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Engineered exosome-mediated delivery of functionally active miR-26a and its enhanced suppression effect in HepG2 cells

Liang, Gaofeng, Kan, Shu, Zhu, Yanliang, Feng, Shuying, Feng, Wenpo, Gao, Shegan·2018·International Journal of Nanomedicine
DOI 10.2147/ijn.s154458PMC PMC5796471
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Figure 1 Schematic representation showing the design, construction, and transfection of pEGFP-CD63-Apo-A1 in HEK293T cells. Notes: (A) Schematic representations of the engineered Apo-A1 expression vector. (B1–B3) Micrographs of fluorescent, bright field, and merged images of untransfected HEK293T cells. (C1–C3) Micrographs of GFP-fluorescence, bright field, and merged images of pEGFP-CD63-Apo-A1-transfected HEK293T cells. Abbreviation: GFP, green fluorescent protein.
Figure 1 Schematic representation showing the design, construction, and transfection of pEGFP-CD63-Apo-A1 in HEK293T cells. Notes: (A) Schematic representations of the engineered Apo-A1 expression vector. (B1–B3) Micrographs of fluorescent, bright field, and merged images of untransfected HEK293T cells. (C1–C3) Micrographs of GFP-fluorescence, bright field, and merged images of pEGFP-CD63-Apo-A1-transfected HEK293T cells. Abbreviation: GFP, green fluorescent protein.CC BY-NC · PubMed Central
Figure 2 Characterization of exosomes. Notes: (A, B) TEM micrographs of Apo-Exo before and after loading with miR-26a, respectively. (C, D) DLS assay corresponding to (A and B). (E) Western blots of 293T cells untransfected (mock) and transfected with pEGFP-CD63-Apo-A1 vector (transfected). (F) Western blots of exosomes obtained from culture supernatants of 293T cells that had been untreated (mock) and transfected with pEGFP-CD63-Apo-A1 vector (transfected; n=3). Abbreviations: Apo-Exo, Apo-A1-Exosomes; DLS, dynamic light scattering; TEM, transmission electron microscope.
Figure 2 Characterization of exosomes. Notes: (A, B) TEM micrographs of Apo-Exo before and after loading with miR-26a, respectively. (C, D) DLS assay corresponding to (A and B). (E) Western blots of 293T cells untransfected (mock) and transfected with pEGFP-CD63-Apo-A1 vector (transfected). (F) Western blots of exosomes obtained from culture supernatants of 293T cells that had been untreated (mock) and transfected with pEGFP-CD63-Apo-A1 vector (transfected; n=3). Abbreviations: Apo-Exo, Apo-A1-Exosomes; DLS, dynamic light scattering; TEM, transmission electron microscope.CC BY-NC · PubMed Central
Figure 3 Uptake of exosome by HepG2 cells. Notes: (A) Confocal images of HepG2 cells after 12 h incubation with 200 μg/mL of DiO-labeled Exo and Apo-Exo-A1 under 37°C 5% CO2 condition. Images of DiO-labeled exosomes (green) and DAPI (blue) stained nuclei were imaged by merging the confocal images. (B) Relative fluorescence intensity of DiO-labeled exosomes internalized in HepG2 cells. Scale bars represent 100 μm. Data are shown as mean ± SD (n=3) with significance value *p<0.05. Abbreviations: Apo-Exo-A1, Apo-A1-modified exosomes; DiO, 3′-dioctadecyloxacarbocyanine perchlorate.
Figure 3 Uptake of exosome by HepG2 cells. Notes: (A) Confocal images of HepG2 cells after 12 h incubation with 200 μg/mL of DiO-labeled Exo and Apo-Exo-A1 under 37°C 5% CO2 condition. Images of DiO-labeled exosomes (green) and DAPI (blue) stained nuclei were imaged by merging the confocal images. (B) Relative fluorescence intensity of DiO-labeled exosomes internalized in HepG2 cells. Scale bars represent 100 μm. Data are shown as mean ± SD (n=3) with significance value *p<0.05. Abbreviations: Apo-Exo-A1, Apo-A1-modified exosomes; DiO, 3′-dioctadecyloxacarbocyanine perchlorate.CC BY-NC · PubMed Central
