摘要
目的:探讨外泌体(EXO)传输Let-7a调控MYC基因在三阴性乳腺癌(TNBC)细胞恶性生物学行为中的作用及其机制。方法:TNBC细胞MDA-MB-231培养完成后,qPCR实验检测TNBC组织和细胞中MYC与Let-7a m RNA的表达水平,WB实验检测MYC与Let-7a蛋白的表达水平。携Let-7a重组慢病毒和敲除MYC的Crisper/Cas-9系统分别转染MDA-MB-231细胞,MTT、Transwell、划痕愈合实验检测MDA-MB-231细胞增殖、侵袭和迁移能力。荧光素酶活性实验验证MYC和Let-7a的作用靶点。分别在野生型和过表达Let-7a的MDA-MB-231细胞中分离EXO,并以透射电镜和WB实验鉴定。qPCR、WB、MTT、Transwell等实验检测两种EXO分别和MDA-MB-231细胞共孵育后Let-7a通过EXO影响MDA-MB-231细胞的生物学功能。结果:Let-7a与MYC在TNBC组织和细胞系中表达呈负相关(P<0.05);MYC促进MDA-MB-231细胞的增殖、迁移与侵袭,Let-7a可以抑制MDA-MB-231细胞的增殖、迁移与侵袭(均P<0.01)。Let-7a通过作用于MYC基因的3’UTR使其沉默,从而减少MYC蛋白的表达(P<0.05)。Let-7a由EXO包裹运输至肿瘤细胞,进而抑制MDA-MB-231细胞的增殖、迁移与侵袭能力(P<0.05)。结论:EXO介导的Let-7a通过作用于MYC基因3’UTR区使得MYC基因沉默,从而抑制MDA-MB-231细胞的增殖、迁移与侵袭。
Objective:To investigate the role of exosome(EXO)transporting Let-7a to regulate MYC gene in the malignant biological behaviors of triple negative breast cancer(TNBC)cell,and to explore the underlying mechanism.Methods:After the completion of cell culture,the gene and protein expressions of MYC and Let-7a in TNBC MDA-MB-231 cells were detected by qPCR and WB,respectively.Recombinant lenti-virus vector carrying Let-7a and Crisper/Cas-9 system with MYC knockdown were transfected into MDA-MB-231 cells;MTT assay,Transwell assay and Scratch healing assay were performed to examine the proliferation,invasion and migration of MDA-MB-231 cells.Luciferase activity assay was performed to validate the binding between MYC and Let-7a.EXO was isolated and identified by transmission electron microscopy and WB assay in wild-type and Let-7a over-expressed MDA-MB-231 cells,respectively.After co-incubation of two types of EXO and MDA-MB-231 cells,the effects of Let-7a on biological behaviors of MDAMB-231 cells via EXO were detected by qPCR,WB,MTT and Transwell etc.Results:Let-7a was negatively correlated with MYC in breast cancer tissues and cell lines(all P<0.05);MYC promoted while Let-7a inhibited the proliferation,migration and invasion of breast cancer cells(all P<0.01);Let-7a silenced MYC by acting on 3’UTR of MYC gene,thereby reducing the expression of MYC protein(P<0.05);Let-7a was enveloped by EXO and transported to cancer cells,there by inhibiting the proliferation,migration and invasion of MDA-MB-231 cells.Conclusion:EXO some mediated Let-7a silences MYC gene by acting on its 3’UTR region,thus inhibiting the proliferation,migration and invasion of MDA-MB-231 cells.
作者
李封
张艳
张琼
陶劲
白殊同
宋娜
孙明令
段亚亭
LI Feng;ZHANG Yan;ZHANG Qiong;TAO Jin;BAI Shutong;SONG Na;SUN Mingling;DUAN Yating(Oncology Department,Chongqing Traditional Chinese Medicine Hospital,Chongqing 400021,China;Gynaecology Departmen,Chongqing Traditional Chinese Medicine Hospital,Chongqing 400021,China;Basic Laboratory,Chongqing Traditional Chinese Medicine Hospital,Chongqing 400021,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2019年第9期962-968,共7页
Chinese Journal of Cancer Biotherapy
基金
国家中医药管理局基金资助项目(No.20140814)~~