Exosomal miR-7 Mediates Bystander Autophagy in Lung after Focal Brain Irradiation in Mice

Abstract

This study investigated whether exosomal microRNA-7 (miR-7) mediates lung bystander autophagy after focal brain irradiation in mice. After 10 Gy or sham irradiation of mice brains, lung tissues were extracted for the detection of autophagy markers by immunohistochemistry, western blotting, and quantitative real-time reverse transcription PCR (qRT-PCR), meanwhile the brains were dissociated, the neuron/astrocyte/microglia/oligodendrocyte were isolated, and the miR-7 expression in each population were detected, respectively. A dual-luciferase reporter assay was developed to identify whether Bcl-2 is a target gene of miR-7. After 10 Gy or sham irradiation of astrocytes, exosomes were extracted, stained with Dil (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate), and added into non-irradiated astrocytes. Meanwhile, Dil-stained exosomes released from 10 Gy or sham irradiated astrocytes were injected into LC3B-GFP mice via the tail vein. Lung tissues were then extracted for western blotting and qRT-PCR. Irradiation of mouse brains increased the LC3B-II/I ratio, Beclin-1 and miR-7 levels, while decreased the Bcl-2 level in non-irradiated lung tissue. Interestingly, brain irradiation remarkably increased the miR-7 expression in astrocyte and oligodendrocyte. MiR-7 significantly inhibited the luciferase activity of the wild-type Bcl-2-3'-untranslated regions (UTR) reporter vector, but not that of the Bcl-2-3'-UTR mutant vector, indicating that Bcl-2 is directly targeted by miR-7. In in vitro study, the addition of irradiated astrocyte-secreted exosomes increased the LC3B-II/I ratio, Beclin-1 and miR-7 levels, while decreased the Bcl-2 level in non-irradiated astrocytes. Further, the injection of irradiated astrocyte-secreted exosomes through the tail vein increased the lung LC3B-II/I ratio, Beclin-1 and miR-7 level, but decreased the Bcl-2 level in vivo. We concluded that exosomal miR-7 targets Bcl-2 to mediate distant bystander autophagy in the lungs after brain irradiation.

Keywords: autophagy.; exosome; miR-7; radiation; radiation induced bystander effect (RIBE).

Figure 1

Brain irradiation induced autophagy in bystander lungs. Panel A: Lung tissues were obtained 0, 2, and 6 h after 10 Gy brain irradiation, and subjected to immunohistochemistry; LC3-B and Beclin-1 positive cells were quantified. Panel B: Lung tissues from GFP-LC3B transgenic mice were obtained 0, 2, and 6 h after 10 Gy brain irradiation. The number of GFP-LC3 dots was counted in five independent visual fields. Panel C: The expressions of LC3-B and Beclin-1 in lung tissues were measured by western blotting; Thirty micrograms of protein was subjected to do and β-actin was used as internal control. Bars represent the mean ± SD, n=3, * P value < 0.05.

Figure 2

Brain irradiation increased miR-7 level, while decreased Bcl-2 level in lungs Panel A: MiR-7 levels in mouse brain, blood, and lung were measured by qRT-PCR at indicated time points after 10 Gy brain irradiation; U6 was used as internal control. Panel B: Mouse lung tissue were obtained and subjected to immunohistochemistry; Bcl-2 positive cells were counted. Panel C: Total RNAs of lung tissue were obtained and subjected to qRT-PCR using a Bcl-2 specific primer, and 18s rRNA was used as internal control. Panel D: Total proteins of lung tissue were extracted and subjected to western blotting using a Bcl-2 primary antibody, and β-actin as the loading control. Bars represent the mean ± SD, n=3, * P value < 0.05.

