RNA-Seq Analysis of UPM-Exposed Epithelium Co-Cultivated with Macrophages and Dendritic Cells in Obstructive Lung Diseases
Abstract
:1. Introduction
2. Results
2.1. RNA-Seq Data Analysis
2.2. GO and KEGG Pathway Enrichment Analysis
2.3. RT-qPCR Analysis
- (a)
- Fold change of mRNA expression between the UPM exposed and nonexposed epithelial cells from the triple co-culture (Figure 5A, Y-axis) was compared to the fold change of mRNA expression between the UPM and no UPM exposed epithelial cells from the monoculture (Figure 5A, X-axis). A separate plot (and gene selection) was prepared for each group (control/asthma/COPD).
- (b)
- Fold change of mRNA expression between the UPM exposed and nonexposed epithelial cells from the triple co-culture in one of the clinical group was plotted against the same value in other clinical groups (three panels: asthma–control, COPD–control, COPD–asthma).
3. Discussion
4. Materials and Methods
4.1. Patient Characteristics
4.2. Flow Cytometry Analysis
4.3. Hematoxylin and Eosin (H&E) Staining
4.4. Cell Culture and Scheme of the Study
- (1)
- Epithelial cells (monoculture);
- (2)
- Epithelial cells + moMφs + moDCs (triple co-culture).
4.5. Particle Preparation
4.6. RNA Isolation
4.7. RNA-Seq Analysis
4.8. Bioinformatic Analysis
4.9. qRT-PCR Measurements
4.10. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Acronym | Full Gene Name |
---|---|
18s rRNA | 18s ribosomal RNA |
AHRR | aryl-hydrocarbon receptor repressor |
ARC | activity regulated cytoskeleton associated protein |
ATP1B2 | ATPase Na+/K+ transporting subunit beta 2 |
BPIFA2 | BPI fold containing family A member 2 |
CCL22 | C-C motif chemokine ligand 22 |
CYP1B1 | cytochrome P450 family 1 subfamily B member 1 |
CYP1B1-AS1 | CYP1B1 antisense RNA 1 |
EDC3 | enhancer of mRNA decapping 3 |
EEF1A2 | eukaryotic translation elongation factor 1 alpha 2 |
ENPEP | glutamyl aminopeptidase |
IRF4 | interferon regulatory factor 4 |
LINC02029 | Long non-coding RNA |
ME1 | malic enzyme 1 |
NCF1 | neutrophil cytosolic factor 1 |
RASD1 | ras related dexamethasone induced 1 |
RMDN2-AS1 | RMDN2 antisense RNA 1 |
TIPARP | TCDD inducible poly(ADP-ribose) polymerase |
Epithelium (mono) + UPM | Epithelium (trio) + UPM | |
---|---|---|
AHRR | 0.640 | 0.144 |
ARC | 0.957 | 0.047 # |
ATP1B2 | 0.154 | 0.597 |
BPIFA2 | 0.025 # | 0.006 # |
CCL22 | 0.214 | 0.109 |
CYP1B1 | 0.224 | 0.167 |
CYP1B1-AS1 | 0.023 * | 0.435 |
EDC3 | 0.065 | 0.655 |
EEF1A2 | 0.745 | 0.318 |
ENPEP | 0.028 * | 0.150 |
IRF4 | 0.407 | 0.913 |
LINC02029 | 0.777 | 0.530 |
ME1 | 0.093 | 0.868 |
NCF1 | 0.806 | 0.353 |
RASD1 | 0.394 | 0.663 |
RMDN2-AS1 | 0.091 | 0.040 |
TIPARP | 0.049 | 0.029 # |
Control n = 8 | Asthma n = 10 | COPD n = 8 | Overall p-Value & | Pairwise p-Value * | |||
---|---|---|---|---|---|---|---|
Asthma vs. Control | COPD vs. Control | Asthma vs. COPD | |||||
Age (years) | 38.5 (32.5–48) | 55 (38–62) | 62 (59.5–72.5) | 0.005 | 0.138 | 0.0002 | 0.138 |
Gender (F/M) | 6/2 | 3/10 | 5/3 | 0.046 | |||
BMI (kg/m2) | 22.1 (20.7–24.1) | 26.9 (26–27.7) | 28 (25.4–30.3) | 0.002 | 0.002 | 0.0003 | 0.696 |
Atopy (n) | 3 | 8 | 2 | 0.03 | |||
Smoking exposure (pack-years) | 0 (0–0) | 0 (0–4) | 32.5 (22.5–50) | 0.0002 | 0.277 | 0.0002 | 0.0003 |
