Bovine Pancreatic RNase A: An Insight into the Mechanism of Antitumor Activity In Vitro and In Vivo
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Cultures
2.2. Mice
2.3. Scratch Assay
2.4. Influence of RNase A on the Migration Activity, Motility, Adhesion, Invasion and Colony Formation of B16 and HeLa Cells
2.5. Conjugation of RNase A with Biotin
2.6. RNA Visualization by Fluorescence Microscopy
2.7. Analysis of Accumulation and Intracellular Localization of RNase A–Biotin Conjugate in HeLa and B16 Cells by Confocal Microscopy
2.8. Analysis of RNase A–Biotin Conjugate Accumulation into B16 and HeLa Cells by Flow Cytometry
2.9. Co-Localization of RNase A–Biotin Conjugate with Ku70/Ku80 in B16 and HeLa Cells
2.10. B16 Implantation and Design of the Animal Experiment
2.11. Sample Processing and RNA Extraction
2.12. RT-qPCR
2.13. Histology and Immunohistochemistry
2.14. Molecular Modeling of RNase A Interaction with Ku70/Ku80
2.15. RNase-A-Susceptible miRNA Target Prediction and Functional Analysis
2.16. Statistics
3. Results
3.1. Intracellular Accumulation and Localization of RNase A–Biotin Conjugate and Its Interaction with RI in B16 and HeLa Cells
3.2. Interaction of RNase A with Ku70/Ku80 Heterodimer: Molecular Modeling and Intracellular Behavior
3.3. The Effect of RNase A on Intracellular RNAs
3.4. The Effect of RNase A on the Migration Activity, Adhesion, Invasion and Colony Formation of B16 and HeLa Cells
3.5. The Influence of RNase A on Metastasis of Melanoma B16 In Vivo
3.6. The Effect of RNase A on the Expression of EMT Markers in Metastatic Foci and Adjacent Lung Tissue of B16 Melanoma-Bearing Mice
3.7. Alteration of miRNA Expression in B16 and HeLa Cells In Vitro and in B16 Metastatic Foci and Adjacent Lung Tissues In Vivo under the Action of RNase A
3.8. RNase-A-Susceptible miRNA: Target Prediction and Functional Analysis
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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miRNA | Cells w/o Any Treatment | Cells Treated with RNase A | ||
---|---|---|---|---|
Absolute Expression Level, a.u. (2) | Fold Changes in miRNA Level (3) | |||
B16 | HeLa | B16 | HeLa | |
mir-21a | 0.7 | 0.9 | n.a. | n.a. |
mir-145a | 0.8 | 0.95 | n.a. | n.a. |
mir-31 | 0.75 | 0.86 | n.a. | n.a. |
mir-10b | 0.8 | 0.78 | 1.1↓–1.5↓ | n.a. |
let-7g | 0.75 | 0.8 | n.a. | n.a. |
miR-155 | 0.56 | 1.0 | 1.6–2.0↓↓ | n.a. |
U6 snRNA (1) | 0.86 | 0.82 | n.a. | n.a. |
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Mohamed, I.S.E.; Sen’kova, A.V.; Markov, O.V.; Markov, A.V.; Savin, I.A.; Zenkova, M.A.; Mironova, N.L. Bovine Pancreatic RNase A: An Insight into the Mechanism of Antitumor Activity In Vitro and In Vivo. Pharmaceutics 2022, 14, 1173. https://doi.org/10.3390/pharmaceutics14061173
Mohamed ISE, Sen’kova AV, Markov OV, Markov AV, Savin IA, Zenkova MA, Mironova NL. Bovine Pancreatic RNase A: An Insight into the Mechanism of Antitumor Activity In Vitro and In Vivo. Pharmaceutics. 2022; 14(6):1173. https://doi.org/10.3390/pharmaceutics14061173
Chicago/Turabian StyleMohamed, Islam Saber Ead, Aleksandra V. Sen’kova, Oleg V. Markov, Andrey V. Markov, Innokenty A. Savin, Marina A. Zenkova, and Nadezhda L. Mironova. 2022. "Bovine Pancreatic RNase A: An Insight into the Mechanism of Antitumor Activity In Vitro and In Vivo" Pharmaceutics 14, no. 6: 1173. https://doi.org/10.3390/pharmaceutics14061173
APA StyleMohamed, I. S. E., Sen’kova, A. V., Markov, O. V., Markov, A. V., Savin, I. A., Zenkova, M. A., & Mironova, N. L. (2022). Bovine Pancreatic RNase A: An Insight into the Mechanism of Antitumor Activity In Vitro and In Vivo. Pharmaceutics, 14(6), 1173. https://doi.org/10.3390/pharmaceutics14061173