Introduction
Methods
Study design
Sample collection
RNA extraction and quality control
Small RNA library preparation and sequencing
Bioinformatics – miRNA-Seq, miRNA target prediction, and miRNA-mRNA target network analysis
Validation of predictions at mRNA level based on RNA sequencing data
Statistical analysis
Interictal (n = 16) | Ictal (n = 8) | Healhy control (n = 12) | P—value | |
---|---|---|---|---|
Age | 34.62 (28.66—40.58) | 36.87 (26.93—46.81) | 30.75 (27.95—33.55) | 0.91 |
Body mass index (BMI) | 21.95 (19.84—24.06) | 21.90 (19.59—24.20) | 23.28 (21.39—25.17) | 0.36 |
Last meal (hours ago) | 6.67 (3.65—9.68) | 6.62 (1.64—11.61) | 3.83 (0.66—6.99) | 0.21 |
Co-morbidities and therapy | ||||
Known other diseases (%) | 50 (24—75) | 50 (12—87) | 1.00 | |
Regular medication (except attack therapy) (%) | 31 (7—54) | 38 (1—73) | 0.76 | |
Hormonal contraceptives (%) | 37 (12—62) | 25 (0—57) | 25 (0—51) | 0.73 |
Antimigraine prophylactic therapy (%) | 0 | 0 | ||
Clinical features of the headache | ||||
Disease duration (years) | 13.18 (8.95—17.41) | 15.5 (7.98—23.01) | 0.51 | |
Attack frequency (attack/year) | 23.93 (16.65—31.22) | 21.87 (10.95—32.79) | 0.62 | |
Visual analogue scale category (VAS) | 2.68 (2.45—2.92) | 2.62 (2.26—2.98) | 0.76 | |
Allodynia (%) | 31 (7—54) | 50 (12—87) | 0.38 | |
Chronic pain (%) | 18 (0—38) | 12 (0—37) | 0.70 | |
Menstruation-headache relationship (sensitive) (%) | 37 (12—62) | 37 (1—73) | 1.00 | |
Migraineurs in the family (%) | 68 (0.45—0.92) | 62 (26—98) | 0.76 | |
Regular exercise (%) | 56 (30—81) | 62 (26—98) | 17 (0—39) | 0.06 |
Features of attacks before samplings | ||||
Number of attacks in the previous month | 2.62 (1.4—3.84) | 0.87 (0.18—1.56) | 0.03 | |
Last attack (days ago) | 20.25 (3.26—37.23) | 36.62 (3.38—69.86) | 0.04 | |
Beginning of attack before ictal sampling (hours) | 6.31 (1.24—11.38) | |||
Actual VAS | 5.62 (4.79—6.45) |
Results
Clinical characteristics of the patient population
microRNA signatures of PBMC samples
ID | Fold change | P-value | Average rank |
---|---|---|---|
hsa-miR-5189-3p | 2,59 | 0,0057 | 1 |
hsa-miR-96-5p | -2,40 | 0,0032 | 2 |
hsa-miR-3613-5p | 2,55 | 0,0101 | 3 |
hsa-miR-99a-3p | 2,37 | 0,0079 | 4 |
hsa-miR-542-3p | 2,40 | 0,0164 | 5 |
hsa-miR-6803-3p | 2,19 | 0,0162 | 6 |
hsa-miR-6731-3p | -2,14 | 0,0084 | 7 |
hsa-miR-577 | -2,17 | 0,0200 | 8 |
hsa-miR-95-3p | -2,06 | 0,0184 | 9 |
hsa-miR-556-3p | -2,18 | 0,0228 | 10 |
hsa-miR-412-5p | -2,36 | 0,0290 | 11 |
hsa-miR-5701 | -2,24 | 0,0263 | 12 |
hsa-miR-3064-5p | 2,10 | 0,0247 | 13 |
hsa-miR-196a-5p | -2,55 | 0,0450 | 14 |
hsa-miR-5189-5p | 1,93 | 0,0222 | 15 |
hsa-let-7i-3p | -1,82 | 0,0067 | 16 |
hsa-miR-1277-5p | 2,07 | 0,0402 | 17 |
hsa-miR-29b-3p | -1,85 | 0,0214 | 18 |
hsa-miR-4676-3p | 1,87 | 0,0261 | 19 |
hsa-miR-548j-3p | 1,91 | 0,0453 | 20 |
hsa-miR-1260b | 1,78 | 0,0361 | 24 |
hsa-miR-326 | 1,62 | 0,0027 | 26 |
hsa-miR-3174 | 1,79 | 0,0480 | 27 |
hsa-miR-210-3p | 1,77 | 0,0461 | 31 |
