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Extracellular Vesicles Derived from Bone Mesenchymal Stem Cells Carrying circ_0000075 Relieves Cerebral Ischemic Injury by Competitively Inhibiting miR-218-5p and Up-regulating E3 Ubiquitin Ligase SMURF2

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Abstract

Extracellular vesicle (EV)–encapsulated circRNAs have the potential role in affecting brain disorders. However, the role of circ_0000075 in cerebral ischemic injury remains unclear. Here, we tried to investigate the mechanism of bone marrow mesenchymal stem cell (BMSC)–derived EVs carrying circ_0000075 in the control of cerebral ischemic injury. Initially, a mouse model with cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO), followed by the determination of circ_0000075 expression. Then, neurons were isolated and subjected to oxygen–glucose deprivation/reperfusion. BMSCs were isolated for extraction of EVs. The correlation among circ_0000075, microRNA (miR)-218-5p, and Smad ubiquitination regulatory factor 2 (SMURF2) was detected with their roles in cerebral ischemic injury analyzed in vivo and in vitro. circ_0000075 was down-regulated in MCAO mice and engineered RVG-EVs were internalized by neurons to up-regulate circ_0000075 expression. Treatment of RVG-circ_0000075-EVs reduced brain tissue damage, increased neuronal count, and significantly curtailed apoptosis rate, suppressing cerebral ischemic injury in vitro and in vivo. miR-218-5p was targeted by circ_0000075 in neurons, which promoted SMURF2 expression. A negative correlation between SMURF2 and transcriptional regulator Yin Yang 1 (YY1) was identified. In vitro experiments further proved that circ_ 00,000 75 could down-regulate the expression of YY1 through SMURF2, and finally relieving cerebral ischemic injury. Collectively, engineered EVs delivered circ_0000075 into brain tissues and increased circ_0000075 expression, which down-regulated miR-218-5p and up-regulated SMURF2, thus alleviating cerebral ischemic injury.

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The data underlying this article will be shared on reasonable request to the corresponding author.

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Authors and Affiliations

  1. Department of Neurosurgery, Jiangxi Province, The First Affiliated Hospital of Nanchang University, No. 17, Yongwaizheng Street, Nanchang, 330006, People’s Republic of China

    Yue Liu, You-Ping Li, Li-Min Xiao, Li-Ke Chen, Su-Yue Zheng, Er-Ming Zeng & Chun-Hua Xu

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  1. Yue Liu
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Contributions

Yue Liu and You-Ping Li conceived and designed research. Li-Min Xiao and Li-Ke Chen performed experiments. Su-Yue Zheng and Er-Ming Zeng interpreted results of experiments. Yue Liu and Chun-Hua Xu drafted paper. You-Ping Li edited and revised manuscript. All authors read and approved the final manuscript.

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Correspondence to Chun-Hua Xu.

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This study was ratified by the Animal Ethics Committee of the First Affiliated Hospital of Nanchang University and all procedures were carried out per the Guide for the Care and Use of Laboratory Animals.

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Supplementary File 1

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Supplementary Fig. 1

Effect of exogenous circ_0000075 on cerebral ischemic injury in MCAO mice. A, The overexpression efficiency of circ_0000075 assayed within BMSCs. B, Foot fault test from 1 day before to 28 days after MCAO. C, Rota-rod test from 1 day before to 28 days after MCAO. D, E, Adhesive removal from 1 day before to 28 days after MCAO. F, Time to locate the mice to the platform (escape latency) measured 22 - 26 days after MCAO. G, Spatial memory assessed by measuring the time spent in the target quadrant during platform removal 27 days after MCAO. H, RT-qPCR of circ_0000075 expression in mouse brain tissues 7 days after MCAO; n = 8. * p < 0.05 vs. the LV-NC group. (PNG 210 kb)

High resolution image (EPS 945 kb)

Supplementary Fig. 2

Extraction and identification of BMSCs and neurons. A, Flow cytometry of surface markers of BMSCs. B, Induction of multifunctional differentiation of BMSCs. i: Alizarin red cytochemical staining for osteogenic differentiation, scale bar = 50 μm; ii: Oil red O cytochemical staining for adipogenic differentiation; iii: Alcian Blue cytochemical staining for chondrogenic differentiation, scale bar = 50 μm. C, Primary Neuronal MAP-2 staining; red: MAP-2, blue: Hoechst (bar = 25 μm). All experiments were repeated 3 times independently. (PNG 717 kb)

High resolution image (EPS 5995 kb)

Supplementary Fig. 3

Target miRNAs of circ_0000075 and target genes of miRNA. A, Volcano map of differentially expressed miRNAs in MCAO mice (n = 10) and sham-operated mice (n = 10). B, Venn diagram displaying the intersection of up-regulated miRNAs in cerebral ischemic injury and target miRNAs of circ_0000075. C, Venn diagram displaying the intersection of target genes of miR-218-5p and cerebral ischemic injury-related genes. (PNG 329 kb)

High resolution image (EPS 2066 kb)

Supplementary Fig. 4

Effect of down-regulated miR-218-5p expression on neuronal damage after OGD/R. A, RT-qPCR to detect miR-218-5p expression in each group of cells after OGD/R treatment. B, CCK-8 assay to detect cell survival. C, TUNEL staining to detect apoptosis. D, LDH release to assess neuronal injury. * p < 0.05 vs. the OGD/R + inhibitor NC group. The cell experiment was repeated 3 times independently. (PNG 82 kb)

High resolution image (EPS 1215 kb)

Supplementary Fig. 5

Effect of SMURF2 overexpression on neuronal damage after OGD/R. A, Western blot assay to detect SMURF2 expression in each group of cells after OGD/R treatment. B, CCK-8 assay for cell survival. C, TUNEL staining for apoptosis. D, LDH release to assess neuronal damage. * p < 0.05 vs. the OGD/R + vector group. The cell experiment was repeated 3 times independently. (PNG 126 kb)

High resolution image (EPS 1500 kb)

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Liu, Y., Li, YP., Xiao, LM. et al. Extracellular Vesicles Derived from Bone Mesenchymal Stem Cells Carrying circ_0000075 Relieves Cerebral Ischemic Injury by Competitively Inhibiting miR-218-5p and Up-regulating E3 Ubiquitin Ligase SMURF2. Mol Neurobiol 60, 2801–2818 (2023). https://doi.org/10.1007/s12035-022-03192-9

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