南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (9): 1525-1535.doi: 10.12122/j.issn.1673-4254.2023.09.10

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JAG1影响单核-巨噬细胞重塑三阴性乳腺癌转移前微环境：基于外泌体中的LncRNA MALAT1

徐梦歧，石宇彤，刘俊平，吴敏敏，张凤梅，何志强，唐 敏   

  1. 重庆医科大学检验医学院//临床检验诊断学教育部重点实验室，重庆 400016
  • 出版日期:2023-09-20 发布日期:2023-09-28

JAG1 affects monocytes-macrophages to reshape the pre-metastatic niche of triple-negative breast cancer through LncRNA MALAT1 in exosomes

XU Mengqi, SHI Yutong, LIU Junping, WU Minmin, ZHANG Fengmei, HE Zhiqiang, TANG Min   

  1. Key Laboratory of Clinical Laboratory and Diagnostics of Ministry of Education, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
  • Online:2023-09-20 Published:2023-09-28

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摘要： 目的 探讨JAG1对单核-巨噬细胞在三阴性乳腺癌（TNBC）中肿瘤转移前微环境（PMN）中作用的影响及其可能的调控机制，以期为TNBC诊疗提供新的思路和靶点。方法 人TNBC细胞MDA-MB-231，MDA-MB-231B体外培养，qRT-PCR检测JAG1的表达。动物实验将雌性裸鼠分为MDA-MB-231组和MDA-MB-231B组，5只/组，分别往乳腺脂肪垫注射细胞5×106，6周取肿瘤组织进行免疫组化分析。人源单核细胞THP-1为研究对象，分别经人源JAG1重组蛋白（rhJAG1）直接处理或经TNBC条件培养基（CM）处理，通过单核-内皮粘附、Transwell、qRT-PCR和Western blot实验检测JAG1对单核细胞的直接作用和在PMN中的影响。实验分为5组：Control组：THP-1正常培养；231-CM组：THP-1+231-CM；231-JAG1-CM组：THP-1+rhJAG1处理后231-CM；231B-CM组：THP-1+231B-CM；231-DAPT-CM组：THP-1+DAPT处理后231-CM。透射电镜（TEM）、纳米颗粒跟踪分析（NTA）检测JAG1对TNBC外泌体分泌影响。生物信息学筛选与LncRNA MALAT1相互作用的miRNA并行qRT-PCR验证。结果 与MDA-MB-231相比，侵袭株MDA-MB-231B的JAG1表达更高（P<0.01），肝转移面积更大；rhJAG1直接处理和TNBC条件培养基处理均能促进单核细胞的黏附、迁移，并影响其分化（均P<0.05）；透射电镜和NTA检测显示JAG1能促进MDA-MB-231外泌体分泌数量增多（P<0.01），外泌体中LncRNA MALAT1含量升高（P<0.0001）。生物信息学分析得到与 LncRNA MALAT1 和 JAG1 相关的 5 个候选 miRNA，实验证实 miR-26a-5p 在 TMN 中的单核-巨噬细胞中受到MALAT1的靶向调控（P<0.0001）。结论 JAG1能促进TNBC的外泌体分泌并影响其中Lnc MALAT1的表达，从而靶向PMN中单核-巨噬细胞miR-26a-5p的表达，从而使单核-巨噬细胞中JAG1表达升高，进而影响单核-巨噬细胞在PMN中的黏附、迁移和破骨分化作用。

关键词: JAG1；三阴性乳腺癌；单核-巨噬细胞；外泌体；肿瘤转移前微环境；LncRNA MALAT1

Abstract: Objective To investigate the effect of JAG1 on the activities of monocytes-macrophages in pre-metastatic niche (PMN) of triple-negative breast cancer (TNBC) and explore the possible regulatory mechanism. Methods JAG1 expression in human TNBC MDA-MB-231 and MDA-MB-231B cells was detected using quantitative real-time PCR (qRT-PCR). Ten female nude mice were inoculated with MDA-MB-231 cells (n=5) or MDA-MB-231B cells (n=5) in the mammary fat pad, and 6 weeks later, the tumor tissues were collected for immunohistochemistry. Human monocytes THP-1 cells were treated with rhJAG1 or conditioned media (CM) of TNBC MDA-MB-231 and MDA-MB-231B cells to assess the direct effect of JAG1 on monocytes and its effect on monocytes in the PMN using monocyte-endothelial adhesion, Transwell assay, qRT- PCR and Western blotting. Transmission electron microscopy and nanoparticle tracking analyses were used to identify the effect of JAG1 on exosome release from the TNBC cells. MiRNAs interacting with lncRNA MALAT1 were identified by bioinformatics and validated using qRT- PCR. Results Compared with MDA-MB-231 cells, the invasive strain MDA-MB- 231B cells showed significantly higher JAG1 expression and greater liver metastasis potential (P<0.01). Both direct treatment with rhJAG1 and treatment with the conditioned media promoted adhesion and migration and affected differentiation of the monocytes (P<0.05). Transmission electron microscopy and nanoparticle tracking analysis showed that JAG1 strongly enhanced exosome secretion from MDA-MB-231 cells (P<0.01) and increased MALAT1 content in the exosomes (P<0.0001). Five candidate miRNAs related to MALAT1 and JAG1 were identified by bioinformatics analysis, and miR-26a-5p was identified as a potential target of MALAT1 in monocytes-macrophages in TMN (P<0.0001). Conclusion JAG1 can promote exocrine secretion of TNBC and increase the expression of MALAT1 to cause targeted downregulation of miR- 26a- 5p in monocytes-macrophages in the PMN, which in turn increases JAG1 expression in monocytes-macrophages to affect their adhesion, migration and osteoclast differentiation in the PMN.

Key words: JAG1; triple- negative breast cancer; monocytes-macrophages; exosomes; pre- metastatic niche; long non-coding RNA MALAT1