hsa-miR-32-5p | -1,65 | 0,0373 | 34 |
hsa-miR-342-3p | -1,60 | 0,0307 | 40 |
hsa-miR-3607-3p | -1,59 | 0,0381 | 44 |
hsa-miR-142-5p | -1,54 | 0,0239 | 54 |
hsa-miR-192-5p | -1,56 | 0,0440 | 57 |
hsa-miR-155-5p | -1,43 | 0,0186 | 74 |
hsa-let-7 g-5p | -1,43 | 0,0351 | 76 |
ID | Fold change | P-value | Average rank |
---|---|---|---|
hsa-miR-3202 | 2,94 | 0,0014 | 1 |
hsa-miR-7855-5p | -2,69 | 0,0033 | 2 |
hsa-miR-6770-3p | 2,90 | 0,0135 | 3 |
hsa-miR-1538 | -2,22 | 0,0023 | 4 |
hsa-miR-409-5p | -2,57 | 0,0072 | 5 |
hsa-miR-501-3p | -2,16 | 0,0014 | 6 |
hsa-miR-1299 | 3,53 | 0,0248 | 7 |
hsa-miR-1271-5p | -2,60 | 0,0187 | 8 |
hsa-miR-4687-3p | -2,21 | 0,0217 | 9 |
hsa-miR-4743-5p | -2,07 | 0,0135 | 10 |
hsa-miR-1277-5p | 2,08 | 0,0184 | 11 |
hsa-miR-3180-3p | 2,51 | 0,0351 | 12 |
hsa-miR-4646-5p | -2,18 | 0,0311 | 13 |
hsa-miR-5581-3p | 2,14 | 0,0251 | 14 |
hsa-miR-6882-5p | 2,11 | 0,0257 | 15 |
hsa-miR-449a | 1,94 | 0,0332 | 16 |
hsa-miR-4473 | 2,01 | 0,0439 | 17 |
hsa-miR-4775 | 1,96 | 0,0422 | 18 |
hsa-miR-33b-3p | -1,92 | 0,0411 | 20 |
hsa-miR-99b-5p | -1,84 | 0,0242 | 21 |
hsa-miR-1270 | 1,87 | 0,0328 | 22 |
hsa-miR-18b-5p | 1,94 | 0,0463 | 23 |
hsa-miR-6864-5p | 1,90 | 0,0374 | 24 |
hsa-miR-211-5p | 1,91 | 0,0493 | 25 |
hsa-miR-590-3p | 1,82 | 0,0392 | 31 |
Predicted targets with the highest absolute node strength values in interictal vs healthy comparison
Interictal vs healthy | Ictal vs interictal | ||||||
---|---|---|---|---|---|---|---|
KEGG pathway | p-value | Number of genes | Number of miRNAs | KEGG pathway | p-value | Number of genes | Number of miRNAs |
ECM-receptor interaction | 6.11E-10 | 47 | 20 | Proteoglycans in cancer | 4.7E-07 | 122 | 18 |
Proteoglycans in cancer | 2.60E-08 | 114 | 23 | Prion diseases | 5.1E-05 | 16 | 11 |
PI3K-Akt signalling pathway | 3.14E-07 | 193 | 24 | Axon guidance | 5.1E-05 | 83 | 18 |
Morphine addiction | 3.14E-07 | 53 | 24 | Hippo signalling pathway | 8.86E-05 | 90 | 19 |
TGF-beta signalling pathway | 1.88E-06 | 49 | 18 | Biosynthesis of unsaturated fatty acids | 1.69E-04 | 12 | 8 |
Axon guidance | 1.88E-06 | 78 | 24 | Adrenergic signalling in cardiomyocytes | 2.52E-04 | 82 | 18 |
Transcriptional misregulation in cancer | 2.61E-06 | 101 | 23 | ErbB signalling pathway | 4.00E-04 | 56 | 17 |
GABAergic synapse | 3.09E-06 | 49 | 22 | TGF-beta signalling pathway | 9.09E-04 | 49 | 15 |
ErbB signalling pathway | 3.42E-06 | 55 | 21 | Fatty acid metabolism | 9.29E-04 | 27 | 11 |
Mucin type O- Glycan biosynthesis | 1.01E-05 | 18 | 13 | Phosphatidylinositol signalling system | 9.29E-04 | 51 | 17 |
FoxO signalling pathway | 1.01E-05 | 79 | 21 | Glutamatergic synapse | 9.29E-04 | 66 | 18 |
Glioma | 5.87E-05 | 40 | 18 | Wnt signalling pathway | 9.29E-04 | 81 | 19 |
Lysine degradation | 5.94E-05 | 29 | 21 | Pathways in cancer | 9.29E-04 | 218 | 23 |
Focal adhesion | 8.27E-05 | 118 | 23 | Mucin type O-Glycan biosynthesis | 9.53E-04 | 17 | 10 |
