Long noncoding RNA BCYRN1 promotes cardioprotection by enhancing human and murine regulatory T cell dynamics

Abstract

Regulatory T cells (Tregs) modulate immune responses and attenuate inflammation. Extracellular vesicles from human cardiosphere-derived cells (CDC-EVs) enhance Treg proliferation and IL-10 production, but the mechanisms remain unclear. Here, we focused on BCYRN1, a long noncoding RNA (lncRNA) highly abundant in CDC-EVs, and its role in Treg function. BCYRN1 acts as a "microRNA sponge," inhibiting miR-138, miR-150, and miR-98. Suppression of these miRs leads to increased Treg proliferation via ATG7-dependent autophagy, CCR6-dependent Treg migration, and enhanced Treg IL-10 production. In a mouse model of myocardial infarction, CDC-EVs, particularly those overexpressing BCYRN1, were cardioprotective, reducing infarct size and troponin I levels even when administered after reperfusion. Underlying the cardioprotection, we verified that CDC-EVs overexpressing BCYRN1 increased cardiac Treg infiltration, proliferation, and IL-10 production in vivo. These salutary effects were negated when BCYRN1 levels were reduced in CDC-EVs or when Tregs were depleted systemically. Thus, we have identified BCYRN1 as a booster of Treg number and bioactivity, rationalizing its cardioprotective efficacy. While we studied BCYRN1 overexpression in the context of ischemic injury here, the same approach merits testing in other disease processes (e.g., autoimmunity or transplant rejection) where increased Treg activity is a recognized therapeutic goal.

Keywords: Autophagy; Cardiology; Gene therapy; T cells; Therapeutics.

Figure 1. CDC-EVs induce human iTreg proliferation, migration, and induction of IL-10.

(A) Experimental outline for in vitro Treg global transcriptomic analysis: total CD4⁺ T cell population isolated from FOXP3 reporter mice, followed by differentiating the isolated naive CD4⁺ T cells into induced Tregs (iTregs). The iTregs were exposed either to vehicle or human CDC-EVs for 5 days and subsequently assessed for transcriptomic changes. (B and C) Functional heatmap depicting enrichment in cell survival (B) and cellular movement (C) categories in the upregulated transcripts in CDC-EVs versus vehicle based on IPA analysis. (D–F) Human iTregs were exposed either to CDC-EVs or fibroblast EVs (NHDF-EVs) at indicated concentrations (0–5,000 EVs/cell) for 24 hours (D), 72 hours (E), and 5 days (F). (G) The migration of human iTregs following exposure to CDC-EVs or NHDF-EVs, toward 500 ng/mL recombinant CCL20, was assessed using a 24-well Transwell plate. (H) Real-time PCR analysis of IL-10 mRNA expression in human iTregs exposed to either CDC-EVs or NHDF-EVs for 72 hours. (I) Protein levels of IL-10 were assayed by ELISA in supernatants of human iTregs exposed to either CDC-EVs or NHDF-EVs for 72 hours. One-way ANOVA followed by Bonferroni’s post hoc test was used to determine statistical significance. All data are presented as mean ± SEM of 3 or 4 individual experiments (biological replicates). *P < 0.05, **P < 0.01, ***P < 0.001 versus control group.

Figure 2. CDC-EVs mediate increased expression of BCYRN1 in Tregs.

(A) RNA-Seq of CDC-EVs shows lncRNA to be plentiful compared with NHDF-EVs. (B) List of top 10 most plentiful lncRNAs, among which BCYRN1 is the highest enriched in CDC-EVs (CDC-EVs [blue bars] and NHDF-EVs [orange bars]). (C) BCYRN1 is expressed more in CDC-EVs than in NHDF-EVs by qPCR. (D) Confocal microscopy shows uptake of PKH26-labeled CDC-EVs by human iTregs. Scale bar: 5 μm. (E) Assessment of BCYRN1 expression by qPCR in human iTregs exposed to CDC-EVs or NHDF-EVs for indicated times. One-way ANOVA followed by Bonferroni’s post hoc test was used to assess statistical significance. All data are presented as mean ± SEM of 3 or 4 individual experiments (biological replicates). *P < 0.05, **P < 0.01, ***P < 0.001 versus control (Ctrl) group.

Figure 3. CDC-EV–mediated Treg proliferation, migration, and induction of IL-10 involves BCYRN1.

(A–C) Effects of overexpression (OE) of BCYRN1 in human iTregs on proliferation (A), migration (B), and IL-10 production (C). (D and E) Transfection of CDCs with siRNA-BCYRN1 results in knockdown of BCYRN1 in both CDCs and CDC-EVs. (F–H) Exposure of human iTregs to CDC-EVs with BCYRN1 knockdown followed by assessments of proliferation (F), migration (G), and IL-10 production (H). One-way ANOVA followed by Bonferroni’s post hoc test was used to assess statistical significance. All data are presented as mean ± SEM of 3 or 4 individual experiments (biological replicates). *P < 0.05, **P < 0.01, ***P < 0.001 versus control group. ^##P < 0.01 versus si-Ctrl-CDC-EVs group.

Figure 4. CDC-EV BCYRN1 induces autophagy by competitively binding miR-138 to regulate ATG7 expression.