Figure 3

MiR-7 targets Bcl-2 directly. Panel A: Complementary sequences between miR-7 and Bcl-2 mRNA 3′-UTR (paired bases are marked in red and mutant bases are indicated in blue). Vector pmirGLO was used to construct the expression vector and mutant vector. Panel B: Relative miR-7 expression after treatment with various concentrations of miR-7 mimics or miR-7 inhibitor. Panel C: HEK 293 cells were transfected with wild-type Bcl-2-3′-UTR or mutant Bcl-2-3′-UTR vector together with miR-7 mimics or miR mimics control, or with miR-7 inhibitor or miR inhibitor control, then luciferase activity was measured. Panel D: Bcl-2 mRNA expression in HEK-293T cells after transfection with Bcl-2 siRNA, miR-7 mimics, or miR-7 inhibitor. 50 nM of miR-7 mimics and 75 nM of miR-7 inhibitor were used. Bars represent the mean ± SD, n=5, * P value < 0.05.

Figure 4

Brain irradiation increased miR-7 expression in astrocytes and oligodendrocytes, but decreased that in neurons and microglia After 10Gy or sham brain irradiation, mice brain were obtained, astrocyte, microglia, neuron and oligodendrocyte were isolated. Then the miR-7 expression in these cells were deceted by qRT-PCR (fig.4A to fig.4D), respectively. Bars represent the mean ± SD, n=5, * P value < 0.05, *** P value < 0.01, NS stands for none significant.

Figure 5

Exosomal miR-7 mediates bystander autophagy in vitro. Panel A: Exosomes were observed under a transmission electron microscope, while exosomal markers TSG101 and CD63 were measured by western blotting. Panel B: miR-7 levels in astrocytes were measured by qRT-PCR after irradiation; U6 was used as internal control. Panel C: miR-7 levels in exosomes secreted by astrocytes were measured by qRT-PCR after irradiation; U6 was used as internal control. Panel D: Expressions of miR-7 and Bcl-2 mRNA in irradiated (rad-exo) or non-irradiated (nc-exo) astrocytes-released exosomes were measured by qRT-PCR; U6 was used as internal control. Panel E: Expression of LC3B, Beclin-1, and Bcl-2 in irradiated (rad-exo) or non-irradiated (nc-exo) astrocytes-released exosomes were measured by western blotting after irradiation. Bars represent the mean ± SD, n=5, * P value < 0.05.

Figure 6

Exosomes from irradiated astrocytes induced bystander autophagy in vitro. Exosomes extracted from Panel A: sham irradiated astrocytes without Dil staining, Panel B: sham irradiated astrocytes with Dil staining, and Panel C: Exosomes from irradiated astrocytes were added into culture medium of non-irradiated primary astrocytes from LC3B-GFP transgenic mice. The recipients astrocytes were observed and pictured under confocal microscopy. Note. Red: exosomes stained with Dil; Green: LC3B-GFP; Blue: nuclei stained with DAPI (4',6-diamidino-2-phenylindole).

Figure 7

Exosomes from irradiated astrocytes induced bystander autophagy in vivo. Exosomes extracted from Panel A: sham irradiated astrocytes without Dil staining, and Panel B: sham irradiated astrocytes with Dil staining, Panel C: Exosomes from irradiated astrocytes were injected into LC3B-GFP transgenic mice via the tail vein several times. 6h after the last injection, mouse lung tissues were obtained and observed under confocal microscopy. Note. Red: exosomes stained with Dil; Green: LC3B-GFP; Blue: nuclei stained with DAPI (4',6-diamidino-2-phenylindole).

Figure 8

Exosomal miR-7 mediates bystander autophagy in vivo. After injected with irradiated (rad-exo) or non-irradiated (nc-exo) astrocytes-released exosomes, Panel A: Expression of miR-7 and Bcl-2 mRNA in mice lung tissue were measured by qRT-PCR; U6 was used as internal control. Panel B: Expression of LC3B, Beclin-1, and Bcl-2 in mice lung tissue were measured by western blotting. Bars represent the mean ± SD, n=5, * P value < 0.05.