MicroRNA-16 Restores Sensitivity to Tyrosine Kinase Inhibitors and Outperforms MEK Inhibitors in KRAS-Mutated Non-Small Cell Lung Cancer
Abstract
:1. Introduction
2. Results
2.1. MicroRNA-16 Is Downregulated in NSCLC Primary Samples and Cell Lines
2.2. MicroRNA-16 Directly Targets MAPK3, MAP2K1, and CRAF
2.3. MicroRNA-16 Restores Sensitivity to Erlotinib in KRAS-Mutated NSCLC Cell Lines
2.4. MicroRNA-16 Restores Sensitivity to Erlotinib by Targeting MAPK3 and MAP2K1
2.5. MicroRNA-16 Outperforms Selumetinib in Reducing NSCLC Growth In Vitro and In Vivo
3. Discussion
4. Materials and Methods
4.1. Patient Samples
4.2. Cell lines and Cell Cultures
4.3. Drugs
4.4. Transfection with microRNA Mimics
4.5. In Vitro Proliferation Assay
4.6. In Vitro Cell Growth Assays
- 4 h post-transfection with erlotinib at a single concentration of 1 μg/mL for 6 h and, following a medium change, allowed for 24–48–72–96 h washout;
- 10 h post-transfection with selumetinib at a single concentration of 1 μM continuously for 24–48–72–96 h.
4.7. In Vitro Cell Migration Assays
- 4 h post-transfection with erlotinib at a single concentration of 1 μg/mL for 6 h and, following a medium change, allowed for 24–48 h washout;
- 10 h post-transfection after erlotinib treatment with selumetinib at a single concentration of 1 μM continuously for 24–48 h.
4.8. DNA, RNA, and Protein Extraction
4.9. Analysis of KRAS Mutation Status
4.10. Evaluation of miRNAs and mRNA Expression
4.11. Protein Expression Analysis
4.12. Luciferase Assay
4.13. CellTiter-Glo® Luminescent Cell Viability Assay
4.14. Annexin-V Assay
4.15. Bromodeoxyuridine (BrdU) Assay
4.16. Animal Models
4.17. Statistical Analyses
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AKT | AKT serine/threonine kinase |
CMS | Medical scientific committee |
CRAF | Raf-1 proto-oncogene, serine/threonine kinase |
DMSO | Dimethyl sulfoxide |
EGFR | Epidermal growth factor receptor |
ERL | Erlotinib |
FBS | Fetal bovine serum |
KRAS | KRAS proto-oncogene, GTPase |
LV16 | LV16 |
LVEV | Lentiviral empty vector |
MAP2K1 | Mitogen-activated protein kinase kinase 1 |
MAPK | Mitogen-activated protein kinases |
MAPK3 | Mitogen-activated protein kinase 3 |
MEK | Mitogen-activated protein kinase kinase |
NSCLC | Non-small cell lung cancer |
PI3K | Phosphoinositide 3-chinasi |
PMSF | Phenylmethylsulphonyl fluoride |
Pre-miRNA | miRNA precursor |
SCR | Scrambled negative control |
SEL | Selumetinib |
TKIs | Tyrosine kinase inhibitors |
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Fanini, F.; Bandini, E.; Plousiou, M.; Carloni, S.; Wise, P.; Neviani, P.; Murtadha, M.; Foca, F.; Fabbri, F.; Vannini, I.; et al. MicroRNA-16 Restores Sensitivity to Tyrosine Kinase Inhibitors and Outperforms MEK Inhibitors in KRAS-Mutated Non-Small Cell Lung Cancer. Int. J. Mol. Sci. 2021, 22, 13357. https://doi.org/10.3390/ijms222413357
Fanini F, Bandini E, Plousiou M, Carloni S, Wise P, Neviani P, Murtadha M, Foca F, Fabbri F, Vannini I, et al. MicroRNA-16 Restores Sensitivity to Tyrosine Kinase Inhibitors and Outperforms MEK Inhibitors in KRAS-Mutated Non-Small Cell Lung Cancer. International Journal of Molecular Sciences. 2021; 22(24):13357. https://doi.org/10.3390/ijms222413357
Chicago/Turabian StyleFanini, Francesca, Erika Bandini, Meropi Plousiou, Silvia Carloni, Petra Wise, Paolo Neviani, Mariam Murtadha, Flavia Foca, Francesco Fabbri, Ivan Vannini, and et al. 2021. "MicroRNA-16 Restores Sensitivity to Tyrosine Kinase Inhibitors and Outperforms MEK Inhibitors in KRAS-Mutated Non-Small Cell Lung Cancer" International Journal of Molecular Sciences 22, no. 24: 13357. https://doi.org/10.3390/ijms222413357