Exosomal miR-1305 in the oncogenic activity of hypoxic multiple myeloma cells: a biomarker for predicting prognosis

Abstract

Background: Exosomes have emerged as important mediators of tumor progression, and a prognostic role for serum exosomal miRNAs has been suggested in multiple myeloma (MM). Given the association of hypoxia with tumor aggressiveness, including cancer stem cell-like phenotypes, we explored exosomal miRNAs from MM cells under hypoxic conditions and analyzed their diverse roles both in promoting oncogenic activity and in predicting prognosis. Methods: The human MM cell line, RPMI 8226, was cultured under hypoxic conditions and their exosome production and exosomal miRNA profiles were compared with those of normoxic parental cells. The survival outcome of myeloma patients was compared using serum levels of exosomal miRNAs, and the effects of exosomal miRNAs on the target genes of MM cells and adjacent immune cells were analyzed. Results: Increased expression of stem cell markers and exosome production were observed in hypoxic MM cells. Exosome miRNA analysis identified a higher expression of miR-1305 in exosomes isolated from hypoxic MM cells than in those of normoxic parental cells. The overall survival of patients with high exosomal miR-1305 was poorer than it was in patients with low exosomal miR-1305. In hypoxic MM cells, an increase of exosomal miR-1305 led to a decrease of cellular miR-1305 and increased expression of the miR-1305 target genes, MDM2, IGF1 and FGF2 resulted in the promotion of oncogenic activity of MM. Exosomal miR-1305 was also transferred from MM cells to macrophages, and miR-1305-transferred macrophages showed tumor-promoting, M2-macrophage phenotypes. Conclusions: Exosome-mediated secretion of miR-1305 in MM cells promoted oncogenic activity of hypoxic MM cells and high serum levels of exosomal miR-1305.

Keywords: exosome; hypoxia; miR-1305; microRNA; multiple myeloma.

Figure 1

Exosomal production is upregulated during long-term culture of myeloma cells under hypoxia. RPMI 8226 cells were cultured under hypoxic conditions (1% or 2% O₂) or normoxic conditions (21% O₂) for the indicated times. (A) mRNA expression of HIF-1α and stem cell markers (Nanog, Oct4 and SOX2) were analyzed by RT-qPCR. Increased colony formation was found in hypoxic RPMI 8226 cells. The data represent the mean ± SEM of three independent experiments, *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant. (B) Heat map of the differentially expressed genes (DEGs) in both hypoxia conditions. (C) Gene ontology (GO) enrichment analysis for DEGs using DAVID. The bar graph shows GO term with the overlap ratio of more than 10% between DEGs and GO genes. (D) Protein concentration of exosomes derived from RPMI 8226 cells cultured under hypoxic or normoxic conditions for the indicated times. Western blot analysis of the exosomal markers Alix and CD63 from RPMI 8226 cells cultured for 4 weeks. (E) Representative particle concentration and size distribution of exosomes derived from RPMI 8226 cells cultured under hypoxic or normoxic conditions. Transmission electron microscopic images of exosomes (Scale bar: 100 nm).

Figure 2

miRNA profiles of hypoxic exosome and its association with overall survival rates in MM patients. (A) Exosomal miRNA profiles of RPMI 8226 cells cultured under hypoxic conditions (1% or 2% O₂). Fold change vs normoxic conditions (21% O₂). (B) Kaplan-Meier curves representing the correlation between miRNA signature based on exosomal miR-1305, miR-30d-5p and miR-21-5p expression levels and overall survival rates in MM patients. (C) MM patients who were initially treated with bortezomib-containing chemotherapy showed a significantly poorer overall survival rate in the high exosomal miR-1305 group. (D) Target gene prediction of differentially expressed miR-1305. Venn diagram showing the overlap of miR-1305 target genes with a high rate of context score by using three different miRNA target analysis programs. Representative pathways of miR-1305 target genes are summarized.

Figure 3

Hypoxia-induced downregulation of cellular miR-1305 and upregulation of miR-1305 target genes in MM cells. RPMI 8226 cells were cultured under normoxic conditions (21% O₂) or hypoxic conditions (2% O₂) for 48 h. (A) Schematic model for comparison of cellular and exosomal miR-1305 from RPMI 8226 cells cultured under hypoxia. (B) The level of cellular and exosomal miR-1305 was analyzed by RT-qPCR. Cellular miR-1305 level was decreased, but exosomal miR-1305 level was increased under hypoxia. (C) Cellular expression of hypoxia markers (GLUT1, HIF2α, PDK1 and PHD2) was upregulated in RPMI 8226 cells cultured under hypoxia. (D) Cellular expression of the miR-1305 candidate targets (FGF2, IGF1 and MDM2) in RPMI 8226 cells cultured under hypoxia. (E) Schematic model for hypothetical consequences caused by myeloma cells cultured under hypoxia. Upregulation of miR-1305 target gene expression was caused by reduced cellular miR-1305 in hypoxic RPMI 8226 cells, which may lead to more aggressive myeloma cells and myeloma patients with poor prognosis.

Figure 4

Transfer of miR-135b derived from myeloma cells to THP-1 macrophages via exosomes. (A) Schematic model of the experiment to confirm the effects of exosomal miR-1305 on the tumor microenvironment. THP-1 macrophage cells were seeded in the lower chamber of a Transwell plate, while miR-1305-transfected RPMI 8226 myeloma cells were seeded on the top of the insert. (B) Internalization of myeloma cell-derived exosomes by THP-1 cells. THP-1 macrophage cells were cultured with PKH67-labeled exosomes derived from RPMI 8226 cells. Representative confocal microscopy image shows the PKH67-labeled exosome (green) signals detected in the cytoplasm of THP-1 macrophage cells (red). Nuclear counterstaining was performed using DAPI (blue). (C) Quantitative RT-PCR for miR-1305 was carried out using RNA isolated from RPMI 8226 and THP-1 cells after 48 h and 72 h of coculture. The data represent the mean ± SEM of three independent experiments. ***p < 0.001. (D) Expression of M2 macrophage markers in THP-1 cells in response to exosomes derived from RPMI 8226/miR-1305_mimic or control (RPMI 8226/miR-NC_mimic). Quantitative RT-PCR for TGF-β, IL10, CCL2 and CD206 was carried out using RNA isolated from THP-1 cells after 48 h or 72 h of coculture. TGF-β and CCL2 expression was upregulated by the addition of RPMI 8226/miR-1305_mimic exosomes compared with control. (E) Proposed model in this study for how myeloma-derived miR-1305 may promote exosome-induced macrophage polarization in multiple myeloma.