Milk Exosome-Derived MicroRNA-2478 Suppresses Melanogenesis through the Akt-GSK3β Pathway
<p>Characterization of milk exosomes. (<b>A</b>) The size distributions of milk exosomes were determined by dynamic light scattering; the inset shows Western blotting of the exosome marker genes CD9, TSG101 and HSP70 in the milk exosome pellet and supernatant after centrifugation during the exosome isolation procedure. (<b>B</b>) An image of milk exosomes captured by cryo-electron microscopy.</p> "> Figure 2
<p>Milk exosomes reduce melanogenesis in mouse B16F10 cells. Cells were cultured with 20 or 50 μg/mL of exosomes for 48 h. (<b>A</b>) Cell viability was measured by a WST assay. (<b>B</b>) Tyrosinase activity was assessed after treatment with milk exosomes; n = 3, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001. (<b>C</b>) Melanin contents were determined in the cells exposed to milk exosomes; n = 3, ** <span class="html-italic">p</span> < 0.01. (<b>D</b>) MITF and TYR mRNA levels in milk exosome-treated cells were measured by qRT-PCR; n = 3, ** <span class="html-italic">p</span> < 0.01. (<b>E</b>) Levels of the proteins MITF and TYR in cells treated with milk exosomes were examined by Western blotting.</p> "> Figure 3
<p>miR-2478 inhibits melanogenesis. (<b>A</b>) MiR-2478 expression levels in cells treated with milk exosomes were measured by qRT-PCR; n = 3, * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001. (<b>B</b>) B16F10 cells were transfected with the miR-2478 mimic or negative control (NC). At 48 h post-transfection, the cell viability assay (WST assay) was performed. (<b>C</b>,<b>D</b>) Tyrosinase activity and melanin contents were measured in the cells transfected with the NC or the miR-2478 mimic; n = 3, ** <span class="html-italic">p</span> < 0.01. (<b>E</b>,<b>F</b>) At 48 h post-transfection of the miR-2478 mimic or NC, the expression of MITF and TYR in the cells was measured by qRT-PCR and Western blotting; n = 3, * <span class="html-italic">p</span> < 0.05.</p> "> Figure 4
<p>Silencing of miR-2478 recovers melanogenesis in milk exosome-treated cells. (<b>A</b>,<b>B</b>) Tyrosinase activity and melanin contents in the cells treated with the negative control (NC) or the miR-2478 inhibitor were analyzed in the presence of 50 μg/mL of exosomes; n = 3, ** <span class="html-italic">p</span> < 0.01. (<b>C</b>,<b>D</b>) Expression of MITF and TYR was measured in NC or miR-2478 inhibitor-transfected cells with milk exosomes, as assessed by qRT-PCR and Western blotting; n = 3, * <span class="html-italic">p</span> < 0.05.</p> "> Figure 5
<p>Rap1a is targeted by miR-2478. (<b>A</b>) Sequence alignment of a putative binding site for miR-2478 in the 3′UTR of Rap1a mRNA. The putative binding site in the Rap1a 3′UTR region was then mutated. (<b>B</b>) Cos7 cells were cotransfected with either the miR-2478 mimic or a negative control (NC) for 48 h and either a wild-type 3′UTR reporter plasmids(pGL3-Rap1a-wt) or a mutant 3′UTR plasmid (pGL3-Rap1a-mut). Luciferase activity was assayed at 48 h post-transfection; n = 3, *** <span class="html-italic">p</span> < 0.001. (<b>C</b>,<b>D</b>) Levels of Rap1a expression in B16F10 cells transfected with either the miR-2478 mimic or NC were measured by qRT-PCR and Western blotting; n = 3, ** <span class="html-italic">p</span> < 0.01.</p> "> Figure 6
<p>Silencing of Rap1a suppresses melanogenesis. (<b>A</b>,<b>B</b>) Levels of Rap1a mRNA and protein in B16F10 cells treated with milk exosomes were examined by qRT-PCR and Western blotting; n = 3, * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01. (<b>C</b>) Levels of Rap1a protein were determined by Western blotting in negative control siRNA (NC-siRNA) or Rap1a siRNA-treated cells. (<b>D</b>) B16F10 cells were transfected with NC-siRNA or Rap1a-siRNA. A cell viability assay (WST assay) was performed at 48 h post-transfection. (<b>E</b>,<b>F</b>) Tyrosinase activity and melanin contents were assayed in the cells transfected with NC-siRNA or Rap1a-siRNA; n = 3, ** <span class="html-italic">p</span> < 0.01. (<b>G</b>) Expression of the proteins MITF and TYR was measured in Rap1a siRNA-treated cells by Western blotting.</p> "> Figure 7
<p>The Akt-GSK3β signaling pathway is affected by milk exosomes. (<b>A</b>) Protein levels of pAkt, total Akt, pGSK3β and total GSK3β in B16F10 cells transfected with NC-siRNA or Rap1a-siRNA were measured by Western blotting. (<b>B</b>) Akt and GSK3β protein levels were analyzed in cells treated with 20 or 50 μg/mL of milk exosomes. (<b>C</b>) After the cells were transfected with a negative control (NC) or a miR-2478 mimic, Akt and GSK3β protein levels were assayed by Western blotting. (<b>D</b>) In the presence of 50 μg/mL of exosomes, cells were transfected with the NC or miR-2478 inhibitor. (<b>E</b>) Cells exposed to 50 μg/mL of exosomes were treated with the Akt inhibitor GSK 690693. The levels of MITF, TYR and GSK3β in B16F10 were analyzed by Western blot analysis.</p> "> Figure 8
