缺氧调节肺腺癌小鼠MDSCs来源外泌体携带的miRNA及其免疫抑制功能

Hypoxia regulates miRNA and immunosuppressive function of MDSCS-derived exosomes

目的观察正常(21%O2)和缺氧(1%O2)条件下,髓源性抑制细胞(myeloid-derived suppressor cells,MDSCs)来源外泌体(exosomes derived from MDSCs,MDSCs-EX)的免疫抑制功能,探究缺氧对MDSCs-EX携带miRNA成分以及免疫抑制能力的影响。方法构建肺腺癌小鼠移植瘤模型,磁珠分选脾脏中的MDSCs,分别在21%O2和1%O2条件下培养MDSCs,提取上清中的外泌体。通过透射电子显微镜观察形态、流式检测颗粒直径、Western bolt检测特异性蛋白鉴定制备的外泌体。观察正常和缺氧条件下MDSCs-EX对CD3+T细胞增殖的影响。对正常和缺氧条件下MDSCs-EX进行miRNA测序分析,观察2者在miRNA表达上的差异,将差异表达前十位的miRNA作为待筛选miRNA。运用siRNA特异性敲减待筛选miRNA,观察缺氧条件下MDSCs-EX携带的miRNA对其免疫抑制功能的影响。结果成功分选出肺腺癌小鼠脾脏中的MDSCs,并制备了正常和缺氧条件下的MDSCs-EX。相比正常条件下制备的MDSCs-EX,缺氧条件下MDSCs-EX对于CD3+T细胞增殖的抑制能力更强。敲减缺氧条件下MDSCs-EX中表达明显上调的miR-43-3p和Let-7c-5p,MDSCs-EX抑制CD3+T细胞增殖的能力减弱。结论相比正常氧含量环境,缺氧条件下制备的MDSCs-EX具有更强的免疫抑制能力,这与缺氧MDSCs-EX中携带更多的miR-43-3p和Let-7c-5p相关。

This study was performed to investigate the effects of hypoxia on miRNA components and immunosuppressive ability of myeloid-derived suppressor cells(MDSCs)by comparing the immunosuppressive function of exosomes(EX)in the culture supernatant of MDSCs under normal(21%O2)and hypoxia(1%O2)conditions.Firstly,the lung adenocarcinoma mouse model was constructed,and MDSCs were sorted by magnetic beads from spleens,and then cultured under normal or hypoxia conditions.Exosomes were extracted from the supernatant,and then identified by transmission electron microscopy,flow particle size analysis and Western blotting.The effects of MDSCs-EX from normal and hypoxia conditions on the proliferation of CD3+T cells was observed.The miRNA sequencing analysis was performed to observe the difference in miRNA expression between MDSCS-EX under normal and hypoxia conditions,and the top 10 miRNA with differential expression were selected as screened miRNA.SiRNA specific knockdown was used to observe the effect of miRNA carried by MDSCS-EX on its immunosuppressive function.Compared with MDSCS-EX prepared under normal condition,MDSCS-EX prepared under hypoxia inhibited the proliferation of CD3+T cells more strongly.After knockdown of miR-43-3p and Let-7c-5p in MDSCsEX prepared under hypoxia,the suppressive ability of MDSCs-EX decreased.In conclusion,MDSCS-EX prepared under hypoxia has stronger immunosuppressive ability compared with normal oxygen content environment,which is associated with higher expression of miR-43-3p and Let-7c-5p.