Deregulated miRNAs Contribute to Silencing of B-Cell Specific Transcription Factors and Activation of NF-κB in Classical Hodgkin Lymphoma
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Lymphoma Cell Lines, GCB Cells and Primary cHL Biopsies
2.2. NGS Based miRNA Profiling
2.3. Real-Time qPCR Based miRNA Expression Analysis
2.4. Identification of Putative Target Genes of the cHL Deregulated miRNAs
2.5. Validation of miRNA Target Genes
2.5.1. Vector Preparation
2.5.2. Luciferase Reporter Assay
- In total, 500 ng of vector containing the 3′UTR WT sequence + 50 µM of the analyzed miRNA mimic (mirVana® miRNA mimic, Invitrogen, Waltham, MA, USA);
- In total, 500 ng of vector containing the 3′UTR WT sequence + 50 µM of the mimic negative control (mirVana™ miRNA Mimic, Negative Control #1, Invitrogen, Waltham, MA, USA);
- In total, 500 ng of vector containing the 3′UTR MUT sequence + 50 µM of the analyzed miRNA mimic (mirVana® miRNA mimic, Invitrogen, Waltham, MA, USA);
- In total, 500 ng of vector containing the 3′UTR MUT sequence + 50 µM of the mimic negative control (mirVana™ miRNA Mimic, Negative Control #1, Invitrogen, Waltham, MA, USA).
2.6. MiRNA Overexpression
2.7. Western Blots
3. Results
3.1. The cHL miRNome and miRNAs Deregulated in cHL
3.2. Biological Processes Deregulated by miRNAs in cHL
3.3. Functional Validation of miRNA-mRNA Interactions for Selected Candidates
3.4. Validation of miRNA Expression in Microdissected Primary HRS Cells
4. Discussion
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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GO Analysis of Target Genes of miRNAs Upregulated in cHL | |||
---|---|---|---|
Top 3 GO Terms (p < 0.05) | PANTHER Database | STRING Database | DAVID Database |
1 | DNA-binding transcription factor activity, RNA polymerase II-specific (GO:0000981) | DNA-binding transcription factor activity (GO:0003700) | DNA-binding transcription factor activity (GO:0003700) |
2 | DNA-binding transcription factor activity (GO:0003700) | molecular function regulator (GO:0098772) | protein binding (GO:0005515) |
3 | molecular function regulator (GO:0098772) | transcription regulator activity (GO:0140110) | regulation of transcription, DNA-templated (GO:0006355) |
GO analysis of target genes of miRNAs downregulated in cHL | |||
Top 3 GO terms (p < 0.05) | PANTHER database | STRING database | DAVID database |
1 | transcription regulatory region sequence-specific DNA binding (GO:0000976) | DNA-binding transcription factor activity, RNA polymerase II-specific (GO:0000981) | transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding (GO:0001077) |
2 | RNA polymerase II regulatory region DNA binding (GO:0001012) | transcription regulatory region sequence-specific DNA binding (GO:0000976) | neuron migration (GO:0001764) |
3 | transcription regulatory region DNA binding (GO:0044212) | RNA polymerase II regulatory region sequence-specific DNA binding (GO:0000977) | RNA polymerase II core promoter proximal region sequence-specific DNA binding (GO:0000978) |
MiRNA in cHL | MiRNA | Target Gene | UTR miRNA Binding Site [Based on TargetScan Database] | Interaction Confirmed in the Luciferase Assay | Gene Function | ||
---|---|---|---|---|---|---|---|
Binding Sites Conservation | Number of Binding Sites | Characteristic of Binding Sites | |||||
UPREGULATED | hsa-miR-27a-5p | SPI1 (PU.1) | poorly conserved | 1 | 7mer-m8 | YES | B-cell related transcription factor |
hsa-miR-330-3p | ELF-1 | poorly conserved | 2 | 7mer-m8, 7mer-m8 | YES | B-cell related transcription factor | |
hsa-miR-542-3p | ELF-1 | poorly conserved | 2 | 7mer-A1, 7mer-m8 | NO | B-cell related transcription factor | |
hsa-miR-450b-5p | ELF-1 | poorly conserved | 1 | 7mer-A1 | YES | B-cell related transcription factor | |
hsa-miR-23a-3p | TNFAIP3 | conserved | 1 | 8mer | YES | negative regulator of NF-kappaB |
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Paczkowska, J.; Janiszewska, J.; Ustaszewski, A.; Bein, J.; Skalski, M.; Dzikiewicz-Krawczyk, A.; Rozwadowska, N.; Hansmann, M.-L.; Hartmann, S.; Giefing, M. Deregulated miRNAs Contribute to Silencing of B-Cell Specific Transcription Factors and Activation of NF-κB in Classical Hodgkin Lymphoma. Cancers 2021, 13, 3131. https://doi.org/10.3390/cancers13133131
Paczkowska J, Janiszewska J, Ustaszewski A, Bein J, Skalski M, Dzikiewicz-Krawczyk A, Rozwadowska N, Hansmann M-L, Hartmann S, Giefing M. Deregulated miRNAs Contribute to Silencing of B-Cell Specific Transcription Factors and Activation of NF-κB in Classical Hodgkin Lymphoma. Cancers. 2021; 13(13):3131. https://doi.org/10.3390/cancers13133131
Chicago/Turabian StylePaczkowska, Julia, Joanna Janiszewska, Adam Ustaszewski, Julia Bein, Marcin Skalski, Agnieszka Dzikiewicz-Krawczyk, Natalia Rozwadowska, Martin-Leo Hansmann, Sylvia Hartmann, and Maciej Giefing. 2021. "Deregulated miRNAs Contribute to Silencing of B-Cell Specific Transcription Factors and Activation of NF-κB in Classical Hodgkin Lymphoma" Cancers 13, no. 13: 3131. https://doi.org/10.3390/cancers13133131