Supplementary MaterialsAdditional file 1: Supplementary Methods. treated with DMSO or GW4869, cell apoptosis (F), cell viability (G, H), IC50 for cDDP (I) was recognized. * em P /em ? ?0.05, ** em P /em ? ?0.01 (TIF 1036 kb) 13046_2019_1095_MOESM5_ESM.tif (1.0M) GUID:?0B33648C-9DC7-4C1C-AE5F-1FE9D358E06E Additional file 6: Figure S4. Exosomic miR-223 mediated cross-talk between macrophages and EOC cells. (A) Related miR-223 level in macrophages under normoxia and hypoxia. (B) Related miR-223 level in normoxic or hypoxic macrophages derived exosomes. (C) Collapse switch of miR-223 in A2780 cells cocultured with macrophages for the indicated time periods under normoxia or CP-690550 distributor hypoxia. (D) Related miR-223 level in A2780 cells treated with PBS, N-Mcr-exo or H-Mcr-exo. (E) Related pre-miR-223 levels in EOC cells cultured only, cocultured with macrophages or treated with H-Mcr-exo. (F-G) Collapse switch of miR-223 in A2780 cells cocultured with TAMs-conditioned, exosome-depleted TAMs-conditioned mass media or TAMs-conditioned mass media pretreated with GW4869 under normoxia (F) and hypoxia (G). (H) Flip transformation of miR-223 in A2780 treated with RNA polymerase II inhibitor or PBS. (I-J) Related miR-223 level in macrophages (I) or exosomes produced from macrophages (J) treated with PBS, DMOG or YC-1 in hypoxia and normoxia. (K-L) A2780 cells had been treated with exosomes produced from the normoxic or hypoxic macrophages that have been transfected with agomir or antagomir respectively, the related miR-223 level was assessed. (M) The amount of cell colonies was discovered in SKOV3 treated as indicated. * em P /em ? ?0.05, ** em P /em ? ?0.01 (TIF 2315 kb) 13046_2019_1095_MOESM6_ESM.tif (2.2M) GUID:?0A0B1CA6-F247-498B-91E3-20FC81AE2F8E Extra file 7: Figure S5. (A-B) Related miR-223 (A) and PTEN proteins (B) level in MCF-7, MBA-MD-231 and Hela cells treated with H-Mcr-exo or PBS, (C) IC50 Rabbit Polyclonal to OR13C4 for cDDP was assessed in SKOV3 cells (pretreated with LY294002) cocultured with exosomes produced from the normoxic or hypoxic macrophages that have been transfected with agomir. * em P /em ? ?0.05, ** em P /em ? ?0.01 (TIF 395 kb) 13046_2019_1095_MOESM7_ESM.tif (396K) GUID:?6683953D-051E-42EA-9D24-712DD0F09771 CP-690550 distributor Extra file 8: Figure S6. (A) Consultant pictures of low (still left) or high (best) HIF-1a appearance in EOC examples by immunohistochemical staining. (magnification, ?200). (B-C) Intertumoral degree of miR-223 (B), and Compact disc163+ cell infiltration (C) (representative of TAMs infiltration) had been assessed with high and low HIF-1a appearance in 28 principal EOC tissue. (D) Representative pictures of Compact disc81, Compact disc63, and CD9 in serum and its derived exosomes from an EOC patient. * em P /em ? ?0.05, ** em P /em ? ?0.01 (TIF 1007 kb) 13046_2019_1095_MOESM8_ESM.tif (1007K) GUID:?BB858759-EBD8-4AA9-BDAB-984C1CE738F1 Data Availability StatementThe datasets encouraging the conclusions of this article are included within the article and its additional documents. Abstract Background How exosomal microRNAs (miRNAs) derived from macrophages contribute to the development of drug resistance in the context of the hypoxic tumor microenvironment in epithelial ovarian malignancy (EOC) remains poorly understood. Methods The miRNA CP-690550 distributor levels were recognized by qRT-PCR. Protein levels of HIF-1, CD163 and PTEN-PI3K/AKT pathway were CP-690550 distributor assessed by Western blot (WB) and Immunohistochemistry (IHC). Exosomes were isolated, and then confirmed by Transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and WB. Internalization of macrophages-secreted exosomes in EOC cells was recognized by Confocal microscope. Subsequently, Dual-luciferase reporter assay verified PTEN was the prospective of miR-223. Gain- and loss-of-function experiments, save experiments, and SKOV3 xenograft models were performed to uncover the underlying mechanisms of miR-223 and PTEN-PI3K/AKT pathway, as well as the exosomal miR-223 in inducing multidrug resistance in vitro and in vivo. Results Here, we showed hypoxic EOC cells induced macrophages recruitment and induced macrophages into a tumor-associated macrophage (TAM)-like phenotype; exosomes derived from hypoxic macrophages enhanced the malignant phenotype of EOC cells, miR-223 was enriched in exosomes released from macrophages under hypoxia, which could be transferred to the co-cultivated EOC cells, accompanied by enhanced drug resistant.