DEF Cell-Derived Exosomal miR-148a-5p Promotes DTMUV Replication by Negative Regulating TLR3 Expression
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Approval and Consent to Participate
2.2. Reagents
2.3. Cell Lines, Birds, Virus, and Infection
2.4. RNA and cDNA Preparation
2.5. Quantitative Real-Time PCR (qRT-PCR)
2.6. Recombinant Plasmid Construction
2.7. TLR3-shRNA, MDA5-shRNA, and Generation of shRNA-Based Knockdown Cell Lines
2.8. Western Blot Analysis of TLR3, MDA5, and Exosome Proteins
2.9. Exosome isolation
2.10. PBMC Preparation
2.11. Interaction of Exosomes with PBMCs
2.12. Enzyme-Linked Immunosorbent Assay (ELISA) to Detect Duck TLR3 Protein
2.13. Dual-Luciferase Reporter Assay
2.14. Statistical Analysis
3. Results
3.1. DTMUV-Infected Ducks Show Up-Regulated the Expression of TLR3, MDA5, and Inflammatory Cytokines
3.2. TLR3 and MDA5 Play Major Immunity Roles in DTMUV-Infected DEF Cells
3.3. MDA5 and TLR3-Dependent Signaling Pathways have Important Roles in Immunity During DTMUV Infection
3.4. Type I IFN Significantly Impairs DTMUV Replication in Dose- and Time-Dependent Manners
3.5. Morphological Characterization and Identification of Exosomes
3.6. DTMUV-Infected DEF Cells and DEF-Derived Exosomes Show Decreased Levels of miR-148a-5p
3.7. miR-148a-5p Targets TLR3
3.8. miR-148a-5p and Exo-miR-148a-5p Negatively Regulate TLR3 and IFN-β Expression in PBMCs
3.9. TLR3 May Be One of the Reasons for the Decrease of miR-148a-5p
4. Discussion
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Primer Name | Oligonucleotide Sequence (5′→3′) | Length | References |
---|---|---|---|
TLR2 | F1: TCCTTCATTCAGCACCAGGC R2: GAAAAACACAGCGCAGATCA | 171 bp | [34] |
TLR3 | F2: ATGTCATGCAAACCTGACCA R2: CCAGGGTCTTGAAAGGATCA | 239 bp | [35] |
TLR4 | F3: ATCTTTCAAGGTGCCACATCC R3: ACTGACCTACCGATTGGACAC | 194 bp | |
MyD88 | F4: TTTACAGCATGAATCCCTTGGC R4: TGGGAGTGTAAAATCCTGGTGT | 184 bp | |
IFNα | F5: TCAGCACCACATCCACCACCTT R5: GGTTCTGGAGGAAGTGTTGGAT | 131 bp | |
IFNβ | F6: TCAGCAGTCCAAGCATCCCT R6: GGAAGTGTTGGATGCTCCTGAAGTA | 186 bp | |
IL-6 | F7: GGTCCAGAACAACCTCAACCTCC R7: CGTTGCCAGATGCTTTGTGC | 202 bp | |
IL-8 | F8: CGGCATCGGTGTTCTTATCT R8: CTGTCCAGTGCCTTCAGTTT | 147 bp | |
MDA5 | F9: GCTACAGAAGATAGAAGTGTCA R9: CAGGATCAGATCTGGTTCAG | 120 bp | [36] |
IPS-1 | F10: TGCGACCGCCTACAAATTCTAT | 139 bp | |
R10: AGGGGTTTGGTAGAGGTCGTAG | |||
NF-κB | F11: ATCAACCCTTTTAACGTGCCT | 142 bp | |
R11: GGTTGGAAATCAAAGGAGGC | |||
TRIF | F12: TCTACTCACTGCTGGCAAAGG | 129 bp | |
R12: CAGCCAGGACGCAGTTTTGTG | |||
IRF7 | F13: ACAACGCCAGGAAGGATGT | 120 bp | |
R13: AGCGAAAGTTGGTCTTCCACT | |||
RIG-I | F14: GCGGATAGAGGCAACAAT | 133 bp | |
R14: AGTTATGCCTGCTGCTTT | |||
DTMUV envelope | F15: AATGGCTGTGGCTTGTTTGG | 207 bp | [37] |
R15: GGGCGTTATCACGAATCTA | |||
U6 | F16: CTCGCTTCGGCACGACA | 73 bp | [38] |
R16: GCGTGTCATCCTTGCGC | |||
β-action | F17: CCCCATTGAACACGGTATTGTC R17: GGCTACATACATGGCTGGGG | 199 bp | [35] |
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Guo, H.; Cheng, A.; Zhang, X.; Pan, Y.; Wang, M.; Huang, J.; Zhu, D.; Chen, S.; Liu, M.; Zhao, X.; et al. DEF Cell-Derived Exosomal miR-148a-5p Promotes DTMUV Replication by Negative Regulating TLR3 Expression. Viruses 2020, 12, 94. https://doi.org/10.3390/v12010094
Guo H, Cheng A, Zhang X, Pan Y, Wang M, Huang J, Zhu D, Chen S, Liu M, Zhao X, et al. DEF Cell-Derived Exosomal miR-148a-5p Promotes DTMUV Replication by Negative Regulating TLR3 Expression. Viruses. 2020; 12(1):94. https://doi.org/10.3390/v12010094
Chicago/Turabian StyleGuo, Hongyan, Anchun Cheng, Xingcui Zhang, YuHong Pan, Mingshu Wang, Juan Huang, Dekang Zhu, Shun Chen, Mafeng Liu, Xinxin Zhao, and et al. 2020. "DEF Cell-Derived Exosomal miR-148a-5p Promotes DTMUV Replication by Negative Regulating TLR3 Expression" Viruses 12, no. 1: 94. https://doi.org/10.3390/v12010094