Cancer-derived exosomal miR-138-5p modulates polarization of tumor-associated macrophages through inhibition of KDM6B

Abstract

Rationale: Differential activation of macrophages correlates closely with tumor progression, and the epigenetic factor lysine demethylase 6B (KDM6B, previously named JMJD3) mediates the regulation of macrophage polarization through an unknown mechanism. Methods: We developed a suspension coculture system comprising breast cancer cells and macrophages and used RT-qPCR and western blotting to measure KDM6B expression. Bioinformatics and luciferase reporter assays were used to identify candidate microRNAs of cancer cells responsible for the downregulation of KDM6B. To determine if exosomes mediated the transfer of miR-138-5p between cancer cells to macrophages, we treated macrophages with exosomes collected from the conditioned medium of cancer cells. The effects of exosomal miR-138-5p on macrophage polarization were measured using RT-qPCR, flow cytometry, and chromatin immunoprecipitation assays. We employed a mouse model of breast cancer, metastatic to the lung, to evaluate the effects on tumor metastasis of macrophages treated with miR-138-5p-enriched exosomes. To develop a diagnostic evaluation index, the levels of exosomal miR-138-5p in samples from patients with breast cancer were compared to those of controls. Results: Coculture of breast cancer cells led to downregulation of KDM6B expression in macrophages. Cancer cell-derived exosomal miR-138-5p inhibited M1 polarization and promoted M2 polarization through inhibition of KDM6B expression in macrophages. Macrophages treated with exosomal miR-138-5p promoted lung metastasis, and the level of circulating exosomal miR-138-5p positively correlated with the progression of breast cancer. Conclusion: Our data suggest that miR-138-5p was delivered from breast cancer cells to tumor-associated macrophages via exosomes to downregulate KDM6B expression, inhibit M1 polarization, and stimulate M2 polarization. Therefore, exosomal miR-138-5p represents a promising prognostic marker and target for the treatment of breast cancer.

Keywords: exosomes; lysine demethylase 6B (KDM6B); macrophage polarization; microRNA-138-5p; tumor-associated macrophages.

Figure 1

miR-138-5p inhibits the expression of KDM6B in macrophages. THP-1 cells were cocultured with MDA-MB-231 cells for 48 h. (A) Schematic of screening for candidate miRNAs. (B) RT-qPCR analysis of miR-138-5p, miR-146a, and miR-20a-3p expression in THP-1 cells. U6 was used as an internal control. (C) RT-qPCR analysis of miR-138-5p and miR-146a expression in THP-1 cells transfected with negative control (NC), miR-138-5p or miR-146a mimics for 48 h. (D) RT-qPCR and western blot analyses of KDM6B expression in THP-1 cells transfected with NC, miR-138-5p or miR-146a mimics. (E) Predicted miR-138-5p binding sites in the 3´-UTR of KDM6B, and the seed sequences of miR-138-5p of different species. (F) The mutant construct with miR-138-5p binding sites in the 3´-UTR of KDM6B (left). The pmirGLO reporter harboring the 3´-UTR of KDM6B with wild-type (wt) or mutated (mt) miR-138-5p binding sites were used to cotransfected THP-1 cells with miR-138-5p mimics or NC. Luciferase activity was analyzed 40 h after transfection (right). The results are shown as the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