Figure 4 Confocal images of Cy5-labeled miR-26a-loaded Apo-Exo in HepG2 cells recorded at different time points. Notes: (A) The uptake of Apo-Exo in different time points (1 h, 3 h, 6 h, 12 h, and 24 h). (B) The relative fluorescence in the Apo-Exo treated HepG2 cells. Scale bar: the first list is 50 μm, and the rest is 10 μm. All data are shown as mean ± SD compared to 1 h group with significance value *p<0.05 (n=3). Abbreviation: Apo-Exo, Apo-A1-Exosomes.
Figure 4 Confocal images of Cy5-labeled miR-26a-loaded Apo-Exo in HepG2 cells recorded at different time points. Notes: (A) The uptake of Apo-Exo in different time points (1 h, 3 h, 6 h, 12 h, and 24 h). (B) The relative fluorescence in the Apo-Exo treated HepG2 cells. Scale bar: the first list is 50 μm, and the rest is 10 μm. All data are shown as mean ± SD compared to 1 h group with significance value *p<0.05 (n=3). Abbreviation: Apo-Exo, Apo-A1-Exosomes.CC BY-NC · PubMed Central
Figure 5 Results of HepG2 cells treated with Apo-Exo/miR-26a in vitro. Notes: (A) Effect of Apo-Exo/miR-26a on HepG2 cells migration. Images were recorded 1, 2, and 4 days after scratching. T1, T2, and T4 represented the first, second, and fourth days. (B) Ratio of migration corresponding to (A). (C) Cell proliferation of HepG2 cells after treatments with different exosome preparations. (D) Relative expression change of miR-26a level in HepG2 cells at different time points (n=3). All data are shown as mean ± SD with significance value *p<0.05. Abbreviation: Apo-Exo, Apo-A1-Exosomes.
Figure 5 Results of HepG2 cells treated with Apo-Exo/miR-26a in vitro. Notes: (A) Effect of Apo-Exo/miR-26a on HepG2 cells migration. Images were recorded 1, 2, and 4 days after scratching. T1, T2, and T4 represented the first, second, and fourth days. (B) Ratio of migration corresponding to (A). (C) Cell proliferation of HepG2 cells after treatments with different exosome preparations. (D) Relative expression change of miR-26a level in HepG2 cells at different time points (n=3). All data are shown as mean ± SD with significance value *p<0.05. Abbreviation: Apo-Exo, Apo-A1-Exosomes.CC BY-NC · PubMed Central
Figure 6 The effects of miR-26a-delivered Apo-Exo on cell cycle and protein expression. Notes: Cell cycle profiles of HepG2 cells after different treatments (A–C) untreated, Exo/miR-26a, and Apo-Exo/miR-26a, respectively. (D) Distribution of the cell cycle untreated and after treated with Exo/miR-26a and Apo-Exo/miR-26a, respectively. (E) Western blot analysis of CCNE2, CCND2, and CDK6 in the HepG2 cells after treatment with CK (untreated), Exo (exo/miR-26a), and Apo-Exo/miR-26a, respectively (n=3). All data are shown as mean ± SD with significance value *p<0.05. Abbreviation: Apo-Exo, Apo-A1-Exosomes.
Figure 6 The effects of miR-26a-delivered Apo-Exo on cell cycle and protein expression. Notes: Cell cycle profiles of HepG2 cells after different treatments (A–C) untreated, Exo/miR-26a, and Apo-Exo/miR-26a, respectively. (D) Distribution of the cell cycle untreated and after treated with Exo/miR-26a and Apo-Exo/miR-26a, respectively. (E) Western blot analysis of CCNE2, CCND2, and CDK6 in the HepG2 cells after treatment with CK (untreated), Exo (exo/miR-26a), and Apo-Exo/miR-26a, respectively (n=3). All data are shown as mean ± SD with significance value *p<0.05. Abbreviation: Apo-Exo, Apo-A1-Exosomes.CC BY-NC · PubMed Central

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Metadata

Authors
Liang, G., Kan, S., Zhu, Y., Feng, S., Feng, W., & Gao, S.
Venue
International Journal of Nanomedicine
Publisher
Informa UK Limited
Published
2018-01
Volume
Volume 13
Pages
585-599
ISSN
1178-2013
DOI
10.2147/ijn.s154458
PMC
PMC5796471
Cited by
276
References
3
Language
en
Licence
creativecommons.org/licenses/by-nc/3.0/ (unspecified)

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