Exosomal transfer of pro-pyroptotic miR-216a-5p exacerbates anthracycline cardiotoxicity through breast cancer-heart pathological crosstalk

Figures

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  Fig. 1. Coincubation with breast cancer cells aggravates DOX-induced pyroptosis in cardiomyocytes. Adult murine ventricular cardiomyocytes (AMVCs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cocultured with breast cancer cells (BCCs) for 24 hours with 1 μM DOX treatment. a Schematic diagram of the coculture of BCCs with cardiomyocytes using transwell chambers. b–e Representative images of and statistics for cell viability after DOX treatment. Scale bar, 50 μm.; (f, g) lactate dehydrogenase (LDH) release and IL-18 levels were assessed using colorimetric methods in AMVCs (n = 5) and in (h and i) hiPSC-CMs (n = 5) (one-way ANOVA was used to compare all experimental groups to the DMSO group). “BCCs” indicates breast cancer cells, Directional arrows indicate exosome transfer between donor cells (4T1 or MDA-MB-231) and recipient cells (hiPSC-CMs or AMVCs). “N-BCC-EXOs” denotes normal breast cancer cell EXOs, and “D-BCC-EXOs” denotes DOX-induced breast cancer cell EXOs. “ns” indicates non-significant, and “DOX” indicates doxorubicin. Data are presented as Mean ± SD

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  Fig. 2. Exosomes from breast cancer cells exacerbate DOX-induced cardiomyocyte pyroptosis in vitro. a EXOs from BCCs were characterized using transmission electron microscopy (TEM). Scale bar: 100 nm. b Particle size distribution was determined using NanoSight tracking analysis. (NTA). EXO particle concentration in the cell culture supernatant with or without DOX. c Western blot analysis of HSP70, CD81, TSG101, GM130, and Calnexin in cell lysate and 4T1-derived EXOs with or without DOX. d In vitro and ex vivo breast cancer EXO uptake analysis showing fluorescence-labeled EXOs internalized by human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) or adult murine ventricular cardiomyocytes (AMVCs) observed under a confocal microscope. For in vitro analysis, PKH67-labeled EXOs were harvested and subsequently incubated with hiPSC-CMs. For ex vivo analysis, AMVCs were isolated from mouse hearts 24 hours after tail vein injection of PKH67-labeled EXOs. Scale bar: 20 μm. e, f Representative images of AMVCs and hiPSC-CMs after DOX + N-BCC-EXO or DOX + D-BCC-EXO treatment for 24 hours and (g, h) assessments of cell viability, (i, j) CASP1 expression levels, and (k, l) LDH release…

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  Fig. 3. Exosomes from breast cancer cells exacerbate DOX-induced cardiomyocyte pyroptosis in vivo. a Schematic illustration of the subchronic DOX exposure model in mice to test the injury efficacy of D-BCC-EXOs (n = 5). b Representative images of M-mode echocardiography and blood flow velocity (obtained using a Doppler system) in mice. c, d The E/A and the E/E’ ratios were quantified using the Doppler echocardiography. e, f Quantification of the left ventricular (LV) ejection fraction and LV fractional shortening via M-mode echocardiography (n = 5). g Representative images of hematoxylin-eosin (H&E) staining (upper), Sirius red staining indicating myocardial fibrosis (middle), and wheat germ agglutinin (WGA) immunofluorescence staining (lower). Scale bar: 50 μm. h Statistics of vacuolization in ventricular tissues. i The fibrotic area per left ventricle was quantified via Sirius red staining (n = 5). j Cell size was quantified via WGA immunofluorescence staining (n = 5). k Mouse plasma brain natriuretic peptide (BNP) was measured using a colorimetric method (n = 5). l–o Pyroptosis-related protein levels in mouse ventricular tissue were assessed via western blotting (n = 5) (one-way ANOVA…

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  Fig. 4. Exosomal miR-216a-5p upregulation induced by DOX aggravates DOX-mediated cardiomyocyte pyroptosis. a Sequencing data of mouse plasma EXO miRNAs were used to identify miRNAs that were differentially expressed. The differentially expressed miRNAs are depicted in a volcano plot. b 4T1 cells were treated with DOX or DMSO, and upregulated exosomal miRNAs were identified by qRT‒PCR (n = 5). c Adult murine ventricular cardiomyocytes (AMVCs) were transfected with a miR-216a-5p mimic/scramble before being treated with DOX. Cell viability was determined by CCK-8 assays (n = 5). d Venn analysis revealed that miR-216a-5p satisfied two conditions. e–h miR-216a-5p knockdown and overexpression in cardiomyocytes were achieved through transfection with miR-216a-5p inhibitors and mimics, respectively. Multiple immunofluorescence stains and statistics were used to quantify AMVC and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) death. DAPI (blue), SYTOX (green), and α-actinin (red) (n = 5). Scale bar: 50 μm. i, j LDH release levels and cell viability were measured (n = 5). k–n The protein expression levels of NLRP3, GSDMD-N, cleaved CASP1, and cleaved IL-1β were detected via…

