Vocal Fold Fibroblasts in Reinke’s Edema Show Alterations Involved in Extracellular Matrix Production, Cytokine Response and Cell Cycle Control
Abstract
:1. Introduction
2. Materials and Methods
2.1. Human Tissue Samples
2.2. Cell Culture
2.3. Proliferation Assay
2.4. Sample Collection
2.4.1. Supernatant for Luminex® Assays, ELISAs and Silver Stain
2.4.2. RNA Isolation
2.4.3. Cell lysates for Protein Analysis
2.5. Sample Analysis
2.5.1. RNA-seq and Enrichment Analysis
2.5.2. Reverse Transcription (RT) and Quantitative Polymerase Chain Reaction (qPCR)
2.5.3. SDS-PAGE and Western Blot Analysis
2.5.4. Pepsin Digestion, SDS-PAGE and Silver Stain
2.5.5. ELISA
2.5.6. Magnetic Luminex® Assay
2.6. Statistical Analysis
3. Results
3.1. RNA-Seq Reveals a Total of 74 Differentially Expressed Genes in RE VFF Cells
3.2. Gene Expression of AQP1, and VWF, But Not Other Endothelial Cell Markers, Is Significantly Upregulated in RE VFF
3.3. Expression of Genes Associated with the ECM Composition, Some of Which Involved in the Interaction with TGF-β Family Members, Are Significantly Upregulated in RE VFF
3.4. MMP1 Is Decreased in RE VFF Leading to Accumulation of COL1α2
3.5. Pathways Involved in ECM Interactions and Turnover, Inflammation and Fibrosis Are Upregulated in RE, While Cell Proliferation Is Downregulated
3.6. RE VFFs Have Increased Levels of Inflammatory Cytokine Receptors IL1R1 and IL6R
3.7. Cell Cycle, DNA Replication and E2F Targets Are Downregulated in RE VFF
4. Discussion
4.1. Cultured RE VFF Reveal Alterations Associated with ECM Remodeling, Fibrosis and Angiogenesis
4.2. RE VFF Display Changes Leading to A Differential Inflammatory Response
4.3. RE VFF Show Cell Cycle Disruption
4.4. Study Limitations
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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Gene Name | Company | Product Number | Sequence Primer 1 | Sequence Primer 2 |
---|---|---|---|---|
CCN5/WISP2 | Bio-Rad | qHsaCED0042203 | ||
GREM2 | Bio-Rad | qHsaCED0036866 | ||
TGM2 | Bio-Rad | qHsaCID0007428 | ||
AQP1 | Bio-Rad | qHsaCED0005827 | ||
SPON2 | Bio-Rad | qHsaCED0005641 | ||
TNXB | Bio-Rad | qHsaCED0038143 | ||
IL1R1 | Bio-Rad | qHsaCID0010015 | ||
IL6R | Bio-Rad | qHsaCED0045907 | ||
STAT3 | Bio-Rad | qHsaCID0010912 | ||
SOCS3 | Bio-Rad | qHsaCED0003543 | ||
AURKA | Bio-Rad | qHsaCID0022123 | ||
UBE2T | Bio-Rad | qHsaCID0011482 | ||
E2F1 | Bio-Rad | qHsaCID0016226 | ||
CSGALNACT2 | Bio-Rad | qHsaCID0016295 | ||
GLB1 | Bio-Rad | qHsaCID0012007 | ||
GALNS | Bio-Rad | qHsaCID0007640 | ||
COL1A1 | Bio-Rad | qHsaCED0002181 | ||
TGFB1 | Microsynth | TACCTGAACCCGTGTTGCTC | GCTGAGGTATCGCCAGGAAT | |
MMP1 | Microsynth | CACGCCAGATTTGCCAAGAG | GTTGTCCCGATGATCTCCCC | |
MMP2 | Microsynth | ACCAAGAACTTCCGTCTGTCC | ATGTCAGGAGAGGCCCCATA | |
MMP14 | Microsynth | CGGAGAATTTTGTGCTGCCC | AACCCTGACTCACCCCCATA | |
COL1A2 | Microsynth | ACCACAGGGTGTTCAAGGTG | CAGGACCAGGGAGACCAAAC | |
VWF | Microsynth | CTCATCGCAGCAAAAGGAGC | ATGCTCATGCACTCCAGGTC | |
PECAM1 | Microsynth | CGTCGAATACCAGTGTGTTGC | TCATCCACCGGGGCTATCA | |
NOS3 | Microsynth | CGAGTGAAGGCGACAATCCT | ACAGGACCCGGGGATCAAAA |
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Grill, M.; Lazzeri, I.; Kirsch, A.; Steurer, N.; Grossmann, T.; Karbiener, M.; Heitzer, E.; Gugatschka, M. Vocal Fold Fibroblasts in Reinke’s Edema Show Alterations Involved in Extracellular Matrix Production, Cytokine Response and Cell Cycle Control. Biomedicines 2021, 9, 735. https://doi.org/10.3390/biomedicines9070735
Grill M, Lazzeri I, Kirsch A, Steurer N, Grossmann T, Karbiener M, Heitzer E, Gugatschka M. Vocal Fold Fibroblasts in Reinke’s Edema Show Alterations Involved in Extracellular Matrix Production, Cytokine Response and Cell Cycle Control. Biomedicines. 2021; 9(7):735. https://doi.org/10.3390/biomedicines9070735
Chicago/Turabian StyleGrill, Magdalena, Isaac Lazzeri, Andrijana Kirsch, Nina Steurer, Tanja Grossmann, Michael Karbiener, Ellen Heitzer, and Markus Gugatschka. 2021. "Vocal Fold Fibroblasts in Reinke’s Edema Show Alterations Involved in Extracellular Matrix Production, Cytokine Response and Cell Cycle Control" Biomedicines 9, no. 7: 735. https://doi.org/10.3390/biomedicines9070735