(A) Human iTregs were exposed to CDC-EVs or not treated (ctrl). The expression of autophagy markers (MAP1LC3B, ATG7, and SQSTM1) was assessed by Western blot (WB). (B) Human iTregs were exposed to ctrl-CDC-EVs or si-BCYRN1 CDC-EVs (EV with BCYRN1 knockdown), or were not treated (ctrl). Expression of autophagy markers (MAP1LC3B, ATG7, and SQSTM1) was assessed by WB. (C) Human iTregs were transfected with vector or OE-BCYRN1 lenti-vector, followed by assessment of autophagy markers by WB. (D) Biotin-labeled BCYRN1 probe was used to pull down BCYRN1-associated RNAs, followed by assessment of miR-138, negative control (U6 and GAPDH), and positive control (BCYRN1) expression by qPCR. (E) The top panel shows putative lncRNA BCYRN1 binding sites in miR-138. The bottom panel shows human iTregs that were cotransfected with WT or mutant luciferase reporters with mimic miR-138 into HEK-293T cells, followed by assessment of relative luciferase activity. (F) Cotransfection of miR-138 and BCYRN1 in Tregs followed by assessments of ATG7 by WB. One-way ANOVA followed by Bonferroni’s post hoc test was used to determine statistical significance. All data are presented as mean ± SEM of 3 or 4 individual experiments (biological replicates). *P < 0.05, **P < 0.01, ***P < 0.001 versus control group.

Figure 5. CDC-EV BCYRN1 induces Treg migration by competitively binding miR-150 to regulate CCR6 expression.

(A) Human iTregs were exposed to CDC-EVs or were not treated (ctrl). The expression of CCR6 was assessed by qPCR. (B) Human Tregs were exposed to ctrl-CDC-EVs or si-BCYRN1 CDC-EVs (EV with BCYRN1 knockdown) or were not treated (ctrl). The expression of CCR6 was assessed by qPCR. (C) Human Tregs were transfected with vector or OE-BCYRN1 lenti-vector, followed by assessment of CCR6 by qPCR. (D) Biotin-labeled BCYRN1 probe was used to pull down BCYRN1-associated RNAs, followed by assessment of miR-150, negative control (U6 and GAPDH), and positive control (BCYRN1) expression by qPCR. (E) The top panels putative lncRNA BCYRN1 binding sites in miR-150. The bottom panel shows human iTregs that were cotransfected with WT or mutant luciferase reporters and mimic miR-150 into HEK-293T cells, followed by the assessment of relative luciferase activity. (F and G). Cotransfection of miR-150 and BCYRN1 in Tregs followed by assessments of CCR6 by WB (F) and cellular migration by trans-well migration assay (G). One-way ANOVA followed by Bonferroni’s post hoc test was used to determine statistical significance. All data are presented as mean ± SEM of 3 or 4individual experiments (biological replicates). **P < 0.01, ***P < 0.001 versus control group. ^###P < 0.001 versus miR-150 group.

Figure 6. BCYRN1 mediates induction of IL-10 in Tregs by competitively binding miR-98 to regulate IL-10 expression.

(A) Biotin-labeled BCYRN1 probe was used to pull down BCYRN1-associated RNAs, followed by assessment of miR-98, negative control (U6 and GAPDH), and positive control (BCYRN1) expression by qPCR. (B) Putative lncRNA BCYRN1 binding sites in miR-98 and luciferase assay results. (C and D) Cotransfection of miR-98 and BCYRN1 in Tregs, followed by assessments of IL-10 by qPCR (C) and ELISA (D). One-way ANOVA followed by Bonferroni’s post hoc test was used to determine statistical significance. All data are presented as mean ± SEM of 3 or 4 individual experiments (biological replicates). **P < 0.01, ***P < 0.001 versus control group. ^##P < 0.01, ^###P < 0.001 versus miR-150 group.

Figure 7. Therapeutic efficacy of CDC-EVs and CDC-EV-BCYRN1 in a mouse myocardial infarction model — the role of Tregs.

(A) Schematic representation of in vivo myocardial infarction (MI) protocol. (B) Representative flow cytometry plots and pooled data of the CD4⁺Foxp3⁺, CD4⁺Foxp3⁺IL-10⁺, and CD4⁺Foxp3⁺Brdu⁺ populations in hearts from animals infused with CDC-EVs, CDC-EVs overexpressing BCYRN1 (CDC-EV-BCYRN1), and IMDM (vehicle) (n = 5 mice per group). (C) Pooled data for percentage of infarct mass (n = 5 mice per group) and representative images of TTC-stained hearts from CDC-EV–, CDC-EV-BCYRN1–, and vehicle-injected animals 72 hours after MI. (D) Plasma cTnI values from CDC-EVs, CDC-EV-BCYRN1– and vehicle-injected animals (n = 5/group) 24 hours after MI. (E) Pooled data for percentage of infarct mass (n = 5/group) of TTC-stained hearts from CDC-EV–, BCYRN1-depleted CDC-EV–, and vehicle-injected animals 72 hours after MI. (F) Plasma cTnI values from CDC-EV–, BCYRN1-depleted CDC-EV–, and vehicle-injected animals (n = 5/group) 24 hours after MI. (G) Echocardiographic analysis of left ventricular ejection fraction (EF) on days 0 (baseline before MI), 7, 14, and 21 after MI with indicated treatment (n = 5/group). (H) Schematic of Treg depletion and in vivo ischemia/reperfusion (I/R) protocol. Mice were injected twice with anti-CD25 antibody (100 μg/mouse/injection i.p.), or isotype control daily for 2 days before I/R. On day 3, TTC assay was used to assess infarct size (n = 5/group). (I) Representative plots showing the percentage of CD25⁺FoxP3⁺ in CD4⁺ T cells (Q2 quadrant). (J) Quantitative measurements of the percentage of infarct mass (n = 5/group) from CDC-EV– or CDC-EV-BCYRN1–injected animals (WT/Treg depletion) at 72 hours after I/R injury. One-way ANOVA followed by Bonferroni’s post hoc test was used to determine the statistical significance among multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control group. ^#P < 0.05 versus CDC-EVs group.