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  Fig. 5. DOX upregulates ATF3 to promote miR-216a-5p transcription in BCCs. a Relative pri-miR-216a expression in mouse cancer tissue and 4T1 cells from DMSO- and DOX-induced mice was analyzed via qRT-PCR. b Protein levels were assessed by Western blotting using DOX/DMSO-treated 4T1 cells with or without siATF3. c The relative pri-miR-216a expression in DOX/DMSO-treated 4T1 cells with ATF3 overexpression or knockdown was analyzed via qRT-PCR. d, e The direct effects of miR-216a-5p on mouse ATF3 were identified using a luciferase assay. 4T1 cells were cotransfected with siATF3/oeATF3 and reporter plasmids for 48 hours. Firefly/Renilla luciferase activity was used to evaluate regulatory effects. In each group, the fold change was calculated by dividing the firefly/Renilla luciferase activity by the value obtained for the DMSO group. f ChIP-PCR was performed using an anti-ATF3 antibody or IgG in 4T1 cells, and PCR of the miR-216a promoter was performed. The values correspond to the ratio of the anti-ATF3 immunoprecipitated DNA relative to the IgG immunoprecipitated DNA. g ATF3 and miR-216a-5p expression levels gradually increased in a time-dependent manner after DOX treatment, and the two…

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  Fig. 6. SF3B4 (sap-49) packages miR-216a-5p into 4T1 exosomes. a Three candidate proteins were screened from RBPBD. b The effect of SF3B4 knockdown in 4T1 cells was assessed via western blotting. c The miR-216a-5p level in cell lysates and EXOs was analyzed via qRT‒PCR. d The SF3B4 binding motif and the matched miR-216a-5p sequence. e The binding of the wild-type and mutated miR-216a-5p probes to SF3B4 in 4T1 cells and EXOs from 4T1 cells was measured using an RNA pull-down assay. f The identified motif was the genuine binding region of miR-216a-5p and SF3B4, and mutated versions of miR-216a-5p were tested for binding to SF3B4 via EMSA. g SF3B4 in 4T1 cells was knocked down, and EXOs were isolated. After adult murine ventricular cardiomyocytes (AMVCs) were incubated with 4T1-derived EXOs containing Cy3-miR-216a-5p, Cy3 fluorescence in AMVCs was detected. Scale bar: 20 μm. h AMVCs were treated with SF3B4-knockdown and DOX-exposed 4T1 BCC-EXOs and exposed to DOX. The viability of AMVCs was assessed using CCK-8 assays (n = 5). i–k LDH release and IL-18 and IL-1β levels in AMVCs were assessed using a colorimetric method (n = 5). “siNC-BCC-EXOs” indicates exosomes from control…

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  Fig. 7. miR-216a-5p aggravates DOX-mediated cardiomyocyte injury by targeting ITCH. a A total of 39 human miR-216a-5p targets were identified via the intersection of six microRNA target prediction databases. b miR-216a-5p mimic-transfected 4T1 cells were cocultured with adult murine ventricular cardiomyocytes (AMVCs) and exposed to DOX, after which, downregulated mRNAs were identified via qRT‒PCR (n = 5). c miR-216a-5p mimic-transfected MDA-MB‒231 cells were cocultured with hiPSC‒CMs and exposed to DOX, after which the downregulated mRNAs were identified via qRT‒PCR. d Venn analysis was performed, and only ITCH decreased under both conditions. e The binding site between ITCH mRNA and the “seed” region of miR-216a-5p is highly conserved among various vertebrates. f The binding and regulatory relationships between miR-216a-5p and the 3’-UTRs of ITCH mRNAs in HL-1 and AC16 cells were identified via luciferase reporter plasmid analysis (n = 5). The reported plasmids containing the ITCH mRNA 3’-UTR regions (including binding sites) are shown above (mutated binding sites were reversed in sequence). g The half-life of ITCH mRNA was measured after actinomycin D treatment in the miR NC and…

