Exosome-mediated microRNA transfer plays a role in radiation-induced bystander effect

Abstract

Bystander effects can be induced through cellular communication between irradiated cells and non-irradiated cells. The signals that mediate this cellular communication, such as cytokines, reactive oxygen species, nitric oxide and even microRNAs, can be transferred between cells via gap junctions or extracellular medium. We have previously reported that miR-21, a well described DDR (DNA damage response) microRNA, is involved in radiation-induced bystander effects through a medium-mediated way. However, the mechanisms of the microRNA transfer have not been elucidated in details. In the present study, it was found that exosomes isolated from irradiated conditioned medium could induce bystander effects. Furthermore, we demonstrated plenty of evidences that miR-21, which is up-regulated as a result of mimic transfection or irradiation, can be transferred from donor or irradiated cells into extracellular medium and subsequently get access to the recipient or bystander cells through exosomes to induce bystander effects. Inhibiting the miR-21 expression in advance can offset the bystander effects to some extent. From all of these results, it can be concluded that the exosome-mediated microRNA transfer plays an important role in the radiation-induced bystander effects. These findings provide new insights into the functions of microRNAs and the cellular communication between the directly irradiated cells and the non-irradiated cells.

Keywords: bystander effect; exosome; miR-21; microRNA; radiation.

Figure 1.

Bystander effects induced by exosomes isolated from conditioned medium of irradiated cells. (A) Frequency of micronuclei (MNF) in bystander cells treated for 48 h with exosomes isolated from conditioned medium harvested from directly irradiated cells. Cells were exposed to 0 Gy and 2 Gy of X-rays. Conditioned medium was harvested 4 h after exposure. (B) Yields of 53BP1 foci in bystander cells treated for 2 h with exosomes isolated from conditioned medium harvested from irradiated cells 4 h after exposure to 0 Gy and 2 Gy of X-rays. Error bars represent means ± standard error of 3 biological replicates and the superscript (*) denote a significant difference between groups (P < 0.05) as determined by Student's t test.

Figure 2.

Exosome-mediated miRNA shuttle between cells. (A) Cel-miR-39 expression of exosomes isolated from the refreshed medium of Cel-miR-39 transfected cells at different time points. (B) The expression of Cel-miR-39 in bystander cells treated with PBS or exosomes from Cel-miR-39 transfected cells. (C) Fluorescent visuals of MRC5 cells transfected with NC (negative control) or Cy3-miR-21. (D) Fluorescent visuals of MRC5 cells that were co-cultured with conditioned medium harvested from NC or Cy3-miR-21 transfected cells or exosomes isolated from conditioned medium of Cy3-miR-21 transfected cells. Nuclei were stained blue (DAPI) while F actin was stained green by fluorescein isothiocyanate labeled phalloidin (Phall) and Cy3-miR-21 displayed red fluorescence (Cy3). Arrows indicated the Cy3-miR-21. Error bars represent means ± standard error of 3 biological replicates and the superscript (**) denote a highly significant difference between groups (P < 0.01) as determined by Student's t test.

Figure 3.

Exosomes mediated the change of miR-21 expression levels in bystander cells. (A) MiR-21 expression in bystander MRC-5 cells after co-cultured with exosomes isolated from 0 and 2 Gy conditioned medium. (B) The expression of pre-miR-21 in bystander cells treated with exosomes isolated from 0 Gy or 2 Gy conditioned medium. (C) The mRNA expression of Bcl⁻2 in bystander cells 24 h after co-cultured with exosomes isolated from 0 Gy or 2 Gy conditioned medium. (D) Bcl^-2 protein expression by Western blotting assay in bystander cells 24 h after co-cultured with exosomes isolated from 0 Gy or 2 Gy conditioned medium. β-actin, loading control. Error bars represent means ± standard error of 3 biological replicates and the superscript (*) denote a significant difference between groups (P < 0.05) as determined by Student's t test.

Figure 4.

Exosomal miR-21 transfer induced bystander-like effects. (A) MiR-21 expression of exosomes isolated from medium of NC or miR-21 mimic transfected cells. (B) The expression of miR-21 in recipient cells treated with exosomes isolated from medium of NC or miR-21 mimic transfected cells. (C) The expression of pre-miR-21 in recipient cells treated with exosomes isolated from medium of NC or miR-21 mimic transfected cells. (D) The mRNA expression of Bcl⁻2 in bystander cells 24 h after co-cultured with NC or miR-21 mimic exosomes. (E) Bcl^-2 protein expression by protein gel blotting assay in recipient cells 24 h after co-cultured with NC or miR-21 mimic exosomes. (F) Frequency of micronuclei (MNF) in recipient cells treated for 48 h with NC or miR-21 mimic exosomes. (G) Yields of 53BP1 foci in recipient cells treated for 2 h with NC or miR-21 mimic exosomes. Error bars represent means ± standard error of 3 biological replicates and the superscript (*) denote a significant difference between groups (P < 0.05) as determined by Student's t test.

Figure 5.

Inhibiting the miR-21 expression in directly irradiated cells suppressed the exosomal miR-21 induced RIBE. (A) The expression of miR-21 in cells at different time points after transfected with miR-21 inhibitor. (B) The expression of miR-21 in irradiated cells previously transfected with NC or miR-21 inhibitor and in exosomes 4 hours after irradiation. (C) MiR-21 expression of bystander cells co-cultured with exosomes in (B). (D) The expression of pre-miR-21 of bystander cells co-cultured with exosomes in (B). (E) The mRNA expression of Bcl⁻2 in bystander cells 24 h after co-cultured with exosomes in (B). (F) Bcl^-2 expression in bystander cells 24 h after co-cultured with exosomes in (B) by western blotting assay. (G) Frequency of micronuclei (MNF) in bystander cells treated for 48 h with exosomes in (B). (H) Yields of 53BP1 foci in bystander cells treated for 2 h with exosomes in (B). Error bars represent means ± standard error of 3 biological replicates and the superscript (*) denote a significant difference between groups (P < 0.05) as determined by Student's t test.

Figure 6.

A proposed model of the exosome-mediated miR-21 transfer in the RIBE. In irradiated cells, the expression of miR-21 is up-regulated and as a response, miR-21 sorting to exosomes is motivated. The exosomes are secreted out from the irradiated cells, diffused into extracellular medium, taken up by non-irradiated cells, and the miR-21 inside the exosomes are released into bystander cells to induce bystander effects.