摘要
目的 研究自然杀伤(NK)细胞来源外泌体的miR-30e-3p对食管鳞状细胞癌(ESCC)细胞增殖、凋亡和侵袭的影响。方法 从健康供体的外周血中分离和扩增NK细胞,并分离NK细胞外泌体(EXO)进行鉴定。NK细胞外泌体与NEC细胞进行共培养,随机分为Exo1和Exo2组。采用透射电子显微镜观察外泌体形态及大小。Western blot法检测外泌体凋亡相关基因2相互作用蛋白X(ALIX)、抗肿瘤易感基因101(TSG101)、 CD81、钙联蛋白(calnexin)表达。将miR-30e-3p模拟物(miR-30e-3p mimic)、 miR-30e-3p抑制物(miR-30e-3p inhibitor)及阴性对照(miR-NC)转入NK细胞,并分离相应的外泌体与NEC细胞进行共培养。CCK-8法检测细胞增殖,流式细胞术检测细胞周期,异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶双染法检测细胞凋亡,Transwell^(TM)侵袭实验检测细胞侵袭能力。实时定量PCR检测NCE细胞和外泌体中miR-23b、 miR-422a、 miR-133b、 miR-124、 miR-30e-3p和miR-99a的表达。结果 NK细胞中CD56+CD3+细胞比例为(0.071±0.008)%,CD56+CD16+细胞比例为(90.6±10.6)%。从NK细胞分离的外泌体直径为30~150 nm,且表达ALIX、 TSG101、 CD81,不表达calnexin。NK细胞来源外泌体抑制NEC细胞增殖和侵袭、增加细胞凋亡,降低S期细胞比例并增加G1期细胞比例。过表达miR-30e-3p的NK细胞来源外泌体抑制NEC细胞增殖和侵袭,并阻滞细胞周期和促进细胞凋亡,敲低miR-30e-3p的NK细胞来源外泌体则相反。结论NK细胞来源外泌体miR-30e-3p阻断人ESCC细胞的细胞周期,抑制其增殖和侵袭并诱导其凋亡。
Objective To investigate the effects of natural killer(NK)-ell-derived miR-30e-3p-containing exosomes(Exo)on esophageal squamous cell carcinoma(ESCC)cell proliferation,apoptosis and invasion.Methods NK cells were isolated and amplified from the peripheral blood of healthy donors,and NK cell-derived Exo was isolated and identified,which were further co-cultured with NEC cells and were randomly grouped into Exol and Exo2 groups.Transmission electron microscopy(TEM)was used to observe the morphology and size of exosomes.Western blot analysis was used to detect the expression levels of exosome markers apoptosis related gene 2-interacting protein X(ALIX),tumor susceptiblity gene 101(TSG101),CD81 and calnexin.The NC plasmids,mimics and inhibitors of miR030e-3p were respectively delivered into the NK cells,and the corresponding NK cells-derived Exo were co-cultured with NEC cells,which were divided into NC,Exo,mimic and inhibitor groups.CCK-8 assay was used to evaluate cell proliferation,flow cytometry was conducted to determine cell cycle,annexin V-FITC/PI double staining was employed to detect cell apoptosis,and TranswellTM assay was performed to detect cell invasion abilities.Real-time quantitative PCR was used to detect the expression of miR-23b,miR-422a,miR-133b,miR-124,miR-30e-3p and miR-99a in NCE cells and exosomes.Results The percentages of CD56^(+)CD3^(+)cells and CD56^(+)CD16^(+)cells in NK cells were(0.071±0.008)%and(90.6±10.6)%,respectively.Exosome isolated from NK cells ranged from 30 nm to 150 nm,and was positive for ALIX,TSG101 and CD81,while negative for calnexin.NK cell-derived Exos inhibited the proliferation,reduced the proportion of S-phase clls and the number of invaded cells of NEC cells,and promoted the apoptosis and the proportion of G1 phase cells.Overexpression of miR-30E-3p in NK cell-derived exosome inhibited the proliferation and invasion of NEC cells,and blocked cell cycle and promoted apoptosis,while knockdown miR-30e-3p in NK cell-derived exosomes did the opposite.Conclusion miR-30e-3p in NK cell-derived exosomes can inhibit the prolifteration and invasion of ESCC cells,block their cell cycle and induce their apoptosis.
作者
孙明月
李洪霖
冯保荣
SUN Mingyue;LI Honglin;FENG Baorong(Oncology Section 2,Second Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450002,China;Tumor ZoneⅡ,Second Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450002,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2023年第4期295-302,共8页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81804057)
河南省中医药管理局基金(2017JDZX016)。
