Abstract P2057: Long Non-coding Rna Bcyrn1 In Cardiosphere-derived Extracellular Vesicles Promotes Regulatory T Cell Proliferation, Migration And Activation: Implications For Cardioprotection
Authors: Liao Ke , Akhmerov Akbarshakh , Li Liang , Liu Weixin , Ibrahim Ahmed , Marbán Eduardo 
Published: November 2022

Background:
Regulatory T (Treg) cells are increasingly implicated as modulators of myocardial ischemia-reperfusion (I/R) injury. We have previously found that extracellular vesicles isolated from human cardiosphere-derived cells (CDC-EVs) could promote Treg proliferation and augment production of the anti-inflammatory cytokine IL-10 in vivo, attenuating cardiac inflammation and functional decline. However, the mechanism by which CDC-EVs regulate Treg is largely unknown. In this study we
assessed the hypothesis
that long non-coding RNA (LncRNA) within CDC-EVs induces Treg proliferation, migration and IL-10 production, which mediates cardioprotection.

Methods:
BCYRN1 (Brain Cytoplasmic RNA 1) was identified as a LncRNA species in CDC-EVs using RNA sequencing. We assessed CDC-EVs and LncRNA BCYRN1 effects on Treg by measuring cell proliferation (Cell Counting Kit-8 assay), cell migration (trans-well migration assay), and the production of IL-10 (qPCR and ELISA assay). Additionally, RNA pulldown and luciferase assays were used to determine whether BCYRN1 functions as a miRNA sponge.
In vivo
, I/R injury was induced in 8-week-old C57BL/6 mouse by left anterior descending coronary artery ligation for 45 minutes followed by reperfusion. Fifteen minutes later, the mice received (i.v., via tail vein) CDC-EVs, CDC-EV BCYRN1 (overexpress BCYRN1 in CDC-EVs), or vehicle (PBS). Infarct size (by TTC staining) and infiltrating Treg (by flow cytometry) were assessed 72 hrs post I/R.

Results:
Exposure of human Treg to CDC-EVs resulted in Treg proliferation (3.00± 0.11 fold increase, N=5), migration (1.77 ± 0.10 fold increase, N=5), and production of IL-10 (qPCR: 2.60 ± 0.35 fold increase, N=6 and ELISA: 6.12 ± 0.78 fold increase, N=5). RNA sequencing of CDC-EV contents showed that BCYRN1 was one of the most highly enriched LncRNAs. RNA pulldown and luciferase assays showed that BCYRN1 contained binding sites for multiple miRNAs, including miR-138, miR-150 and miR-98 that repress the expression of ATG7, CCR6 and IL-10 mRNAs, respectively. These results reveal that BCYRN1 serves as a miRNA sponge, leading to Treg cell proliferation (via induction of ATG-7 dependent autophagy), CCR-6 dependent migration and production of IL-10. In the mouse I/R model (N=5, each group), CDC-EVs and CDC-EV BCYRN1 (but not vehicle) induced Treg infiltration, proliferation and IL10 production in the heart, with a concomitant decrease of infarct size.

Conclusions:
We have pinpointed BCYRN1 as a bioactive constituent of CDC-EVs, promoting Treg cell-mediated cardioprotection. BCYRN1 serves as a miRNA sponge to mediate its beneficial effects on Treg infiltration, proliferation, and IL-10 production in the heart. Thus, a single LncRNA mimics the effects of CDC-EV on Treg
in vitro
and
in vivo
. Our study provides a novel approach, targeting Treg by a defined LncRNA mined from CDC-EV, to treat myocardial infarction.