摘要
目的:明确宫颈癌细胞分泌的外泌体源性miR-23a-3p对肿瘤相关巨噬细胞极化方向的影响,探究宫颈癌细胞迁移及侵袭行为的机制。方法:纳米颗粒跟踪分析、透射电镜观察和Western blot鉴定宫颈癌细胞分泌的外囊泡是否为外泌体。建立佛波酯(PMA)诱导急性单核细胞白血病细胞(THP-1)成巨噬细胞模型,免疫荧光检测巨噬细胞摄取外泌体情况。巨噬细胞添加SiHa/HeLa分泌的外泌体处理或转染miR-23a-3p mimics/miR-23a-3p inhibitors处理后,RT-qPCR、Western blot检测巨噬细胞标志物表达水平;荧光素酶报告基因验证miR-23a-3p与PTEN 3’-UTR的结合能力。宫颈癌细胞与巨噬细胞利用Transwell小室共培养,检测宫颈癌细胞的迁移及侵袭能力在SiHa/HeLa分泌的外泌体处理或转染miR-23a-3p mimics前后的差异。结果:经过纳米颗粒跟踪分析、透射电镜和Western blot检测外泌体标志蛋白后确认宫颈癌细胞分泌的外囊泡为外泌体,并且发现SiHa/HeLa分泌的外泌体源性miR-23a-3p表达较细胞内高。利用PMA诱导THP-1细胞分化成巨噬细胞,通过免疫荧光观察到巨噬细胞能够摄取外泌体。RT-qPCR检测巨噬细胞摄取外泌体后,M2型标志物TGF-β、CD206、Argians-1表达较非摄取组明显升高(P<0.05),而M1标志物iNOS表达较之减少(P<0.05)。巨噬细胞转染miR-23a-3p mimics处理后,得到同样的结果。通过体外宫颈癌细胞和巨噬细胞共培养发现,肿瘤源性外泌体或转染miR-23a-3p mimics诱导的M2型巨噬细胞可使宫颈癌细胞迁移及侵袭增加(P<0.05)。双荧光素酶报告基因检测发现,外泌体源性miR-23a-3p能结合到巨噬细胞内PTEN的3’-UTR,影响PTEN表达。Western blot检测发现,巨噬细胞转染miR-23a-3p mimics后PTEN表达水平下降(P<0.05),且下游p-AKT增加(P<0.05);反之,转染miR-23a-3p inhibitors后PTEN表达增加(P<0.05),且p-AKT磷酸化降低(P<0.05)。结论:宫颈癌细胞分泌的外泌体源性miR-23a-3p诱导巨噬细胞M2型极化,通过下调M2型巨噬细胞PTEN激活AKT信号通路,从而增强宫颈癌细胞迁移及侵袭能力。
Objective:To clarify the effect of exosome-derived miR-23a-3p secreted by cervical cancer cells on the polarization direction of tumor-associated macrophages,and thus exploring the mechanism of migration and invasion behaviors of cervical cancer cells.Methods:Nanoparticle tracking analysis and transmission electron microscopy were used to identify whether the extracellular vesicles secreted by cervical cancer cells were exosomes.A model of acute monocytic leukemia cells(THP-1)induced by phorbol ester(PMA)to form macrophages was established,and immunofluorescence was used to detect the uptake of exosomes by macrophages.After macrophages were treated with exosomes secreted by SiHa/HeLa or transfected with miR-23a-3p mimics/miR-23a-3p inhibitors,the expression levels of macrophage markers,as well as phosphatase and tensin homolog(PTEN),protein kinase B(AKT),and phosphorylated protein kinase B(p-AKT)were detected.Luciferase reporter gene was used to verify the binding ability of miR-23a-3p to the 3’-UTR of PTEN.Cervical cancer cells and macrophages were co-cultured in Transwell chambers,and the differences in the migration and invasion abilities of cervical cancer cells before and after treatment with exosomes secreted by SiHa/HeLa or transfection were compared for analysis.Results:After detecting the exosomal marker proteins,it was confirmed that the extracellular vesicles secreted by cervical cancer cells were exosomes,and it was found that the expression of exosome-derived miR-23a-3p secreted by SiHa/HeLa was higher than that in cells.THP-1 cells were induced by PMA to differentiate into macrophages.Immunofluorescence showed that macrophages could take up exosomes.RT-qPCR detection showed that after macrophages took up exosomes,the expressions of M2 markers such as TGF-β,CD206,and Argians-1 were significantly increased compared with the non-uptake group(P<0.05);while the expression of M1 marker iNOS comparatively decreased(P<0.05).The same results were obtained after macrophages were transfected with miR-23a-3p mimics.Through in vitro co-culture of cervical cancer cells and macrophages,it was found that tumor-derived exosomes or M2 macrophages induced by transfection with miR-23a-3p mimics could increase the migration and invasion of cervical cancer cells(P<0.05).Mechanistically,dual luciferase reporter gene detection showed that exosome-derived miR-23a-3p could bind to the 3’-UTR of PTEN in macrophages and affect the expression of PTEN.Western blot detection showed that after macrophages were transfected with miR-23a-3p mimics,the expression level of PTEN decreased(P<0.05),and the downstream p-AKT increased(P<0.05);conversely,after transfection with miR-23a-3p inhibitors,the expression of PTEN increased(P<0.05),and the phosphorylation of p-AKT decreased(P<0.05).Conclusion:Exosome-derived miR-23a-3p secreted by cervical cancer cells induces M2 polarization of macrophages,activates the AKT signaling pathway by downregulating PTEN in M2 macrophages,thus enhancing the migration and invasion abilities of cervical cancer cells.
作者
徐梦伟
吕单单
林晨
XU Mengwei;LYU Dandan;LIN Chen(Department of Pathology,School of Basic Medicine,Xinjiang Medical University,Urumqi 830054,China;Xinjiang Key Laboratory of Molecular Biology of Endemic Diseases,Urumqi 830017,China)
出处
《温州医科大学学报》
2025年第8期628-637,共10页
Journal of Wenzhou Medical University
基金
新疆维吾尔自治区自然科学基金青年基金项目(2021D01C292)。
