The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential
Abstract
:1. Introduction
2. Results
2.1. Generation of Wild Type (WT) and Mutated circSMARCA5 Constructs
2.2. RNA Immunoprecipitation (RIP) Assay Demonstrates That Mut1 circSMARCA5 Is Significantly Less Able to Interact with SRSF1 than WT, Mut2 and Mut3 circSMARCA5
2.3. In Silico Analysis Predicts the GAUGAA Motif as Critically Involved in the Interaction between circSMARCA5 and SRSF1
2.4. U87MG and CAS-1 Transfected with Mut1 circSMARCA5 Migrate Significantly More than Controls Transfected with WT circSMARCA5
2.5. Conditioned Medium (CM) from U87MG or CAS-1 Transfected with Mut1 circSMARCA5 Increases Immortalized Human Brain Microvascular Endothelial Cell (IM-HBMEC) Sprouting as Compared to CM from U87MG or CAS-1 Transfected with WT circSMARCA5
2.6. Pro- to Anti-Angiogenic VEGFA mRNA Isoform Ratio and the Amount of Extracellular Total VEGFA mRNA Increase in CAS-1 Transfected with Mut1 circSMARCA5 as Compared to CAS-1 Transfected with WT circSMARCA5
2.7. Interplay between circSMARCA5, Its Negative Regulator DExH-Box Helicase 9 (DHX9), and VEGFA
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. GBM Biopsies
4.3. Cloning and Transfection
4.4. RNA Extraction, Northern Analysis and qRT-PCR
4.5. RIP Assay
4.6. Cell Migration Assay
4.7. Tube Formation Assay
4.8. In Silico Analysis
4.9. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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# | Mutated circSMARCA5 ID | Mutated Sequence of Predicted SRSF1 BS | Predicted SRSF1 BS Genomic Localization (GRCh38/hg38 Assembly) | Localization within circSMARCA5 (nt) |
---|---|---|---|---|
1 | Mut 1 | TCTACTT | chr4:143543546–143543552 | 38–44 |
2 | ACTACTTCTA | chr4:143543615–143543624 | 107–116 | |
3 | Mut 2 | CGATCCA | chr4:143543627–143543633 | 119–125 |
4 | TCCACGT | chr4:143543645–143543651 | 137–143 | |
5 | Mut 3 | TCTACTTACT | chr4:143543860–143543869 | 157–166 |
6 | GCTTCCTCTTCTG | chr4:143543942–143543954 | 239–251 |
# | SRSF1 Binding Motif | p-Value | q-Value | Mutated circSMARCA5 |
---|---|---|---|---|
1 | UGAAGAU | 6 × 10−5 | 0.0011 | Mut1 |
2 | UGAUGAA | 6 × 10−5 | 0.0011 | Mut1 |
3 | GAAGGA | 6 × 10−5 | 0.0011 | Mut3 |
Sample | n | Mean Age (Years ± StdDev) | Sex | |
---|---|---|---|---|
Male | Female | |||
Fresh frozen GBM biopsies | 28 | 62.9 ± 11.1 | 14 | 14 |
Fresh frozen unaffected brain parenchyma | 20 | 64 ± 10.3 | 8 | 12 |
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Barbagallo, D.; Caponnetto, A.; Barbagallo, C.; Battaglia, R.; Mirabella, F.; Brex, D.; Stella, M.; Broggi, G.; Altieri, R.; Certo, F.; et al. The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential. Int. J. Mol. Sci. 2021, 22, 1678. https://doi.org/10.3390/ijms22041678
Barbagallo D, Caponnetto A, Barbagallo C, Battaglia R, Mirabella F, Brex D, Stella M, Broggi G, Altieri R, Certo F, et al. The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential. International Journal of Molecular Sciences. 2021; 22(4):1678. https://doi.org/10.3390/ijms22041678
Chicago/Turabian StyleBarbagallo, Davide, Angela Caponnetto, Cristina Barbagallo, Rosalia Battaglia, Federica Mirabella, Duilia Brex, Michele Stella, Giuseppe Broggi, Roberto Altieri, Francesco Certo, and et al. 2021. "The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential" International Journal of Molecular Sciences 22, no. 4: 1678. https://doi.org/10.3390/ijms22041678