显著(P<0.05);let-7b-5p mimic联合FTO 3'-UTR使转染的THP-1细胞荧光素酶活性显著降低,而联合3'-UTR结合位点突变使转染的THP-1细胞荧光素酶活性显著增高,与阴性对照组的差异显著(P<0.05)。let-7b-5p inhibitor下调c-MYC mRNA m^6A富集水平,上调PBMNC表达FTO,差异显著(P<0.05);然而,let-7b-5p mimic上调c-MYC mRNA m^6A富集水平,下调THP-1表达FTO,细胞增殖率显著降低,与空载体组(阴性对照组)差异显著(P<0.05)。结论:人急性髓系白血病细胞系THP-1低表达let-7b-5p,通过let-7b-5p-/FTO-/m^6A轴调节c-MYC表达,进而促进白血病细胞增殖。Objective:To investigate the down-regulation effect of let-7b-5p on the expression of FTO in acute myeloid leukemia cell line THP-1 and inhibitory effect on THP-1 proliferation via m^6A/MYC signaling pathway.Methods:The acute myeloid leukemia cell line THP-1 and the normal human peripheral blood mononuclear cells(PBMNC)were selected as subjects.The expression of let-7b-5p and FTO mRNA in those cells was detected by qPCR,further the expression of FTO protein in those cells was detected by Western blot.And,the luciferase reporter gene assay was used to verify the targeting effect of let-7b-5p on FTO.Finally,THP-1 cells were transfected respectively with let-7b-5p mimic,and PBMNC with let-7b-5p inhibitor,there after the C-MYC mRNA m^6A enrichment level in transfected cells was analyzed by dot blot,the expression levels of let-7b-5p,FTO and c-MYC were assayed by RT-PCR,the expressions of FTO and c-MYC protein were verified by Western blot,and the proliferation level of cells after transfection was detected by MTT assay.Results:Compared with PBMNC,the expression of let-7b-5p in THP-1 significantly decreased,while the expression of FTO was significantly increased(P<0.05).After transfection with let-7b-5p mimic combined with FTO 3'-UTR,the luciferase activity of transfected THP-1 cells significantly decreased,but the luciferase activity significantly increased after transfection with mutant 3'-UTR,which was significantly different from the negative control group(blank vector)(P<0.05).Let-7b-5p inhibitor down-regulated c-MYC mRNA m^6A enrichment,and then up-regulated the expression of FTO in transfected PBMNC cells,the effects of which were significant(P<0.05).However,let-7b-5p mimic up-regulated c-MYC mRNA m^6A enrichment level and down-regu-lated the expression of FTO in the transfected THP-1 cells,and the cell proliferation rate was significantly lower than that in the negative control group(blank vector)(P<0.05).Conclusion:Human acute myeloid leukemia cell line THP-1 low expresses the let-7b-5p,which regulates c-MYC

关 键 词：急性白血病 表观转录组学 N6-甲基腺苷 信号通路 脂肪与肥胖相关基因 

分 类 号：R733.71[医药卫生—肿瘤]