Analysis of the Differential Exosomal miRNAs of DC2.4 Dendritic Cells Induced by Toxoplasma gondii Infection
Abstract
:1. Introduction
2. Results
2.1. Evaluation of the Efficiency of T. gondii RH Tachyzoites Invading DC2.4 Cells
2.2. Qualification and Quantification of the Exosomes
2.3. miRNA Sequencing and Analysis of the Differential Exosomal miRNAs (DEmiRs) from the DC2.4 Cells Uninfected and Infected with T.gondii RH Tachyzoites
2.4. Verification of the Stably Enriched DEmiRs with RT-qPCR
2.5. Prediction and Analysis of the Target Genes of the Stably Enriched DEmiRNAs
3. Discussion
4. Materials and Methods
4.1. Parasites and Cell Lines
4.2. Exosome Isolation
4.3. Detection of the Quality of Exosomes with a Transmission Electron Microscope (TEM)
4.4. Detection of the Size and Quantity of Exosomes by Nanoparticle Tracking Analysis (NTA)
4.5. Verification of Exosomal Proteins by Western Blotting
4.6. Construction of Small RNA Library, Sequencing of the miRNA, and Bioinformatics Analysis
4.7. The Screening of the Differentially Enriched miRNAs
4.8. Verification of the Differential miRNAs with RT-qPCR
4.9. Bioinformatics Analysis of the Differential Enriched miRNA Target Genes
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
DCs | Dendritic cells |
DEmiR | Differential exosomal miRNA |
APCs | Antigen-presenting cells |
PPI | Protein–protein interaction network |
KDA | Key driver analysis |
GO | Gene ontology |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
MOI | Multiplicity of infection |
TEM | Transmission electron microscope |
NTA | Nanoparticle tracking analysis |
FBS | Fetal bovine serum |
PVDF | Polyvinylidene fluoride |
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miRNA ID | Sequence |
---|---|
mmu-miR-151-3p | CUAGACUGAGGCUCCUUGAGG |
mmu-miR-199a-3p | ACAGUAGUCUGCACAUUGGUUA |
mmu-miR-99b-5p | CACCCGUAGAACCGACCUUGCG |
mmu-miR-125b-2-3p | ACAAGUCAGGUUCUUGGGACCU |
mmu-miR-129-5p | CUUUUUGCGGUCUGGGCUUGC |
mmu-miR-132-5p | AACCGUGGCUUUCGAUUGUUAC |
mmu-miR-196b-5p | UAGGUAGUUUCCUGUUGUUGGG |
mmu-miR-212-3p | UAACAGUCUCCAGUCACGGCCA |
mmu-miR-3109-3p | UAGGGCCAUCUCAUCCAGAUA |
mmu-miR-32-5p | UAUUGCACAUUACUAAGUUGCA |
mmu-miR-324-3p | CCACUGCCCCAGGUGCUGCU |
mmu-miR-369-5p | AGAUCGACCGUGUUAUAUUCGC |
miRNA ID | Reverse Transcription Primer (5′ to 3′) | qPCR Forward Primer (5′ to 3′) |
---|---|---|
mmu-miR-151-3p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG CCTCAAGG | ACACTCCAGCTGGGCTAGACTGAGGCTCC |
mmu-miR-199a-3p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG TAACCAAT | ACACTCCAGCTGGGACAGTAGTCTGCACAT |
mmu-miR-99b-5p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG CGCAAGGT | ACACTCCAGCTGGGCACCCGTAGAACCGAC |
mmu-miR-125b-2-3p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG AGGTCCCA | ACACTCCAGCTGGGACAAGTCAGGTTCTTG |
mmu-miR-129-5p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG GCAAGCCC | ACACTCCAGCTGGGCTTTTTGCGGTCTGG |
mmu-miR-132-5p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG GTAACAAT | ACACTCCAGCTGGGAACCGTGGCTTTCGAT |
mmu-miR-196b-5p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG CCCAACAA | ACACTCCAGCTGGGTAGGTAGTTTCCTGTT |
mmu-miR-212-3p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG TGGCCGTG | ACACTCCAGCTGGGTAACAGTCTCCAGTCA |
mmu-miR-3109-3p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG TATCTGGA | ACACTCCAGCTGGGTAGGGCCATCTCATC |
mmu-miR-32-5p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG TGCAACTT | ACACTCCAGCTGGGTATTGCACATTACTAA |
mmu-miR-324-3p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG AGCAGCAC | ACACTCCAGCTGGGCCACTGCCCCAGGT |
mmu-miR-369-5p | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG GCGAATAT | ACACTCCAGCTGGGAGATCGACCGTGTTAT |
Universal Reverse primer for qPCR (URP) | TGGTGTCGTGGAGTCG | |
U6 F | CTCGCTTCGGCAGCACA | |
U6 R | AACGCTTCACGAATTTGCGT | AACGCTTCACGAATTTGCGT |
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Li, D.-L.; Zou, W.-H.; Deng, S.-Q.; Peng, H.-J. Analysis of the Differential Exosomal miRNAs of DC2.4 Dendritic Cells Induced by Toxoplasma gondii Infection. Int. J. Mol. Sci. 2019, 20, 5506. https://doi.org/10.3390/ijms20215506
Li D-L, Zou W-H, Deng S-Q, Peng H-J. Analysis of the Differential Exosomal miRNAs of DC2.4 Dendritic Cells Induced by Toxoplasma gondii Infection. International Journal of Molecular Sciences. 2019; 20(21):5506. https://doi.org/10.3390/ijms20215506
Chicago/Turabian StyleLi, Dong-Liang, Wei-Hao Zou, Sheng-Qun Deng, and Hong-Juan Peng. 2019. "Analysis of the Differential Exosomal miRNAs of DC2.4 Dendritic Cells Induced by Toxoplasma gondii Infection" International Journal of Molecular Sciences 20, no. 21: 5506. https://doi.org/10.3390/ijms20215506