miR-9-Mediated Inhibition of EFEMP1 Contributes to the Acquisition of Pro-Tumoral Properties in Normal Fibroblasts
Abstract
:1. Introduction
2. Materials and Methods
2.1. In-Silico Analysis to Define Caf and NF EFEMP1 Expression Portraits
2.2. Cell Culture and Primary Fibroblasts Isolation
2.3. MiRNA Mimics and siRNA Transient Transfection
2.4. Cloning and Mutagenesis
2.5. Luciferase Reporter Assay
2.6. Motility Assays
2.7. Protein Extraction and Western Blot
2.8. Immunohistochemistry
2.9. Tumor Cell Conditioning and Resistance Test
2.10. Mining Data to Evaluate Correlation of MiR-9 Expression and Cisplatin Response
3. Results
3.1. In-Silico Evaluation of EFEMP1 Levels in CAFs
3.2. MiR-9 Directly Targets EFEMP1 and Affects Protein Levels In Vitro and In Vivo
3.3. EFEMP1 Silencing Recapitulates miR-9-Induced CAF-Like Features in Normal Fibroblasts
3.4. CAF-Like Properties Induced by miR-9/si-EFEMP1-Transfection Reduce MDA-MB-468 Cell Sensitivity to Cisplatin
3.5. Characterization of miR-9/CAF Axis on TNBC Biology and Chemotherapy Response by Mining mRNA and miRNA Expression Data
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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3′UTR EFEMP1 Forward | 5′-AATTGCTAGCTTGACAATAATAGTGGGGCCA-3′ |
3′UTR EFEMP1 Reverse | 5′-AATTTCTAGATGCCCACTTTATACCATGG-3′ |
pmirGLO Forward | 5′-CGCGAGATTCTCATTAAGGCC-3′ |
pmirGLO Reverse | 5′-CAACTCAGCTTCCTTTCGG-3′ |
MiR-9 binding site | 5′-CCAAAGA-3′ |
3′UTR EFEMP1 MUT Forward | 5′-ATAAAATAGTGCTTTAAGGTAACAATATCGTGTCGCTGACTTAAA TGCCTGTGGTTGACTCT-3′ |
3′UTR EFEMP1 MUT Reverse | 5′-AGAGTCAACCACAGGCATTTAAGTCAGCGACACGATATTGTTAC CTTAAAGCACTATTTTAT-3′ |
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Cosentino, G.; Romero-Cordoba, S.; Plantamura, I.; Cataldo, A.; Iorio, M.V. miR-9-Mediated Inhibition of EFEMP1 Contributes to the Acquisition of Pro-Tumoral Properties in Normal Fibroblasts. Cells 2020, 9, 2143. https://doi.org/10.3390/cells9092143
Cosentino G, Romero-Cordoba S, Plantamura I, Cataldo A, Iorio MV. miR-9-Mediated Inhibition of EFEMP1 Contributes to the Acquisition of Pro-Tumoral Properties in Normal Fibroblasts. Cells. 2020; 9(9):2143. https://doi.org/10.3390/cells9092143
Chicago/Turabian StyleCosentino, Giulia, Sandra Romero-Cordoba, Ilaria Plantamura, Alessandra Cataldo, and Marilena V. Iorio. 2020. "miR-9-Mediated Inhibition of EFEMP1 Contributes to the Acquisition of Pro-Tumoral Properties in Normal Fibroblasts" Cells 9, no. 9: 2143. https://doi.org/10.3390/cells9092143