A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA)
Abstract
:1. Introduction
2. Results
2.1. SMNΔ7 Stem Cells Derived from E13.5 Spinal Cords form Neurospheres and Express Specific Regional Markers
2.2. SMNΔ7 Neural Stem Cells Show a Perturbed Cell Cycle Phase Distribution and Increased Proliferation Rate Compared to Wild Type Cells
2.3. The Expression of Specific microRNAs Is Affected in SMNΔ7 Neural Stem Cells
Underexpressed in SMA vs. WT | ||
miRNA | p-Value | Log2 (SMA/WT) |
mmu-miR-20b-5p | 4.73 × 10−3 | −1.81 |
mmu-miR-6944-5p | 2.79 × 10−2 | −1.78 |
mmu-miR-100-5p | 3.22 × 10−2 | −1.74 |
mmu-miR-335-5p | 1.70 × 10−2 | −1.69 |
mmu-let-7k | 4.76 × 10−2 | −1.31 |
mmu-miR-6931-5p | 3.88 × 10−2 | −1.15 |
mmu-miR-1224-5p | 3.77 × 10−2 | −1.13 |
mmu-miR-3960 | 1.00 × 10−2 | −0.81 |
mmu-miR-26b-5p | 1.65 × 10−2 | −0.76 |
mmu-miR-2861 | 3.47 × 10−2 | −0.72 |
mmu-miR-7047-5p | 2.01 × 10−2 | −0.52 |
mmu-miR-106a-5p | 4.90 × 10−2 | −0.52 |
mmu-miR-23b-3p | 3.35 × 10−2 | −0.51 |
Overexpressed in SMA vs. WT | ||
miRNA | p-Value | Log2 (SMA/WT) |
mmu-miR-466q | 3.98 × 10−2 | 0.80 |
mmu-miR-1187 | 1.32 × 10−2 | 0.64 |
mmu-miR-466g | 2.58 × 10−2 | 0.64 |
3. Discussion
4. Experimental Section
4.1. Animal Models
4.2. Isolation and Culture of Neural Stem Cells (NSCs)
4.3. RT-PCR Analysis
Gene | Forward | Reverse |
---|---|---|
Gapdh | CATGGCCTTCCGTGTTCCTA | GCGGCACGTCAGATCCA |
Hoxb4 | TCACGTGAGCACGGTAAACCC | GCGTCAGGTAGCGATTGTAGTGAA |
Hoxb9 | ATTTGCGAAGGAAGCGAGGACA | TAGCTCCAGCGTCTGGTATTTGGT |
Olig2 | GTTCTCCTCCGCAGCGAG | CCTTCTTTTTTCAACCTTCCGA |
Pax6 | CAGCTCCAGCATGCAGAACA | CCGCCCGTTGACAAAGAC |
Sox2 | AGAAGAGGAGAGAGAAAGAAAGGGAGAGA | GAGAGAGGCAAACTGGAATCAGGATCAAA |
Nestin | AGGCTGAGAACTCTCGCTTGC | GGTGCTGGTCCTCTGGTATCC |
U1 | CCTGGCAGGGGAGATACCATGAT | TGCAGTCGAGTTTCCCGCATTT |
U2 | CGGCCTTTTGGCTAAGATCAAGTG | TCCTCGGATAGAGGACGTATCAGA |
U4 | GAGGTTTATCCGAGGCGCGATTAT | CACGGCGTATTGGGAAAAGTT |
U11 | CGTGCGGAATCGACATCAAGAGA | CAACGATCACCAGCTGCCCAATTA |
U12 | AACTTATGAGTAAGGAAAATAACGATTCG | CCGCYCAAAAATTCTTCTCACA |
U4atac | TTTCTTGGGGTTGCGCTACTGT | AAAGCAGAGCTCTAACCGATGCAG |
5.8S | GCGCTAGCTGCGAGAATTAAT | CAAGTGCGTTCGAAGTGTCGA |
SMN FL | TGCTCCGTGGACCTCATTTCT | TGGCTTTCCTGGTCCTAATCC |
4.4. Expression Analyses by RT-qPCR
4.5. MTS Cell Viability Assay
4.6. Flow Cytometry
4.7. miRNA Microarray Assay
4.8. Validation of miRNA Expression
4.9. snRNP (Small Nuclear Ribonucleoproteins) Level Analysis
4.10. Immunofluorescence
4.11. Western Blot Analysis
4.12. Statistical Analyses
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
References
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Luchetti, A.; Ciafrè, S.A.; Murdocca, M.; Malgieri, A.; Masotti, A.; Sanchez, M.; Farace, M.G.; Novelli, G.; Sangiuolo, F. A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA). Int. J. Mol. Sci. 2015, 16, 18312-18327. https://doi.org/10.3390/ijms160818312
Luchetti A, Ciafrè SA, Murdocca M, Malgieri A, Masotti A, Sanchez M, Farace MG, Novelli G, Sangiuolo F. A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA). International Journal of Molecular Sciences. 2015; 16(8):18312-18327. https://doi.org/10.3390/ijms160818312
Chicago/Turabian StyleLuchetti, Andrea, Silvia Anna Ciafrè, Michela Murdocca, Arianna Malgieri, Andrea Masotti, Massimo Sanchez, Maria Giulia Farace, Giuseppe Novelli, and Federica Sangiuolo. 2015. "A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA)" International Journal of Molecular Sciences 16, no. 8: 18312-18327. https://doi.org/10.3390/ijms160818312