This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
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Research
Chengheng Hu
The First Affiliated Hospital of Sun Yat-sen University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-4243-0618
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Accumulating evidence has shown that endothelial progenitor cell-derived exosomes (EPC-Exos) can ameliorate myocardial fibrosis. The purpose of the present study was to investigate the effects of EPC-Exos-derived microRNAs (miRNAs) on myocardial infarction (MI).
Methods
A miRNA-Seq dataset of miRNAs differentially expressed between EPCs and exosomes was collected. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the miRNA expression indicated by miRNA-Seq. Immunofluorescence, cell proliferation and angiogenesis assays were employed to investigate the effects of miRNAs on cardiac fibroblasts (CFs) in vitro. Interactions between miRNAs and their respective targets were examined via immunoblotting, qRT-PCR and luciferase reporter assays. An MI rat model was constructed, and various staining and immunohistochemical assays were performed to explore the mechanisms underlying the miRNA-mediated effects on MI.
Results
miR-363-3p and miR-218-5p were enriched in EPC-Exos, and miR-218-5p and miR-363-3p mimic or inhibitor enhanced or suppressed CF proliferation and angiogenesis, respectively. miR-218-5p and miR-363-3p regulated P53 and junction-mediating and regulatory protein (JMY) by binding to the promoter region of P53 and the 3’ untranslated region of JMY. Additionally, treatment of CFs with exo-miR-218-5p or miR-363-3p mimic upregulated P53 and down-regulated JMY expression, promoted mesenchymal-endothelial transition and inhibited myocardial fibrosis. Administration of exosomes containing miR-218-5p mimic or miR-363-3p mimic ameliorated left coronary artery ligation-induced MI and restored myocardial tissue integrity in the MI model rats.
Conclusions
In summary, these results show that the protective ability of EPC-Exos against MI was mediated by the shuttled miR-218-5p or miR-363-3p via targeting of the P53/JMY signaling pathway.
Keywords
exosomes, myocardial fibrosis, myocardial infarction, microRNA, endothelial progenitor cells, P53/JMY
Figure 1
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This is a list of supplementary files associated with this preprint. Click to download.
- SupplementalFigure1.tif
Characterization of EPC-Exos. A). Transmission electron microscopy analysis of EPC-Exos in sparse regions. Scale bar: 100 nm. B). The particle diameter size distribution of EPC-Exos. C). Western analysis of the surface proteins of exosomes (CD63, Alix, TSG101). D). PKH67 staining of EPC-Exos.
- SupplementalFigure2.tif
Reads statistics for miRNA sequencing data in EPCs and EPC-Exos. A). Length distribution of small RNAs in EPCs and EPC-Exos. B). New miRNAs were discovered using miRDeep2.
- SupplementalFigure3.tif
Gene Ontology (GO) enrichment and KEGG pathway analysis of differentially expressed miRNAs in EPC-Exos vs EPCs. A). KEGG pathway analysis of the upregulated differentially expressed miRNAs (left panel) and downregulated miRNAs (right panel) in EPC-Exos vs EPCs. The P-value (ease-score, Fisher’s P-value, or hypergeometric P-value, cutoff at 0.05) denotes the significance of the pathway correlated with the conditions. The lower the P-value is, the more significant the pathway is. B). Gene Ontology (GO) enrichment analysis of upregulated differentially expressed miRNAs in EPC-Exos vs EPCs. The top ten enrichment score counts in the GO biological process classification for biological process, cellular components and molecular function are listed. C). Gene Ontology (GO) enrichment analysis of downregulated differentially expressed miRNAs in EPC-Exos vs EPCs.
- SupplementalFigure4.tif
Characterization of EPC-Exos treated with GW4869. A). Transmission electron microscopy analysis of EPC-Exos and EPC-Exos treated with GW4869 in sparse regions. Scale bar: 100 nm. B). The particle diameter size distribution and concentration of EPC-Exos with and without GW4869 treatment. C-D). The relative expression of miR-218-5p and miR-363-3p in EPCs and EPC-Exos with and without GW4869 treatment. **P < 0.01, GW4869 versus Control.
- Supplementalfigure5.tif
Function analysis of EPC-exo to CF proliferation and angiogenesis treated with or without GW4869. A) CCK-8 assays to cell viability of CFs treated with EPC-exo or GW4869-exo. B) Capillary-like structures and the tube length in CFs treated with EPC-exo or GW4869-exo. The data are presented as the mean ± SD (n = 3). Control group, without any treatment; Exo group, CFs co- incubation with EPC-Exosomes; GW4869-Exo, CFs co- incubation with EPC-Exosomes which treated with GW4869. **P < 0.01 Exo versus control; ## P < 0.01 GW4869-Exo versus Exo.
- SupplementalFigure6.tif
Angiogenesis and proliferation of CFs treated with miR-218-5p and miR-363-3p directly. A). The relative expression levels of miR-218-5p and miR-363-3p were measured with qRT-PCR in overexpressing and suppressing miR-218-5p and miR-363-3p groups, respectively. The data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, mimic or inhibitor versus NC. B). The expression of P53 and JMY detected by western blotting in overexpressing and suppressing miR-218-5p and miR-363-3p groups, respectively. C and D). The tube formation capability and relative tube length were increased in the mimic-218 and mimic-363 groups. The data are presented as the mean ± SD (n = 3). **P < 0.01 mimic-218 or mimic-363 groups versus NC. E and F). BrdU labelling and flow cytometry assay to detect the effect of miR-218-5p and miR-363-3p on cell proliferation. The data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, mimic or inhibitor group versus NC.
