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. 2021 Jul 2;27(1):67.
doi: 10.1186/s10020-021-00324-0.

MSCs‑derived exosomes attenuate ischemia-reperfusion brain injury and inhibit microglia apoptosis might via exosomal miR-26a-5p mediated suppression of CDK6

Chang Cheng  1 , Xiuying Chen  1 , Yuhan Wang  1 , Wenchao Cheng  1 , Xuzheng Zuo  1 , Weiju Tang  1 , Wen Huang  2
Affiliations

MSCs‑derived exosomes attenuate ischemia-reperfusion brain injury and inhibit microglia apoptosis might via exosomal miR-26a-5p mediated suppression of CDK6

Chang Cheng et al. Mol Med. .

Abstract

Background: This study aimed to explore the role of mesenchymal stromal cells (MSCs)-derived exosomes (MSCs-Exo) in the cerebral ischemia-reperfusion (I/R) injury.

Methods: Exosomes were isolated from MSCs of adult C57BL/6J mice by the gradient centrifugation method. The expression of miR-26a-5p and CDK6 in MSCs-Exo and mice brain tissues were evaluated by qRT-PCR and western blot. miR-26a-5p mimics and miR-NC were transfected into MSCs, and exosomes were isolated from the MSCs stably expressing miR-26a-5p. Then MSCs-Exo-miR-26a-5p mimics or MSCs-Exo-miR-NC was injected into mice through the tail vein, or added into medium to stimulate BV-2 cells. Cell viability was evaluated by CCK-8 assay. Cell apoptosis was detected by flow cytometry. The apoptosis in brain tissues was evaluated by TUNEL staining assay. Bioinformatics analysis and luciferase reporter assay were performed to determine the binding relationship between miR-26a-5p and CDK6.

Results: miR-26a-5p was downregulated and CDK6 was upregulated in MSCs-Exo of MCAO-mice and OGD-induced MSCs. MSCs-Exo-miR-26a-5p mimics significantly reduced cell apoptosis of OGD-injured BV-2 cells. MSCs-Exo-miR-26a-5p mimics significantly reduced the infarct volume of MCAO-induced mice. Luciferase reporter assay revealed that CDK-6 was a target of miR-26a-5p. In addition, MSCs-Exo-miR-26a-5p mimics significantly decreased the expression of CDK6 in both OGD-induced BV-2 cells and the brain tissues of MCAO-treated mice.

Conclusion: Our results indicated that MSCs‑Exo attenuated I/R injury in mice by inhibiting microglia apoptosis might via exosomal miR-26a-5p mediated suppression of CDK6. Our study shed light on the application of MSC-Exo as a potential therapeutic tool for cerebral I/R injury.

Keywords: CDK6; Exosomes; Ischemia–reperfusion injury; Mesenchymal stromal cells; miR-26a-5p.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Characterization of MSCs‑derived exosomes. A Morphological change of MSCs-derived exosomes by TEM. B FACS analysis of exosome surface markers CD63 and CD81 in MSCs-derived exosomes. C The protein expression of exosome surface markers (CD9, CD63, CD81, HSP70, Calnexin) in MSCs and MSCs-derived exosomes was evaluated by western blot
Fig. 2
Fig. 2
MiR-26a-5p was downregulated and CDK6 was upregulated in MSCs-derived exosomes of MCAO and OGD model. A and B The mRNA expression of miR-26a-5p in MSCs-derived exosomes (A) and brain tissues (B) of MCAO-induced mice was evaluated by qRT-PCR. C The level of miR-26a-5p in exosomes of OGD-treated MSCs at indicated time was evaluated by qRT-PCR. D The protein expression of CDK6 in MSCs-derived exosomes of MCAO-induced mice was evaluated by western blot. E The protein expression of CDK6 in exosomes of OGD-treated MSCs at indicated time was evaluated by western blot. *p < 0.05, **p < 0.01
Fig. 3
Fig. 3
miR-26a-5p reversed the effects of MSCs-derived exosomes in reducing cell apoptosis of OGD-injured microglia. BV-2 cells were treated by OGD for the given time, and then treated with 200 μg/mL Exo-miR‐26a-5p mimics or Exo-miR-NC. A Cell viability was evaluated by CCK-8 assay. B Cell apoptosis was detected by flow cytometry. C The protein expression of apoptosis-related makers (cleaved caspase-3, total caspase-3, cleaved RARP and total RARP) was evaluated by western blot. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 4
Fig. 4
MSCs-Exo-miR-26a-5p mimic attenuated ischemia–reperfusion injury in MCAO/R model. Mice were treated with MCAO/R, and injected with 200 μL/mice MSCs-exosomes-miR-NC or MSCs-exosomes-miR-26a-5p mimics. A The mRNA level of miR-26a-5p in brain tissues was evaluated qRT-PCR. B Representative brain sections of MCAO/R induced mice were detected by TTC staining. C TUNEL staining in brain tissues. D The protein level of apoptosis-related markers (cleaved caspase-3, total caspase-3, cleaved RARP and total RARP) in brain tissues was detected by western blot. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 5
Fig. 5
The effect of miR-26a-5p was partially mediated by CDK6. A The putative binding site between miR-26a-5p and CDK6 was predicted by Targetscan. B 293 T cells were transfected with miR-26a-5p mimics or miR-NC, and the mRNA level of miR-26a-5p was detected by qRT-PCR. C 293 T cells were co-transfected with miR-26a-5p mimics or miR-NC and Luc-CDK6-WT or Luc-CDK6-MUT, and the relative luciferase reporter activity was evaluated by dual luciferase reporter system. D and E BV-2 cells were transfected with miR-26a-5p or miR-NC, and the expression of CDK6 was detected by qRT-PCR (D) and western blot (E). F BV-2 cells were treated by OGD/R, and then treated with 200 μg/mL Exo-miR-26a-5p mimics or Exo-miR-NC, and the protein level of CDK6 was evaluated by western blot. G Mice were induced by MCAO/R, and then injected with 200 μL/mice Exo-miR-NC or Exo-miR-26a-5p mimics. The protein level of CDK6 in brain tissues was detected by western blot. *p < 0.05 **p < 0.01
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