Theranostics 2020; 10(15):6728-6742. doi:10.7150/thno.42259

Research Paper

Stem cell-derived exosomes prevent pyroptosis and repair ischemic muscle injury through a novel exosome/circHIPK3/ FOXO3a pathway

Bing Yan1#, Yu Zhang1#, Chun Liang2#, Bin Liu3#, Fengzhi Ding1, Yanli Wang1, Bao Zhu1, Ranzun Zhao4, Xi-Yong Yu5, Yangxin Li1,✉

1. Institute for Cardiovascular Science and Department of Cardiovascular Surgery, First Affiliated Hospital and Medical College of Soochow University, Suzhou, Jiangsu 215123, P. R. China
2. Department of Cardiology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, P. R. China
3. Department of Cardiology, the Second Hospital of Jilin University, Changchun, Jilin 130041, P. R. China
4. The First Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou, 563000, P. R. China
5. Key Laboratory of Molecular Target & Clinical Pharmacology and the State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou 511436, P. R. China
#These authors contributed equally.

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Citation:
Yan B, Zhang Y, Liang C, Liu B, Ding F, Wang Y, Zhu B, Zhao R, Yu XY, Li Y. Stem cell-derived exosomes prevent pyroptosis and repair ischemic muscle injury through a novel exosome/circHIPK3/ FOXO3a pathway. Theranostics 2020; 10(15):6728-6742. doi:10.7150/thno.42259. Available from http://www.thno.org/v10p6728.htm

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Abstract

Rational: Ischemic injury of the skeletal muscle remains a serious clinical problem and currently there is no effective therapy. The aim of the present study is to determine whether human umbilical cord mesenchymal stem cells- derived exosomes (UMSC-Exo) could repair ischemic injury by releasing circular RNA.

Methods and Results: To create hindlimb ischemia, we surgically ligated the left femoral artery in C57BL/6 mice. Using circRNA-seq analyses of total RNA from ischemic and control muscles, we found reduced expression of circHIPK3 in the ischemic muscle. To explore the role of circHIPK3 in ischemic injury, the mice were randomly assigned into three groups after surgery: 1) vehicle; 2) UMSC-Exo; 3) UMSC-Exo and siRNA targeting circHIPK3 (UMSC-Exo /si-circHIPK3). UMSC-Exo treatment significantly increased expression of circHIPK3 and improved blood perfusion, running distance and muscle force, which were reversed by injection of UMSC-Exo /si-circHIPK3, suggesting that UMSC-Exo improve muscle function by releasing circHIPK3. UMSC-Exo treatment also inhibited ischemia induced pyroptosis - cell death caused by inflammasome as evidenced by activation of NLRP3, cleaved caspase-1, and subsequent increase of IL-1β and IL-18, and the effects were reversed by injection UMSC-Exo /si-circHIPK3. Bioinformatic analysis identified miR-421/FOXO3a as a potential target for circHIPK3, which was confirmed by luciferase reporter assay. Knockdown of circHIPK3 in C2C12 cells resulted in increased expression of miR-421. We established an in vitro model of pyroptosis by stimulating C2C12 cells with LPS and ATP. LPS and ATP treatment resulted in reduced expression of circHIPK3 and increased expression of miR-421, which was prevented by UMSC-Exo. Western blot analysis showed reduced levels of NLRP3 and cleaved caspase-1 when cells were treated by UMSC-Exo. The expression of FOXO3a in C2C12 cells was increased in the presence of miR-421 inhibitor, and the expression was reduced when cells were treated by LPS and ATP. Importantly, the expression of FOXO3a was upregulated by UMSC-Exo but was reduced when si-circHIPK3 was present.

Conclusions: Using loss/gain-of function method, we demonstrated that miR-421/FOXO3a is the direct target of circHIPK3, and UMSC-Exo prevent ischemic injury by releasing circHIPK3, which in turn down regulate miR-421, resulting in increased expression of FOXO3a, leading to inhibition of pyroptosis and release of IL-1β and IL-18.

Keywords: circRNA, pyroptosis, exosome, inflammasome, NLRP3