FEV1 (% predicted) | 103 (81–111) | 81 (75–94) | 61.5 (51.5–76.5) | 0.006 | 0.043 | 0.004 | 0.034 |
FEV1/VC (%) | 106 (81.8–112) | 82 (75–86) | 54 (50–68) | 0.0003 | 0.02 | 0.0006 | 0.0004 |
FeNO (ppb) | 11.0 (9.3–12.6) | 52.3 (31.3–77.6) | 17.4 (12.6–26.1) | 0.0047 | 0.03 | 0.333 | 0.003 |
ACT (points) | N.A. | 20.5 (17–25) | N.A. | N.A | N.A. | N.A. | N.A. |
ICS treatment (n) | N.A. | 6 | 1 | N.A | N.A. | N.A. | N.A. |
CAT (points) | N.A. | N.A. | 10.5 (8–15) | N.A | N.A. | N.A. | N.A. |
mMRC (points) | N.A. | N.A. | 1.5 (1–3) | N.A | N.A. | N.A. | N.A. |
Gene Symbol | Forward Primer | Reverse Primer | Probe | Product Size |
---|---|---|---|---|
LINC02029 | TGCCCCCACG AGGTACAC | CAGGACCCAAA GAAGGAATGAT | 6-FAM-TCCCGGGA AACAAA-MGB | 58 |
Gene symbol | Entrez Gene ID | |||
18s rRNA | Hs99999901_s1 | 187 | ||
AHRR | Hs01005075_m1 | 98 | ||
ARC | Hs01045540_g1 | 92 | ||
ATP1B2 | Hs01020302_g1 | 81 | ||
BPIFA2 | Hs00395980_m1 | 68 | ||
CCL22 | Hs01574247_m1 | 88 | ||
CYP1B1 | Hs00164383_m1 | 118 | ||
CYP1B1-AS1 | Hs00381672_m1 | 80 | ||
EDC3 | Hs00257810_m1 | 122 | ||
EEF1A2 | Hs00951278_m1 | 80 | ||
ENPEP | Hs00989749_m1 | 70 | ||
IRF4 | Hs00180031_m1 | 88 | ||
ME1 | Hs00159110_m1 | 73 | ||
NCF1 | Hs00165362_m1 | 113 | ||
RASD1 | Hs02568415_s1 | 159 | ||
RMDN2-AS1 | Hs04409587_s1 | 86 | ||
TIPARP | Hs00296054_m1 | 80 |
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Control n = 4 | Asthma n = 4 | COPD n = 4 | Overall p-Value | |
---|---|---|---|---|
Age (years) | 36 (27–44.5) | 61 (38–71) | 67 (62–72.5) | 0.06 |
Gender (F/M) | 4/0 | 1/3 | 2/2 | 0.09 |
BMI (kg/m2) | 22.4 (20.3–23.1) | 27.2 (26–30.1) | 28 (25.8–30.9) | 0.025 * |
Atopy (n) | 2 | 3 | 0 | 0.03 |
Smoking exposure (pack-years) | 0 (0–3.5) | 0 (0–0.75) | 25 (20–52) | 0.015 * |
FEV1 (% predicted) | 105.5 (101–109.5) | 84 (81–100) | 53 (47–61) | 0.018 * |
FEV1/VC (%) | 100.5 (98.5–106.5) | 76.3 (70–80.8) | 53 (47–61) | 0.013 * |
FeNO (ppb) | 9.3 (9.3–9.3) | 47.5 (29.6–67.7) | 22.4 (13.9–37.3) | 0.124 |
ACT (points) | N.A. | 19 (10–22) | N.A. | N.A. |
ICS treatment (n) | N.A. | 2 | 0 | N.A. |
CAT (points) | N.A. | N.A. | 11 (8–17) | N.A. |
mMRC (points) | N.A. | N.A. | 3 (1–3) | N.A. |
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Misiukiewicz-Stępien, P.; Mierzejewski, M.; Zajusz-Zubek, E.; Goryca, K.; Adamska, D.; Szeląg, M.; Krenke, R.; Paplińska-Goryca, M. RNA-Seq Analysis of UPM-Exposed Epithelium Co-Cultivated with Macrophages and Dendritic Cells in Obstructive Lung Diseases. Int. J. Mol. Sci. 2022, 23, 9125. https://doi.org/10.3390/ijms23169125
Misiukiewicz-Stępien P, Mierzejewski M, Zajusz-Zubek E, Goryca K, Adamska D, Szeląg M, Krenke R, Paplińska-Goryca M. RNA-Seq Analysis of UPM-Exposed Epithelium Co-Cultivated with Macrophages and Dendritic Cells in Obstructive Lung Diseases. International Journal of Molecular Sciences. 2022; 23(16):9125. https://doi.org/10.3390/ijms23169125
Chicago/Turabian StyleMisiukiewicz-Stępien, Paulina, Michał Mierzejewski, Elwira Zajusz-Zubek, Krzysztof Goryca, Dorota Adamska, Michał Szeląg, Rafał Krenke, and Magdalena Paplińska-Goryca. 2022. "RNA-Seq Analysis of UPM-Exposed Epithelium Co-Cultivated with Macrophages and Dendritic Cells in Obstructive Lung Diseases" International Journal of Molecular Sciences 23, no. 16: 9125. https://doi.org/10.3390/ijms23169125