Role of miR-34a-5p in Hematopoietic Progenitor Cells Proliferation and Fate Decision: Novel Insights into the Pathogenesis of Primary Myelofibrosis
Abstract
:1. Introduction
2. Results
2.1. miR-34a-5p Expression Levels in CD34+ Cells from PMF Patients and HDs
2.2. Effects of miR-34a-5p Overexpression on HPCs Proliferation and Clonogenic Efficiency
2.3. Effects of miR-34a-5p Overexpression on HPCs Lineage Choice
2.4. Gene Expression Profile (GEP) of miR-34a-5p-Overexpressing Cells and Identification of miR-34a-5p Targets
2.5. Effects of LEF1 and NR4A2 Silencing on HPCs Proliferation and Clonogenic Efficiency
2.6. Effects of LEF1 and NR4A2 on the HPCs Lineage Choice
3. Discussion
4. Materials and Methods
4.1. Ethics Statement
4.2. miR-34a-5p Expression Analysis in PMF and HD CD34+ Cells
4.3. Human CD34+ Hematopoietic Progenitor Cells (HPCs) Purification
4.4. miR-34a-5p, LEF1 and NR4A2 Kinetics during the Erythroid, Megakaryocyte, Granulocyte and Mono-Macrophage Differentiation of CD34+ HPCs
4.5. Nucleofection of CD34+ Cells
4.6. Methylcellulose- and Collagen-Based Clonogenic Assays
4.7. Cell Cycle Analysis
4.8. Morphological and Immunophenotypic Analysis
4.9. RNA Extraction
4.10. Gene Expression Profiling (GEP)
4.11. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)
4.12. Western Blot
4.13. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Probeset ID | Gene Symbol | Gene Title | RefSeq Transcript ID | FC miR-34a-5p Mimic vs. NegCTR Mimic CD34+ Cells | TargetScan-Predicted miR-34a-5p Target | Modulation in PMF vs. HD CD34+ Cells |
---|---|---|---|---|---|---|
11734171_at | PRTN3 | proteinase 3 | NM_002777 | −3.81 | − | − |
11728026_x_at | IGLL1 | immunoglobulin lambda-like polypeptide 1 | NM_020070; NM_152855 | −3.22 | − | − |
11756600_a_at | TPD52 | tumor protein D52 | NM_001025252;NM_001025253; NM_005079 | −2.93 | + | − |
11729643_s_at | TPD52 | tumor protein D52 | NM_001025252; NM_001025253; NM_005079 | −2.89 | + | − |
11754659_x_at | TPD52 | tumor protein D52 | NM_001025252;NM_001025253;NM_005079 | −2.69 | + | − |
11727965_at | ELANE | elastase, neutrophil expressed | NM_001972 | −2.65 | − | − |
11715306_s_at | AREG; AREGB | amphiregulin /// amphiregulin B | NM_001657; XM_001125684 | −2.61 | + | D |
11745205_s_at | TPD52 | tumor protein D52 | NM_001025252;NM_001025253; NM_005079 | −2.59 | + | − |
11723339_at | CTSG | cathepsin G | NM_001911 | −2.29 | − | − |
11726333_s_at | LEF1 | lymphoid enhancer-binding factor 1 | NM_001130713;NM_001130714;NM_001166119; NM_016269 | −2.29 | + | D |
11718477_a_at | STAR | steroidogenic acute regulatory protein | NM_000349; NM_001007243 | −2.24 | − | − |
11720051_at | SPOCK1 | sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1 | NM_004598 | −2.17 | − | − |
11718479_x_at | STAR | steroidogenic acute regulatory protein | NM_000349; NM_001007243 | −2.17 | − | − |
11715245_s_at | IGLL1 | immunoglobulin lambda-like polypeptide 1 | NM_020070; NM_152855 | −2.15 | − | − |
11729641_a_at | TPD52 | tumor protein D52 | NM_001025252; NM_001025253; NM_005079 | −2.06 | + | − |
11725632_at | NR4A2 | nuclear receptor subfamily 4, group A, member 2 | NM_006186; NM_173171; NM_173172; NM_173173 | −2.03 | + | D |
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Bianchi, E.; Ruberti, S.; Rontauroli, S.; Guglielmelli, P.; Salati, S.; Rossi, C.; Zini, R.; Tagliafico, E.; Vannucchi, A.M.; Manfredini, R. Role of miR-34a-5p in Hematopoietic Progenitor Cells Proliferation and Fate Decision: Novel Insights into the Pathogenesis of Primary Myelofibrosis. Int. J. Mol. Sci. 2017, 18, 145. https://doi.org/10.3390/ijms18010145
Bianchi E, Ruberti S, Rontauroli S, Guglielmelli P, Salati S, Rossi C, Zini R, Tagliafico E, Vannucchi AM, Manfredini R. Role of miR-34a-5p in Hematopoietic Progenitor Cells Proliferation and Fate Decision: Novel Insights into the Pathogenesis of Primary Myelofibrosis. International Journal of Molecular Sciences. 2017; 18(1):145. https://doi.org/10.3390/ijms18010145
Chicago/Turabian StyleBianchi, Elisa, Samantha Ruberti, Sebastiano Rontauroli, Paola Guglielmelli, Simona Salati, Chiara Rossi, Roberta Zini, Enrico Tagliafico, Alessandro Maria Vannucchi, and Rossella Manfredini. 2017. "Role of miR-34a-5p in Hematopoietic Progenitor Cells Proliferation and Fate Decision: Novel Insights into the Pathogenesis of Primary Myelofibrosis" International Journal of Molecular Sciences 18, no. 1: 145. https://doi.org/10.3390/ijms18010145