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