Synergistic Autophagy Effect of miR-212-3p in Zoledronic Acid-Treated In Vitro and Orthotopic In Vivo Models and in Patient-Derived Osteosarcoma Cells
Abstract
:1. Introduction
2. Results
2.1. ZOL Decreased OS Cell Proliferation in a Dose-Dependent Manner
2.2. ZOL Induced Accumulation of Acidic Vacuoles (AVOs)
2.3. ZOL Treatment Induced Autophagy
2.4. Inhibition of ZOL-Induced Autophagy Decreased Cell Death
2.5. ZOL Suppressed the mTOR/p70S6K Signaling Pathway
2.6. miR-212-3p Directly Targeted and, Thus, Positively Regulated Autophagy after ZOL Treatment
2.7. The Autophagic Effects of Combined miR-212-3p and ZOL Treatment in In Vivo Orthotopic Model
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Tissue Samples
4.2. Reagents
4.3. MTT Assay
4.4. Colony-Formation Assay
4.5. Detection of Apoptotic Cells by Annexin V Staining
4.6. Western Blotting
4.7. Morphology
4.8. 5-Bromo-2′-Deoxyuridine (BrdU)-Labeling Assay
4.9. Orthotopic Model and Histological Analysis
4.10. Quantification of Acidic Vacuoles (AVOs) by Acridine Orange (AO) Staining
4.11. Immunohistochemical Staining
4.12. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Assays
4.13. Elisa Assay
4.14. miRNA and Transient Transfection
4.15. Quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR) Experiments
4.16. In Vivo Tumor Model and Administration of ZOL and miR-212-3p Mimic
4.17. Positron Emission Tomography (PET)/Computed Tomography (CT) Acquisition
4.18. Statistical Analysis
4.19. Data Availability
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
[18F]-FDG | 18F-fluoro-2-deoxy-d-glucose |
AO | acridine orange |
AVOs | acidic vacuoles |
BrdU | 5-bromo-2′-deoxyuridine |
CT | computed tomography |
FACS | fluorescence-activated cell sorting |
i.p. | intraperitoneal |
MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
NSCLC | non-small cell lung cancer |
OS | osteosarcoma |
PBS | phosphate-buffered saline |
PET | positron emission tomography |
qRT-PCR | quantitative reverse-transcriptase polymerase chain reaction |
SUV | standardized uptake value |
TUNEL | terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling |
VOI | volumes of interest |
ZOL | zoledronic acid |
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Oh, J.Y.; Kim, E.H.; Lee, Y.-J.; Sai, S.; Lim, S.H.; Park, J.W.; Chung, H.K.; Kim, J.; Vares, G.; Takahashi, A.; et al. Synergistic Autophagy Effect of miR-212-3p in Zoledronic Acid-Treated In Vitro and Orthotopic In Vivo Models and in Patient-Derived Osteosarcoma Cells. Cancers 2019, 11, 1812. https://doi.org/10.3390/cancers11111812
Oh JY, Kim EH, Lee Y-J, Sai S, Lim SH, Park JW, Chung HK, Kim J, Vares G, Takahashi A, et al. Synergistic Autophagy Effect of miR-212-3p in Zoledronic Acid-Treated In Vitro and Orthotopic In Vivo Models and in Patient-Derived Osteosarcoma Cells. Cancers. 2019; 11(11):1812. https://doi.org/10.3390/cancers11111812
Chicago/Turabian StyleOh, Ju Yeon, Eun Ho Kim, Yeon-Joo Lee, Sei Sai, Sun Ha Lim, Jang Woo Park, Hye Kyung Chung, Joon Kim, Guillaume Vares, Akihisa Takahashi, and et al. 2019. "Synergistic Autophagy Effect of miR-212-3p in Zoledronic Acid-Treated In Vitro and Orthotopic In Vivo Models and in Patient-Derived Osteosarcoma Cells" Cancers 11, no. 11: 1812. https://doi.org/10.3390/cancers11111812