Macrophage exosomes modified by miR-365-2-5p promoted osteoblast osteogenic differentiation by targeting OLFML1

Abstract

In the bone immune microenvironment, immune cells can regulate osteoblasts through a complex communication network. Macrophages play a central role in mediating immune osteogenesis, exosomes derived from them have osteogenic regulation and can be used as carriers in bone tissue engineering. However, there are problems with exosomal therapy alone, such as poor targeting, and the content of loaded molecules cannot reach the therapeutic concentration. In this study, macrophage-derived exosomes modified with miR-365-2-5p were developed to accelerate bone healing. MC3T3-E1 cells were incubated with the culture supernatants of M0, M1 and M2 macrophages, and it was found that the culture medium of M2 macrophages had the most significant effects in contributing to osteogenesis. High-throughput sequencing identified that miR-365-2-5p was significantly expressed in exosomes derived from M2 macrophages. We incubated MC3T3-E1 with exosomes overexpressing or knocking down miR-365-2-5p to examine the biological function of exosome miR-365-2-5p on MC3T3-E1 differentiation. These findings suggested that miR-365-2-5p secreted by exosomes increased the osteogenesis of MC3T3-E1. Moreover, miR-365-2-5p had a direct influence over osteogenesis for MC3T3-E1. Sequencing analysis combined with dual luciferase detection indicated that miR-365-2-5p binded to the 3'-UTR of OLFML1. In summary, exosomes secreted by M2 macrophages targeted OLFML1 through miR-365-2-5p to facilitate osteogenesis.

Keywords: exosomes; macrophage; osteogenesis; osteoimmunology.

Graphical abstract

Figure 1.

Effects of conditioned medium on MC3T3-E1 osteogenesis. (A) The expression of CD86 (M1 marker) and CD206 (M2 marker) in macrophages was measured by qRT-PCR. (B) The schematic diagram of co-culture for macrophages and MC3T3-E1. (C) The osteogenesis of MC3T3-E1 was analyzed by ALP staining after 7 days in the co-culture system. (D) The ability of MC3T3-E1 to form mineralized nodules was analyzed by ARS staining after 14 days in the co-culture system. (E) The mRNA expression of COL-1, Runx2 and ALP in MC3T3-E1 was analyzed by qRT-PCR after 7 days in the co-culture system. ^*P<0.05, ^**P<0.01, ^***P<0.01.

Figure 2.

Extraction and characterization of exosomes secreted by M2 macrophage. (A) Exosomes preparation process. (B) TEM diagram of exosomes (scale bar: 100 nm). (C) Particle size analysis of exosomes. (D) Concentration information of exosomes. (E) Analysis of exosome uptake by MC3T3-E1. (F) Effects of different concentrations of M2-exos on MC3T3-E1 proliferation.

Figure 3.

Identification of differentially expressed miRNAs of M2-exos. (A) Expression of miRNAs in M1-exos and M2-exos. (B) The number of differentially expressed miRNAs in M2-exos. (C) Differentially expressed miRNAs in M2-exos. (D) Expression profiles of differential miRNAs highly expressed in M2-exos. (E) Differential miRNAs highly expressed in M2-exos were confirmed by qRT-PCR.^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 4.

Influence of miR-365-2-5p secreted by M2-exos on MC3T3-E1 osteogenesis. (A) Fluorescence microscopy for GFP expression. (B) MiR-365-2-5p expression in M2 macrophage was confirmed by qRT-PCR. (C) Expression of miR-365-2-5p in cells following co-culture of MC3T3-E1 with M2-exos overexpressing and knocking down miR-365-2-5p by qRT-PCR. (D) ALP staining to detect the impact of M2-exos secreted miR-365-2-5p on MC3T3-E1 osteogenic differentiation (scale bar: 100 µm). (E) ARS staining was employed to observe the influence of miR-365-2-5p secreted by M2-exos on MC3T3-E1 mineralization (scale bar: 100 µm). (F) Influence of miR-365-2-5p secreted by M2-exos on the expression of osteogenesis-related genes (ALP, Runx2 and BMP2) in MC3T3-E1 cells. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 4.

(Continued).

Figure 5.

The effect of miR-365-2-5p on osteogenic differentiation of MC3T3-E1. (A) A cell model of MC3T3-E1 overexpressing miR-365-2-5p was constructed. (B) CCK-8 was used to examine the efficacy of miR-365-2-5p on the proliferation of MC3T3-E1. (C) The effects of miR-365-2-5p on mRNA expression of OPN, COL-1, OCN and Runx2 were measured by qRT-PCR. (D) The influence of miR-365-2-5p on Runx2, BMP2 and OPN protein expression was measured by WB. (E) The influence of miR-365-2-5p on extracellular matrix mineralization was observed by ARS staining (scale bar: 100 µm). (F) The influence of miR-365-2-5p on osteogenesis of MC3T3-E1 was observed by ALP staining (scale bar: 100 µm). ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 6.

The mechanism of miR-365-2-5p mediating MC3T3-E1 osteogenic differentiation was analyzed by bioinformatics. (A) Volcano map of DEs in OE-NC group and OE-miR-365-2-5p group. (B) Histogram of DEs in OE-NC and OE-miR-365-2-5p groups. (C) Heatmap of DEs in OE-NC and OE-miR-365-2-5p groups. (D) The target genes of miR-365-2-5 p were analyzed by Venn diagram. (E) The expression of OLFML1 and GNB4 in MC3T3-E1 cells was determined by qRT-PCR. (F) Heatmap of down-regulated gene expression profiles in the OE-miR-365-2-5p group. ^**P<0.01, ^***P<0.001.

Figure 7.

MiR-365-2-5p Promoted MC3T3-E1 bone formation by targeting OLFML1. (A) Diagram of miR-365-2-5p and OLFML1 binding sequence. (B) The construction of 293 T cell model overexpressing miR-365-2-5p. (C) The targeting relationship between miR-365-2-5p and OLFML1 was detected by dual luciferase assay.^**P<0.01.

Figure 8.

MiR-365-2-5p derived from M2 macrophage exosomes promotes osteogenesis of MC3T3-E1 by targeting OLFML1.