Role of Site-Specific Glycosylation in the I-Like Domain of Integrin β1 in Small Extracellular Vesicle-Mediated Malignant Behavior and FAK Activation
Abstract
:1. Introduction
2. Results
2.1. N-Glycosylation Site Mutation of Integrin β1
2.2. Influence of Integrin β1 Mutants on the Cell Adhesion, Mobility, and Signaling Pathway
2.3. Effects of Secreted Components from β1 Mutants on Behavior of Recipient Cells
2.4. Effects of β1 Mutants on Biological Function of sEVs
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Plasmid Construction
4.3. Stable Transfection of WT or Mutant Integrin β1
4.4. Whole-Cell Lysate Extraction and Western Blotting
4.5. Cell Apoptosis Assay
4.6. Cell Adhesion
4.7. Cell Migration by Transwell Assay
4.8. Conditioned Medium Extraction, sEV Isolation, and sEV-Free FBS Preparation
4.9. Co-Culture System
4.10. Nanoparticle Tracking Analysis
4.11. Transmission Electron Microscopy (TEM)
4.12. sEV Uptake by Flow Cytometry
4.13. Data Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
sEVs | Small extracellular vesicles |
ECM | Extracellular matrix |
FAK | Focal adhesion kinase |
AKT | protein kinase B |
PSI | Plexin-semaphorin-integrin domain |
I-EGF | Integrin-epidermal growth factor domain |
FN | Fibronectin |
CM | Conditioned medium |
GFP | green fluorescent protein |
FBS | fetal bovine serum |
BSA | bovine serum albumin |
PBS | phosphate buffer saline |
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Oligonucleotide (Integrin β1) | Primer Sequence (5′-3′) | |
---|---|---|
Full length-sense: | CCGGAATTCATGAATTTACAACCAATTTTCTGGATTGGACT | |
Full length-antisense: | GCTCTAGATCATTTTCCCTCATACTTCGGATTGAC | |
Δ1–3 mutant | 50-sense: | TGTTGAATCTGTGCACCACCCACAATTTGG |
50-anti-sense | TGGTGCACAGATTCAACATTTTTACAGGAAGG | |
94 and 97-sense: | GCTACGGTCGGTTACATCTTTATTTTTCTTTAT | |
94 and 97-anti-sense: | AATAAAGATGTAACCGACCGTAGCAAAGGAAC | |
Δ4–6 mutant | 212-sense: | CTGGTGCAGTCCTGTTCACTTGTGC |
212-anti-sense: | TGAAC AGGAC TGCAC CAGCC CATT | |
269-sense: | TGTAACATCCCTCCAGCCAATCAGTG | |
269-anti-sense: | TGGAGGGATGTTACACGGCTGC | |
363-sense: | CATTGCTAGAATCTGCAGATAATGTTCC | |
363-anti-sense: | TATCTGCAGATTCTAGCAATGTAATTCAGTT | |
Δ7–8 mutant | 406-sense: | TTCCATCCACCCCGTTCTTGCAGTAAG |
406-anti-sense: | CGGGGTGGATGGAACAGGGGAAAAT | |
417-sense: | GGAAATATCGGAACATTTTCTTCC | |
417-anti-sense: | TGTTCCGATATTTCCATTGGAGATGAGG | |
Δ9–12 mutant primer | 481-sense: | AATGTCCCATCTCCTTCATGACACTT |
481-anti-sense: | CATGAAGGAGATGGGACATTTGAGTG | |
520-sense: | CTGAACTGTCTTCTTTCCTGCAGTAAGC | |
520-anti-sense: | GGAAAGAAGACAGTTCAGAAATCT | |
584-sense: | GTGTAGTCGGGGTTGCACTCACAC | |
584-anti-sense: | GCAACCCCGACTACACTGGC | |
669-sense: | CTACCTTGGTAATGTCAAAATAGGAACATTC | |
669-anti-sense: | CCTATTTTGACATTACCAAGGTAGAAAGT |
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Cao, L.; Wu, Y.; Wang, X.; Li, X.; Tan, Z.; Guan, F. Role of Site-Specific Glycosylation in the I-Like Domain of Integrin β1 in Small Extracellular Vesicle-Mediated Malignant Behavior and FAK Activation. Int. J. Mol. Sci. 2021, 22, 1770. https://doi.org/10.3390/ijms22041770
Cao L, Wu Y, Wang X, Li X, Tan Z, Guan F. Role of Site-Specific Glycosylation in the I-Like Domain of Integrin β1 in Small Extracellular Vesicle-Mediated Malignant Behavior and FAK Activation. International Journal of Molecular Sciences. 2021; 22(4):1770. https://doi.org/10.3390/ijms22041770
Chicago/Turabian StyleCao, Lin, Yurong Wu, Xiuxiu Wang, Xiang Li, Zengqi Tan, and Feng Guan. 2021. "Role of Site-Specific Glycosylation in the I-Like Domain of Integrin β1 in Small Extracellular Vesicle-Mediated Malignant Behavior and FAK Activation" International Journal of Molecular Sciences 22, no. 4: 1770. https://doi.org/10.3390/ijms22041770