Signalling pathways regulating pluripotency of stem cells | 9.09E-05 | 83 | 23 | FoxO signalling pathway | 1.17E-03 | 80 | 17 |
TermID | Name | Frequency | p-value | Number of genes | Number of miRNAs |
---|---|---|---|---|---|
Interictal vs healthy | |||||
GO:0048011 | neurotrophin TRK receptor signalling pathway | 0.07 | 1.22E-51 | 161 | 24 |
GO:0038095 | Fc-epsilon receptor signalling pathway | 0.14 | 6.77E-39 | 104 | 24 |
GO:0035666 | TRIF-dependent toll-like receptor signalling pathway | 0.04 | 6.03E-15 | 43 | 20 |
GO:0034166 | toll-like receptor 10 signalling pathway | 0.01 | 4.80E-14 | 38 | 19 |
GO:0,038,123 | toll-like receptor TLR1:TLR2 signalling pathway | 0.01 | 1.63E-13 | 39 | 20 |
GO:0034146 | toll-like receptor 5 signalling pathway | 0.01 | 1.82E-11 | 38 | 19 |
GO:0018279 | protein N-linked glycosylation via asparagine | 0.13 | 3.52E-07 | 47 | 19 |
GO:0006921 | cellular component disassembly involved in execution phase of apoptosis | 0.12 | 2.29E-06 | 23 | 19 |
GO:0002576 | platelet degranulation | 0.05 | 2.34E-05 | 32 | 18 |
GO:0050690 | regulation of defense response to virus by virus | 0.01 | 8.27E-04 | 16 | 13 |
GO:0061418 | regulation of transcription from RNA polymerase II promoter in response to hypoxia | 0.07 | 8.27E-04 | 16 | 16 |
GO:0035872 | nucleotide-binding domain. leucine rich repeat containing receptor signalling pathway | 0.08 | 1.04E-03 | 18 | 16 |
GO:0034199 | activation of protein kinase A activity | 0.02 | 4.86E-04 | 10 | 8 |
GO:0071377 | cellular response to glucagon stimulus | 0.06 | 5.90E-03 | 18 | 16 |
GO:0036109 | alpha-linolenic acid metabolic process | 0.06 | 6.51E-03 | 7 | 6 |
Ictal vs interictal | |||||
GO:0048011 | neurotrophin TRK receptor signalling pathway | 0.07 | 2.39E-44 | 157 | 21 |
GO:0038095 | Fc-epsilon receptor signalling pathway | 0.14 | 2.95E-34 | 102 | 19 |
GO:0006921 | cellular component disassembly involved in execution phase of apoptosis | 0.12 | 3.28E-10 | 29 | 16 |
GO:0018279 | protein N-linked glycosylation via asparagine | 0.13 | 1.91E-09 | 54 | 16 |
GO:0038123 | toll-like receptor TLR1:TLR2 signalling pathway | 0.01 | 2.20E-09 | 35 | 15 |
GO:0038124 | toll-like receptor TLR6:TLR2 signalling pathway | 0.04 | 2.20E-09 | 35 | 15 |
GO:0034166 | toll-like receptor 10 signalling pathway | 0.01 | 4.66E-09 | 33 | 15 |
GO:0034146 | toll-like receptor 5 signalling pathway | 0.01 | 3.68E-07 | 33 | 15 |
GO:0006369 | termination of RNA polymerase II transcription | 0.07 | 1.35E-04 | 23 | 14 |
GO:0002576 | platelet degranulation | 0.05 | 3.17E-04 | 31 | 15 |
GO:0022400 | regulation of rhodopsin mediated signalling pathway | 0.06 | 4.62E-04 | 16 | 9 |
GO:1900740 | positive regulation of protein insertion into mitochondrial membrane involved in apoptotic signalling pathway | 0.02 | 2.12E-03 | 18 | 15 |
GO:0007603 | phototransduction. visible light | 0.07 | 4.18E-03 | 38 | 14 |
GO:0050690 | regulation of defense response to virus by virus | 0.01 | 6.52E-03 | 13 | 8 |