<p>Milk exosomes suppress melanogenesis in human melanoma cells and melanocytes. (<b>A</b>) MNT-1 human melanoma cells and NHEM human melanocytes were exposed to 20 or 50 μg/mL of milk exosomes for 48 h, and cell viability was evaluated by a WST assay. (<b>B</b>,<b>C</b>) Tyrosinase activity and melanin contents in the cells treated with milk exosomes were measured; n = 3, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001. (<b>D</b>) Human skin tissues (MelanoDerm) were treated with PBS as a control, or 50 or 100 μg/mL of exosomes, and then photographed. The level of pigmentation in the skin was measured by the L value. (<b>E</b>) Paraffin-embedded tissue sections were stained using hematoxylin and eosin (H&E). The melanin pigment of MelanoDerm was visualized by Fontana–Masson staining (F&M). (<b>F</b>) Melanin contents were determined in the MelanoDerm tissues exposed to milk exosomes; n = 3, * <span class="html-italic">p</span> < 0.05.</p> "> Figure 9
<p>miR-2478 inhibits melanogenesis in human melanoma cells and melanocytes. (<b>A</b>) MiR-2478 expression levels in cells treated with milk exosomes were measured by qRT-PCR; n = 3, * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001. (<b>B</b>) The Rap1a protein level in milk exosome-treated cells was assessed by Western blotting. (<b>C</b>,<b>D</b>) Tyrosinase activity and melanin contents were examined in the negative control (NC) or miR-2478 inhibitor-treated cells with 50 μg/mL of exosomes; n = 3, * <span class="html-italic">p</span> < 0.05. ** <span class="html-italic">p</span> < 0.01. (<b>E</b>) Protein levels of MITF, TYR, pAkt, total Akt, pGSK3β and total GSK3β in NC or miR-2478 inhibitor-transfected cells exposed to milk exosomes were measured by Western blotting.</p> "> Figure 10
<p>The proposed model explaining how milk exosomes inhibit melanin production in melanoma cells. In the absence of milk exosomes, Rap1a maintains the active form of GSK3β by inhibiting Akt phosphorylation. The dephosphorylation of GSK3β upregulates the melanogenesis-related protein MITF, which promotes melanin production by driving the expression of tyrosinase. However, in the presence of milk exosomes, miR-2478-carrying milk exosomes inhibit Rap1a expression in melanoma cells, and thus promote the activation of its downstream mediator Akt (via phosphorylation), resulting in GSK3β phosphorylation at Ser9; this change inactivates GSK3β and represses the expression of melanogenesis-related genes such as MITF and TYR, thereby inhibiting melanin production.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Exosomes Purification
2.3. Cryo-Electron Microscopy
2.4. Cell Viability Assay (WST Assay)
2.5. Melanin Content Measurement
2.6. Tyrosinase Activity Assay
2.7. Quantitative rReverse-Transcription Polymerase Chain Reaction (qRT-PCR)
2.8. Transfection and Luciferase Reporter Assay
2.9. Western Blotting
2.10. Human Skin Tissues
2.11. Histological Analysis
2.12. Statistical Analysis
3. Results
3.1. Characterization of the Exosomes Isolated from Milk
3.2. Tyrosinase Activity and Melanin Production Suppressed by Milk Exosomes in B16F10 Cells
3.3. Enrichment of Bovine-Specific miR-2478 in Milk Exosomes
3.4. Bovine-Specific miR-2478 from Milk Exosomes Suppresses Melanogenesis
3.5. Rap1a Is Directly Targeted by miR-2478
3.6. Rap1a Promotes Melanogenesis
3.7. Milk Exosomes Suppress Melanogenesis through the Akt-GSK3β Pathway
3.8. Milk Exosomes Inhibited Melanogenesis in Human Melanoma Cells, Melanocytes and MelanoDerm Tissue
3.9. miR-2478 Inhibited Melanogenesis in Human MNT-1 Melanoma Cells and NHEM
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Bae, I.-S.; Kim, S.H. Milk Exosome-Derived MicroRNA-2478 Suppresses Melanogenesis through the Akt-GSK3β Pathway. Cells 2021, 10, 2848. https://doi.org/10.3390/cells10112848
Bae I-S, Kim SH. Milk Exosome-Derived MicroRNA-2478 Suppresses Melanogenesis through the Akt-GSK3β Pathway. Cells. 2021; 10(11):2848. https://doi.org/10.3390/cells10112848
Chicago/Turabian StyleBae, In-Seon, and Sang Hoon Kim. 2021. "Milk Exosome-Derived MicroRNA-2478 Suppresses Melanogenesis through the Akt-GSK3β Pathway" Cells 10, no. 11: 2848. https://doi.org/10.3390/cells10112848