- SupplementalTable1.docx
- SupplementalTable2.docx
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Chengheng Hu
The First Affiliated Hospital of Sun Yat-sen University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-4243-0618
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
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History
CURRENT STATUS: Posted
Version 1
Posted 18 Jan, 2021
No community comments so far
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Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cell Communication and Signaling
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Accumulating evidence has shown that endothelial progenitor cell-derived exosomes (EPC-Exos) can ameliorate myocardial fibrosis. The purpose of the present study was to investigate the effects of EPC-Exos-derived microRNAs (miRNAs) on myocardial infarction (MI).
Methods
A miRNA-Seq dataset of miRNAs differentially expressed between EPCs and exosomes was collected. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the miRNA expression indicated by miRNA-Seq. Immunofluorescence, cell proliferation and angiogenesis assays were employed to investigate the effects of miRNAs on cardiac fibroblasts (CFs) in vitro. Interactions between miRNAs and their respective targets were examined via immunoblotting, qRT-PCR and luciferase reporter assays. An MI rat model was constructed, and various staining and immunohistochemical assays were performed to explore the mechanisms underlying the miRNA-mediated effects on MI.
Results
miR-363-3p and miR-218-5p were enriched in EPC-Exos, and miR-218-5p and miR-363-3p mimic or inhibitor enhanced or suppressed CF proliferation and angiogenesis, respectively. miR-218-5p and miR-363-3p regulated P53 and junction-mediating and regulatory protein (JMY) by binding to the promoter region of P53 and the 3’ untranslated region of JMY. Additionally, treatment of CFs with exo-miR-218-5p or miR-363-3p mimic upregulated P53 and down-regulated JMY expression, promoted mesenchymal-endothelial transition and inhibited myocardial fibrosis. Administration of exosomes containing miR-218-5p mimic or miR-363-3p mimic ameliorated left coronary artery ligation-induced MI and restored myocardial tissue integrity in the MI model rats.
Conclusions
In summary, these results show that the protective ability of EPC-Exos against MI was mediated by the shuttled miR-218-5p or miR-363-3p via targeting of the P53/JMY signaling pathway.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementalFigure1.tif
Characterization of EPC-Exos. A). Transmission electron microscopy analysis of EPC-Exos in sparse regions. Scale bar: 100 nm. B). The particle diameter size distribution of EPC-Exos. C). Western analysis of the surface proteins of exosomes (CD63, Alix, TSG101). D). PKH67 staining of EPC-Exos.
- SupplementalFigure2.tif
Reads statistics for miRNA sequencing data in EPCs and EPC-Exos. A). Length distribution of small RNAs in EPCs and EPC-Exos. B). New miRNAs were discovered using miRDeep2.
- SupplementalFigure3.tif
Gene Ontology (GO) enrichment and KEGG pathway analysis of differentially expressed miRNAs in EPC-Exos vs EPCs. A). KEGG pathway analysis of the upregulated differentially expressed miRNAs (left panel) and downregulated miRNAs (right panel) in EPC-Exos vs EPCs. The P-value (ease-score, Fisher’s P-value, or hypergeometric P-value, cutoff at 0.05) denotes the significance of the pathway correlated with the conditions. The lower the P-value is, the more significant the pathway is. B). Gene Ontology (GO) enrichment analysis of upregulated differentially expressed miRNAs in EPC-Exos vs EPCs. The top ten enrichment score counts in the GO biological process classification for biological process, cellular components and molecular function are listed. C). Gene Ontology (GO) enrichment analysis of downregulated differentially expressed miRNAs in EPC-Exos vs EPCs.
- SupplementalFigure4.tif
Characterization of EPC-Exos treated with GW4869. A). Transmission electron microscopy analysis of EPC-Exos and EPC-Exos treated with GW4869 in sparse regions. Scale bar: 100 nm. B). The particle diameter size distribution and concentration of EPC-Exos with and without GW4869 treatment. C-D). The relative expression of miR-218-5p and miR-363-3p in EPCs and EPC-Exos with and without GW4869 treatment. **P < 0.01, GW4869 versus Control.
- Supplementalfigure5.tif
Function analysis of EPC-exo to CF proliferation and angiogenesis treated with or without GW4869. A) CCK-8 assays to cell viability of CFs treated with EPC-exo or GW4869-exo. B) Capillary-like structures and the tube length in CFs treated with EPC-exo or GW4869-exo. The data are presented as the mean ± SD (n = 3). Control group, without any treatment; Exo group, CFs co- incubation with EPC-Exosomes; GW4869-Exo, CFs co- incubation with EPC-Exosomes which treated with GW4869. **P < 0.01 Exo versus control; ## P < 0.01 GW4869-Exo versus Exo.
- SupplementalFigure6.tif
Angiogenesis and proliferation of CFs treated with miR-218-5p and miR-363-3p directly. A). The relative expression levels of miR-218-5p and miR-363-3p were measured with qRT-PCR in overexpressing and suppressing miR-218-5p and miR-363-3p groups, respectively. The data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, mimic or inhibitor versus NC. B). The expression of P53 and JMY detected by western blotting in overexpressing and suppressing miR-218-5p and miR-363-3p groups, respectively. C and D). The tube formation capability and relative tube length were increased in the mimic-218 and mimic-363 groups. The data are presented as the mean ± SD (n = 3). **P < 0.01 mimic-218 or mimic-363 groups versus NC. E and F). BrdU labelling and flow cytometry assay to detect the effect of miR-218-5p and miR-363-3p on cell proliferation. The data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, mimic or inhibitor group versus NC.
- SupplementalTable1.docx
- SupplementalTable2.docx
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