The miRNA-21-5p Payload in Exosomes from M2 Macrophages Drives Tumor Cell Aggression via PTEN/Akt Signaling in Renal Cell Carcinoma
Abstract
:1. Introduction
2. Results
2.1. M2 Macrophages Promote RCC Migration and Invasion
2.2. Characterization and Internalization of M2-Exos
2.3. M2-Exos Promote RCC Migration and Invasion
2.4. MiR-21-5p in M2-Exos Directs RCC Aggressive Behaviors In Vitro and Metastasis In Vivo
2.5. MiR-21-5p in M2-Exos Promotes RCC Metastasis through PTEN/Akt Signaling
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Exosomes Isolation and TEM
4.3. Internalization of PKH67-Labeled M2-Exos
4.4. RNA Isolation and RT-qPCR
4.5. Western Blotting
4.6. Transwell and Wound-Healing Assays
4.7. Cell Proliferation Assay
4.8. Cell Transfection and miRNA Targeted Luciferase Reporter Assay
4.9. IHC and IF
4.10. CAM Model and Quantitative PCR (qPCR) Analysis of Genomic DNA
4.11. Statistics Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
RCC | Renal cell carcinoma |
ccRCC | clear cell renal cell carcinoma |
miRNA | microRNA |
TAMs | Tumor-associated macrophages |
THP-1-Mφ | Mφ subtype differentiated from THP-1 cell |
THP-1-M2 | M2 subtype differentiated from THP-1 cell |
M2-Exos | Exosomes derived from M2 macrophages |
miR-21-Inh | miR-21-5p inhibitor |
miR-21-NC | miR-21-5p negative control |
miR-21-Inh-Exos | Exosomes derived from M2 macrophages transfected with miR-21-Inh |
miR-21-NC-Exos | Exosomes derived from M2 macrophages transfected with miR-21-NC |
CAM | Chorioallantoic membrane |
DLS | Dynamic light scattering |
DAPI | 4′,6-diamidino-2-phenylindole |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
H&E | Hematoxylin and Eosin |
IHC | Immunohistochemistry |
IF | Immunofluorescence |
3′UTRs | the 3′-untranslated regions of mRNA |
qPCR | quantitative PCR |
RT-qPCR | Reverse transcription-qPCR |
PTEN | Phosphatase and tensin homolog |
CD163 | Cluster of differentiation 163 |
CD206 | Cluster of differentiation 206 |
HLA-DR | Human leukocyte antigen-DR isotype |
TNF-α | Tumor necrosis factor alpha |
MMP-9 | Matrix metalloproteinase 9 |
EMT | Epithelial-mesenchymal transition |
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Zhang, Z.; Hu, J.; Ishihara, M.; Sharrow, A.C.; Flora, K.; He, Y.; Wu, L. The miRNA-21-5p Payload in Exosomes from M2 Macrophages Drives Tumor Cell Aggression via PTEN/Akt Signaling in Renal Cell Carcinoma. Int. J. Mol. Sci. 2022, 23, 3005. https://doi.org/10.3390/ijms23063005
Zhang Z, Hu J, Ishihara M, Sharrow AC, Flora K, He Y, Wu L. The miRNA-21-5p Payload in Exosomes from M2 Macrophages Drives Tumor Cell Aggression via PTEN/Akt Signaling in Renal Cell Carcinoma. International Journal of Molecular Sciences. 2022; 23(6):3005. https://doi.org/10.3390/ijms23063005
Chicago/Turabian StyleZhang, Zhicheng, Junhui Hu, Moe Ishihara, Allison C. Sharrow, Kailey Flora, Yao He, and Lily Wu. 2022. "The miRNA-21-5p Payload in Exosomes from M2 Macrophages Drives Tumor Cell Aggression via PTEN/Akt Signaling in Renal Cell Carcinoma" International Journal of Molecular Sciences 23, no. 6: 3005. https://doi.org/10.3390/ijms23063005