miR-138-5p mediates cancer cell-induced inhibition of KDM6B expression in macrophages. THP-1 cells were cocultured with MDA-MB-231 cells for 48 h. (A) RT-qPCR analysis of miR-138-5p expression in MCF7 and T47D cells transfected with NC or miR-138-5p mimics. (B) RT-qPCR analysis of miR-138-5p expression in THP-1 cells treated with conditioned medium (CM) of cultures of MDA-MB-231 cells for 48 h. THP-1 cells were cocultured for 48 h with MCF7 or T47D cells transfected NC or miR-138-5p mimics. (C) RT-qPCR analysis of levels of miR-138-5p in THP-1 cells. (D, E) The analysis of KDM6B mRNA and protein levels in THP-1 cells. The results are shown as the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Cancer cell-derived exosomal miR-138-5p downregulates KDM6B expression. THP-1 cells were cocultured with MDA-MB-231 cells for 48 h. (A) RT-qPCR analysis of pri-miR-138 and pre-miR-138 expression in THP-1 cells. (B) RT-qPCR analysis of the expression levels of miR-138-5p in the CM of MDA-MB-231 cells treated with RNase A alone or in combination with Triton X-100. (C) RT-qPCR analysis of miR-138-5p levels in the CM of MDA-MB-231 cells depleted of exosomes using GW4869 or ultracentrifugation. CM of MDA-MB-231 cells treated with 10 μM GW4869 were collected. (D) RT-qPCR analysis of miR-138-5p expression in THP-1 cells treated with the indicated conditioned medium for 48 h. (E) Western blot analysis of KDM6B expression in THP-1 cells. (F) RT-qPCR analysis of miR-138-5p expression in THP-1 cells treated with exosomes from MDA-MB-231 cells. (G) THP-1 cells transfected with NC or an miR-138-5p inhibitor were treated with PBS or exosomes from MDA-MB-231 cells for 48 h. RT-qPCR and western blot analyses KDM6B expression in the indicated THP-1 cells. (H) RT-qPCR analysis of miR-138-5p levels in the exosomes derived from T47D cells transfected with NC or miR-138-5p mimics for 48 h. (I) RT-qPCR analysis of miR-138-5p expression in THP-1 cells treated with PBS or exosomes derived from T47D cells transfected with NC or miR-138-5p mimics. (J) RT-qPCR and western blot analysis of KDM6B expression in THP-1 cells treated with PBS or its levels in exosomes from T47D cells transfected with NC or miR-138-5p mimics. The results are shown as the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Exosomal miR-138-5p regulates macrophage polarization via inhibiting KDM6B expression. (A) RT-qPCR analysis of M1- and M2-associated genes, (B) western blot analysis of CD163 and TNF-α expression, and (C) Flow cytometry of CD163-positive cells in KDM6B-overexpressing THP-1 cells cocultured with MDA-MB-231 cells for 48 h. THP-1 cells overexpressing miR-138-5p or NC were transfected with the KDM6B expression plasmid or control for 48 h. (D) RT-qPCR analysis of M1- and M2-associated genes expressed in THP-1 cells. (E) Western blot analysis of CD163 and TNF-α expression in THP-1 cells. (F) Flow cytometric analysis of CD163-positive cells. (G) RT-qPCR analysis of M1- and M2-associated genes, (H) western blot analysis of CD163 and TNF-α expression, and (I) CD163-positive cells in THP-1 cells incubated with PBS or with exosomes derived from T47D cells transfected with NC or miR-138-5p mimics. (J) RT-qPCR analysis of M1- and M2-associated gene expression, (K) western blot analysis of CD163 and TNF-α expression and (L) flow cytometric analysis of CD163-positive cells in THP-1 cells. THP-1 cells transfected with NC or an miR-138-5p inhibitor were treated with PBS or exosomes from MDA-MB-231 cells for 48 h. The results are shown as the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

KDM6B demethylase activity increases the expression of M1-related genes. THP-1 cells overexpressing KDM6B were cocultured with MDA-MB-231 cells for 48 h. (A) Dual-luciferase reporter assay analysis of the promoter activities of M1-associated genes in THP-1 cells overexpressing KDM6B and controls and in THP-1-KDM6B-knockdown and scrambled-sequence control THP-1 cells. (B) Western blot analysis of KDM6B and H3K27me3 expression in THP-KDM6B and THP-shKDM6B cells, respectively. ChIP array analysis of the enrichment of KDM6B at the promoter regions of the genes encoding IL-6, TNF-α, and IL-1β in THP-1 cells overexpressing KDM6B overexpressing and control THP-1 cells (C) or THP-1-KDM6B-knockdown and scrambled-sequence control THP-1 cells (D). ChIP array analysis of the enrichment of H3K27me3 on the promoter regions of the genes encoding IL-6, TNF-α, and IL-1β in THP-1 cells overexpressing KDM6B and control THP-1 cells (E) or THP-1-KDM6B-knockdown and scrambled sequence control THP-1 cells (F) Data are shown as the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

Exosomal miR-138-5p promotes lung metastasis of breast cancer. Macrophages were depleted using clodronate liposomes (CL). The mice were then intravenously injected with Raw264.7 cells treated with exosomes followed by tail-vein injection of 4T1-luciferase cells. (A) Schematic of the lung metastasis assay. (B) Representative images of lung luminescence of mice (left) and the analysis of the results of luminescent photon flux (right). (C) Representative images of HE-stained lung tissues (right panel). (D) Representative images of immunohistochemical analysis of CD206 expression in lung tissues (left) and analysis (right). (E) Representative images of immunohistochemical analysis of Arg1 expression in lung tissues (left) and analysis (right). Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.