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  Fig. 8. The miR-216a-5p/ITCH axis reduces TXNIP ubiquitination, aggravating DOX-induced cardiomyocyte pyroptosis. To determine whether miR-216a-5p inversely regulates TXNIP ubiquitination to promote TXNIP expression in DOX-treated adult murine ventricular cardiomyocytes (AMVCs), (a) TXNIP ubiquitination levels were assessed via Co-IP. AMVCs were transfected with a miR-216a-5p inhibitor or mimic for 24 hours and exposed to DOX or DMSO for another 24 hours. The protein lysates were immunoprecipitated with an anti-TXNIP antibody and immunoblotted with the indicated antibodies. To explore whether this effect could be regulated by ITCH knockdown or overexpression, (b) we assessed TXNIP ubiquitination via co-IP after AMVCs were transfected with adenovirus-shITCH or overexpression (oe) ITCH and transfected with the miR-216a-5p mimic. The reverse effect of TXNIP knockdown on DOX-induced pyroptosis was verified by (c) cell viability (n = 5), (d–f) LDH release; IL-18 and IL-1β levels; (g–k) and levels of pyroptosis-related proteins (NLRP3, GSDMD-N, cleaved-caspase-1, and cleaved-IL-1β) (n = 5). “ns” indicates non-significant, and “DOX” indicates doxorubicin. Data are presented as means ± SD

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  Fig. 9. The cardiomyocyte-specific miR-216a-5p sponges reduce the damaging impact of breast cancer EXOs on DOX-induced cardiomyocyte pyroptosis in vivo. a miR-216a-5p sponges and negative control (NC) sponges were carried by adeno-associated virus with serotype 9 (AAV9) with the cTnT promoter. Four weeks after the injection, the breast cancer model mice were treated with DOX (n = 5). b, c The E/A and -E/E′ ratios were quantified using the Doppler echocardiography. d, e Quantification of the left ventricular (LV) ejection fraction and LV fractional shortening via M-mode echocardiography. f A representative image of H&E staining, Sirius red staining indicating myocardial fibrosis, and WGA staining indicating myocardial atrophy are shown. Scale bar: 50 μm. g Statistics of vacuolization in ventricular tissues. h The fibrotic area per left ventricle was quantified. i Cell size was quantified. j Plasma BNP levels (marker of heart failure) were measured. k Western blot analysis of pyroptosis-related protein levels in mouse ventricular tissue. “E” indicates early diastolic transmitral flow velocity, “E′” indicates early diastolic mitral annular velocity, “A” indicates late (atrial) diastolic…

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  Fig. 10. Treatment with TXNIP/NLRP3/Caspase-1 pathway inhibitors attenuates DOX-induced cardiotoxicity in PDX models. a–c Western blot analysis of target proteins in DOX (1 μM), 24 h)-treated adult murine ventricular cardiomyocytes (AMVCs) co-cultured with 4T1 cells upon VX765, MCC950, or SRI-37330 treatment. d Schematic diagram showing the treatment protocol of PDX model mice with DOX and inhibitor. e–i Cardiac function parameters including E/A ratio, -E/E′ ratio, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and plasma BNP levels. j Representative image of H&E staining. Scale bar: 50 μm. k Quantification of cardiomyocyte vacuolation. Scale bar: 50 μm. l Representative image of Sirius red staining. Scale bar: 50 μm. m Quantification of cardiac fibrosis area. n Representative image of wheat germ agglutinin (WGA) staining. o Quantification of cardiomyocyte size. “i.p.(q.o.d.)” indicates intraperitoneal injection every other day; “p.o.(q.d.)” indicates oral administration once daily, “E” indicates early diastolic transmitral flow velocity, “E′” indicates early diastolic mitral annular velocity, “A” indicates late (atrial) diastolic transmitral…

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  Fig. 11. Human exosomes aggravate DOX-induced cardiomyocyte pyroptosis, an effect attenuated by a miR-216a-5p inhibitor. a EXOs were isolated from cardiomyopathy patients with adriamycin (Dis-EXOs) and matched healthy donors (N-EXOs). Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were transfected with a miR-216a-5p inhibitor/NC for 24 hours and then treated with Dis-EXOs/N-EXOs or DOX/DMSO for 24 hours. b Cell viability was determined (n = 5). c LDH release (d) the levels of pyroptosis-related proteins (GSDMD-N, cleaved-CASP1, and NLRP3) were assessed by western blotting. e, f The levels of pyroptosis-related cytokines (IL-1β and IL-18) were assessed (n = 5). g Western blotting was used to assess ITCH and TXNIP levels after cells were treated with Dis-EXOs (n = 5). “DOX” indicates doxorubicin. The schematic diagram part was created using SMART - Servier Medical Art by Servier. Data are presented as means ± SD