MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Culture and Reagents
2.2. Cell Transfection
2.3. Epidermal Growth Factors (EGF) Cells Treatments
2.4. Gefitinib Cells Treatments
2.5. hMSC Viability (WST-1 Test)
2.6. RNA Extraction and Real-Time PCR
2.7. Western Blot Analysis
2.8. OsteoImage™ Bone Mineralization Assay
2.9. Statistical Analysis
3. Results
3.1. MiR-33a Family is Involved in the Maintenance of hMSCs and Osteoblast Phenotypes
3.2. MiR-33a Family Can Promote hMSCs Osteoblast Commitments
3.3. Modulation of miR-33a-5p and miR-33a-3p Expression Regulates the Activation of EMT Signaling in Pre-Osteoblast Cells
3.4. YAP/TAZ: Possible Mediators of miR-33a Effects During hMSCs Osteoblast Differentiation Process
3.5. EGF Treatment Induce the Expression of miR-33a-3p and YAP/TAZ Modulation on hMSCs
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Gene | Qiagen Primers QuantiTect Primer Assay | Catalog Number |
---|---|---|
RUNX-2 “Runt-related transcription factor 2” | Hs_RUNX2_1_SG | QT00020517 |
ALPL “Alkaline phosphatase” | Hs_ALPL_1_SG | QT00012957 |
BGLAP “bone gamma-carboxyglutamic acid-containing protein” | Hs_BGLAP_1_SG | QT00232771 |
SPP1 “Secreted Phosphoprotein 1” | Hs_SPP1_1_SG | QT01008798 |
Gene | Custom Made Primers Forward | Custom Made Primers Reverse |
SNAIL “Snail family transcriptional repressor 1” | GCGAGCTGCAGGACTCTAAT | CCCGCAATGGTCCACAAAAC |
SLUG “Snail family transcriptional repressor 2” | CATGCCTGTCATACCACAAC | GGTGTCAGATGGAGGAGGG |
TWIST “Twist family bHLH transcription factor 1” | CGGCCAGGTACATCGACTTC | CAGAGGTGTGAGGATGGTGC |
TGF-B “Transforming growth factor beta 1” | TGGTGGAAACCCACAACGAA | ACACAGAGATCCGCAGTCCT |
YAP “Yes associated protein 1” | CCTCGTTTTGCCATGAACCAG | GTTCTTGCTGTTTCAGCCGCAG |
TAZ “WW domain containing transcription regulator” | TGGACCAAGTACATGAACCACC | TCCACAGCCGACTTGTTCTC |
HMGA-2 “High mobility group AT-hook 2” | GCGCCTCAGAAGAGAGGAC | GTCTTCCCCTGGGTCTCTTAG |
EGFR “Epidermal growth factor receptor” | CCTATGTGCAGAGGAATTATGATCTTT | CCACTGTGTTGAGGGCAATG |
ERK-2 “Extracellular signal-regulated kinases -2” | GCGCTACACTAATCTCTCGT | CTGAGGTGCTGTGTCTTCAA |
ERK-3 “Extracellular signal-regulated kinases -3” | GAATGGCAAATCTGCTCAATT | ACAGTCCTCCCCACCACTCA |
Reference Gene | Forward | Reverse |
ACTB “Beta-Actin” | ATCAAGATCATTGCTCCTCCTGA | CTGCTTGCTGATCCACATCTG |
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Costa, V.; Carina, V.; Raimondi, L.; De Luca, A.; Bellavia, D.; Conigliaro, A.; Salamanna, F.; Alessandro, R.; Fini, M.; Giavaresi, G. MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation. Cells 2019, 8, 1495. https://doi.org/10.3390/cells8121495
Costa V, Carina V, Raimondi L, De Luca A, Bellavia D, Conigliaro A, Salamanna F, Alessandro R, Fini M, Giavaresi G. MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation. Cells. 2019; 8(12):1495. https://doi.org/10.3390/cells8121495
Chicago/Turabian StyleCosta, Viviana, Valeria Carina, Lavinia Raimondi, Angela De Luca, Daniele Bellavia, Alice Conigliaro, Francesca Salamanna, Riccardo Alessandro, Milena Fini, and Gianluca Giavaresi. 2019. "MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation" Cells 8, no. 12: 1495. https://doi.org/10.3390/cells8121495
APA StyleCosta, V., Carina, V., Raimondi, L., De Luca, A., Bellavia, D., Conigliaro, A., Salamanna, F., Alessandro, R., Fini, M., & Giavaresi, G. (2019). MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation. Cells, 8(12), 1495. https://doi.org/10.3390/cells8121495