Elevated Pro-Inflammatory Cell-Free MicroRNA Levels in Cerebrospinal Fluid of Premature Infants after Intraventricular Hemorrhage
Abstract
:1. Introduction
2. Results
2.1. Baseline Characteristics of Study Participants
2.2. Elevated miRNA Levels in CSF after the Onset of Preterm IVH
2.3. Time-Dependent Alteration in miRNAs in Post-IVH CSF Samples
2.4. Relationship between the Levels of Oxidized Hb Forms and miRNAs in CSF after IVH
2.5. Correlations between Pro-Inflammatory Protein Biomarkers and miRNAs in Post-IVH CSF Samples
2.6. Impact of Hemolysis on miRNAs Based on In Vitro Controlled Hemolysis Experiments
2.7. miRNA Levels in the Supernatant of Human Choroid Plexus Epithelial Cell Cultures after Treatment with Oxidized Hb Forms and Heme
3. Discussion
4. Materials and Methods
4.1. Study Participants
4.2. CSF Sample Collection and Preparation
4.3. Human Choroid Plexus Epithelial Cell Culture
4.4. Treatment of Human Choroid Plexus Epithelial Cells with Oxidative Hb Forms and Heme
4.5. Extraction of miRNAs from CSF Samples and HCPEpiC Supernatants
4.6. miRNA-Specific Stem-Loop RT-qPCR Analysis
4.7. Determination of Hemoglobin Forms and Heme Levels in CSF Samples
4.8. Measurement of Soluble Adhesion Molecule Concentrations
4.9. Other Laboratory Analyses
4.10. In Vitro Controlled Hemolysis Experiments
4.11. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
CSF | cerebrospinal fluid |
CNS | central nervous system |
CRP | C-reactive protein |
DCI | delayed cerebral ischemia |
EC | endothelial cell |
ELISA | enzyme-linked immunosorbent assay |
Hb | hemoglobin |
HCPEpiC | human choroid plexus epithelial cell |
ICAM-1 | intercellular adhesion molecule 1 |
IL | interleukin |
IVH | intraventricular hemorrhage |
LDH | lactate dehydrogenase |
miRNA | microRNA |
NF-κB | nuclear factor kappa-B |
Ox. Hb | oxidized hemoglobin |
PCT | procalcitonin |
RBC | red blood cell |
RT | room temperature |
RT-qPCR | real-time quantitative polymerase chain reaction |
S100B | S100 calcium-binding protein B |
SAH | subarachnoid hemorrhage |
TLR4 | Toll-like receptor 4 |
TNF-α | tumor necrosis factor alpha |
UPL | Universal ProbeLibrary |
VCAM-1 | vascular cell adhesion molecule 1 |
WBC | white blood cell |
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Characteristics | IVH Patients (n = 47) | Clinical Controls (n = 14) | p Value |
---|---|---|---|
Gestational age (weeks) | 28.2 ± 3.2 | 32.0 ± 6.8 | 0.3427 |
Gender (male/female) | 26/21 | 7/7 | 0.7259 |
Birth weight (g) | 1235 ± 610 | 2046 ± 1281 | 0.240 |
Severity of IVH (grade III/IV) | 21/26 | ||
Whole blood WBC count (G/L) | 12.0 (9.77–13.80) | 10.26 (8.64–12.33) | 0.0940 |
Whole blood Hb (g/L) | 120 (102–135) | 107 (99–130) | 0.4474 |
Whole blood platelet count (G/L) | 384 (286–484) | 370 (288–509) | 0.963 |
Serum CRP (mg/L) | 1.34 (0.60–3.76) | 0.50 (0.50–4.51) | 0.2192 |
Serum PCT (µg/L) | 0.29 (0.19–0.50) | 0.15 (0.10–0.19) | 0.0066 |
CSF RBC count (cells/µL) | 4267 (1536–10400) | 2 (1–518) | <0.0001 |
CSF WBC count (cells/µL) | 52 (23–207) | 8 (4–26) | 0.0005 |
CSF total protein (mg/L) | 1799 (1376–2498) | 758 (448–1510) | 0.0007 |
CSF S100B (µg/L) | 7.49 (5.01–10.40) | 3.23 (1.12–5.46) | 0.0003 |
CSF lactate (mmol/L) | 3.47 (2.58–3.70) | 2.16 (1.55–2.45) | 0.0002 |
CSF oxidized Hb (µmol/L) | 36.98 ± 54.64 | 1.82 ± 5.84 | 0.0005 |
CSF total heme (µmol/L) | 226.3 ± 262.9 | 0.78 ± 2.81 | <0.0001 |
CSF RBC | CSF WBC | E-selectin | ICAM-1 | VCAM-1 | IL-8 | Ox. Hb | Total Heme | |
---|---|---|---|---|---|---|---|---|
miR-223 | r = 0.5293 p < 0.0001 | r = 0.5135 p < 0.0001 | r = 0.4244 p = 0.0002 | r = 0.3683 p = 0.0009 | r = 0.5101 p < 0.0001 | r = 0.5049 p < 0.0001 | r = 0.5931 p < 0.0001 | r = 0.6281 p < 0.0001 |
miR-155 | r = 0.4241 p < 0.0001 | r = 0.2874 p = 0.0107 | r = 0.2670 p = 0.0110 | r = 0.2721 p = 0.0070 | r = 0.3733 p = 0.0036 | r = 0.3160 p = 0.0097 | r = 0.4735 p < 0.0001 | r = 0.5394 p < 0.0001 |
miR-181b | r = 0.3554 p = 0.0014 | r = 0.2254 p = 0.0487 | r = 0.2680 p = 0.0111 | r = 0.2397 p = 0.0187 | r = 0.2994 p = 0.0212 | r = 0.3486 p = 0.0041 | r = 0.4198 p = 0.0001 | r = 0.4510 p < 0.0001 |
miR-126 | r = 0.2975 p = 0.0057 | r = 0.0865 p = 0.4933 | r = 0.3588 p = 0.0014 | r = 0.2133 p = 0.0514 | r = 0.1832 p = 0.2030 | r = 0.3303 p = 0.0129 | r = 0.2432 p = 0.0323 | r = 0.3558 p = 0.0010 |
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Fejes, Z.; Erdei, J.; Pócsi, M.; Takai, J.; Jeney, V.; Nagy, A.; Varga, A.; Bácsi, A.; Bognár, L.; Novák, L.; et al. Elevated Pro-Inflammatory Cell-Free MicroRNA Levels in Cerebrospinal Fluid of Premature Infants after Intraventricular Hemorrhage. Int. J. Mol. Sci. 2020, 21, 6870. https://doi.org/10.3390/ijms21186870
Fejes Z, Erdei J, Pócsi M, Takai J, Jeney V, Nagy A, Varga A, Bácsi A, Bognár L, Novák L, et al. Elevated Pro-Inflammatory Cell-Free MicroRNA Levels in Cerebrospinal Fluid of Premature Infants after Intraventricular Hemorrhage. International Journal of Molecular Sciences. 2020; 21(18):6870. https://doi.org/10.3390/ijms21186870
Chicago/Turabian StyleFejes, Zsolt, Judit Erdei, Marianna Pócsi, Jun Takai, Viktória Jeney, Andrea Nagy, Alíz Varga, Attila Bácsi, László Bognár, László Novák, and et al. 2020. "Elevated Pro-Inflammatory Cell-Free MicroRNA Levels in Cerebrospinal Fluid of Premature Infants after Intraventricular Hemorrhage" International Journal of Molecular Sciences 21, no. 18: 6870. https://doi.org/10.3390/ijms21186870