miR-519d/Twist1轴介导BrMC抑制SMMC-7721源性肝癌干样细胞体外致癌能力  

Inhibition of in vitro carcinogenicity by 8-bromo-7-methoxychrysin mediated by modulating miR-519d/TWIST1 axis in liver cancer stem-like cells from SMMC-7721 cell line

作　　者：罗祎敏[1] 曹晓诚 李翔[2,3] 崔迎红 许畅[2,3] 陈阿[2,3] 曹建国 LUO Yi-min;CAO Xiao-cheng;LI Xiang;CUI Ying-hong;XU Chang;CHEN A;CAO Jian-guo(Pathology Dept,Medical College,University of South China,Hengyang,Hunan 421001,China;Pharmaceutical Science,Medical College,Hunan Normal University,Changsha 410013,China;Key Lab of Study and Discover of Small Targeted Molecules of Hunan Province（Medical College,Hunan Normal Univeristy）,Changsha410013,China)

机构地区：[1]南华大学医学院病理学教研室,湖南衡阳421001 [2]湖南师范大学医学院药学系,湖南长沙410013 [3]湖南师范大学湖南省小分子靶向药物研制与创制重点实验室,湖南长沙410013

出　　处：《中国药理学通报》2018年第7期1005-1012,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81172375)

摘　　要：目的探讨8-溴-7-甲氧基白杨素(8-bromo-7-methoxychrysin,BrMC)是否通过上调miR-519d,下调Twist1表达,进而抑制SMMC-7721源性肝癌干样细胞(liver cancer stem cell-like cells,LCSLCs)体外致癌能力。方法用球培养法从SMMC-7721细胞系得到第2代球细胞,命名为LCSLCs。不同浓度BrMC(1.0、3.0、10.0μmol·L^(-1))处理LCSLCs,实时定量PCR检测miR-519d表达水平;球形成和琼脂集落形成实验评价体外致癌能力;荧光素酶报告基因实验和Western blot分析Twist1转录活性和蛋白表达;miR-519d模拟物或Twist1基因转导探讨BrMC作用的分子机制。结果与SMMC-7721细胞比较,LCSLCs的miR-519d低表达,而Twist1蛋白高表达。BrMC(1.0、3.0、10.0μmol·L^(-1))浓度依赖性降低LCSLCs球形成和琼脂集落形成率;并上调miR-519d表达、下调Twist1蛋白表达。荧光素酶报告基因实验证实,miR-519d可以直接靶向Twist1 mRNA 3'非翻译区,并调节蛋白表达。miR-519d模拟物增强BrMC(3.0μmol·L^(-1))的作用,然而,Twist1基因转导有效逆转BrMC(3.0μmol·L^(-1))的效应。结论 BrMC通过调节miR-519d/Twist1信号轴,抑制SMMC-7721源性LCSLCs体外致癌能力。Aim To determine whether 8-bromo-7-methoxychrysin（ BrMC） inhibits in vitro carcinogenicity via up-regulating miR-519d expression and down-regulating Twist1 expression in liver cancer stem-like cells （ LCSLCs） derived from SMMC-7721 cell line.Methods The second generation spheroids derived from SMMC-7721 cell line were obtained by sphere-forming assay and were considered as LCSLCs.Then LCSLCs were treated with various concentrations（ 1.0,3.0,10.0 μmol·L^（-1）） of BrMC.The expression level of miR-519d was detected using real-time PCR.And in vitro carcinogenicity was investigated by sphere-forming assay and clone-forming assay in agar.The transcriptional activity and protein expression of Twist1 were analyzed using luciferase reporter assay and Western blot.Moreover,the molecular mechanism of BrMC was elucidated via miR-519 mimic transfection and Twist1 gene transduction,respectively.Results Compared with SMMC-7721 cells, miR-519d-3p was low-expressed and Twist1 was over expressed in LCSLCs.And the sphere-forming ratio and the clone-forming ratio decreased by treatment with BrMC（ 1.0,3.0,10.0 μmol·L^（-1）） in a dose-dependent manner.Furthermore,luciferase reporter assay demonstrated miR-519d could directly target the 3＇ untranslated region of Twist1 mRNA and regulate protein expression.miR-519d mimic enhanced the effects of BrMC（ 3.0 μmol·L^（-1））.However,Twist1 gene transduction effectively reversed the effects of BrMC（ 3.0 μmol·L^（-1））.Conclusion BrMC inhibits in vivo carcinogenicity via regulating miR-519/Twist1 signal axis in LCSLCs derived from SMMC-7721 cell line.

关 键 词：肝细胞癌 肿瘤干细胞 8-溴-7-甲氧基白杨素 治疗作用 miR-519d TWIST1 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R394.2[医药卫生—基础医学]