GO:0034199 | activation of protein kinase A activity | 0.02 | 7.95E-03 | 10 | 8 |
Predicted targets with the highest absolute node strength values in ictal vs interictal comparison
Interictal vs Healthy | Ictal vs Interictal | ||||
---|---|---|---|---|---|
Official Symbol | Official Full Name | Node strength | Official Symbol | Official Full Name | Node Strength |
Targets predicted to be downregulated | |||||
CADM2 | cell adhesion molecule 2 | 4 | NR3C1 | nuclear receptor subfamily 3 group C member 1 | 7 |
PLEKHM3 | pleckstrin homology domain containing M3 | 4 | GRIA2 | glutamate ionotropic receptor AMPA type subunit 2 | 6 |
MEF2C | myocyte enhancer factor 2C | 4 | MLLT3 | MLLT3 super elongation complex subunit | 6 |
BBX | BBX high mobility group box domain containing | 4 | |||
RIMKLB | ribosomal modification protein rimK like family member B | 4 | |||
HACE1 | HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 | 4 | |||
Targets predicted to be upregulated | |||||
CCNT2 | cyclin T2 | -6 | 32 targets with a node strength of -2 | ||
KLHL15 | kelch like family member 15 | -8 |
Gene Ontology enrichment analysis of predicted mRNAs
Interictal vs Healthy | Ictal vs Interictal | ||||
---|---|---|---|---|---|
GO biological process | Fold Enrichment | FDR | GO biological process | Fold Enrichment | FDR |
Targets predicted to be downregulated | |||||
flavonoid glucuronidation (GO:0052696) | 10.28 | 7.09E-04 | postsynaptic specialization organization (GO:0099084) | 3.47 | 3.09E-02 |
negative regulation of cellular glucuronidation (GO:2001030) | 10.28 | 2.04E-03 | regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay (GO:1900151) | 3.47 | 3.09E-02 |
negative regulation of glucuronosyltransferase activity (GO:1904224) | 10.28 | 2.03E-03 | postsynaptic density organization (GO:0097106) | 3.47 | 3.08E-02 |
regulation of glucuronosyltransferase activity (GO:1904223) | 10.28 | 2.02E-03 | negative regulation of smooth muscle cell migration (GO:0014912) | 3.30 | 1.49E-02 |
regulation of cellular glucuronidation (GO:2001029) | 9.14 | 3.21E-03 | hippo signalling (GO:0035329) | 2.85 | 3.17E-02 |
xenobiotic glucuronidation (GO:0052697) | 8.41 | 1.88E-03 | regulation of mesenchymal cell proliferation (GO:0010464) | 2.72 | 1.91E-02 |
flavonoid metabolic process (GO:0,009,812) | 6.43 | 3.10E-03 | roof of mouth development (GO:0060021) | 2.47 | 7.20E-05 |
cellular glucuronidation (GO:0052695) | 6.28 | 1.67E-03 | regulation of smooth muscle cell migration (GO:0014910) | 2.41 | 4.74E-03 |
uronic acid metabolic process (GO:0006063) | 5.36 | 2.17E-03 | regulation of dendritic spine morphogenesis (GO:0061001) | 2.41 | 1.85E-02 |
glucuronate metabolic process (GO:0019585) | 5.36 | 2.16E-03 | positive regulation of epithelial to mesenchymal transition (GO:0010718) | 2.36 | 2.89E-02 |
Targets predicted to be upregulated | |||||
negative regulation of platelet activation (GO:0010544) | 3.35 | 3.79E-02 | regulation of cell projection organization (GO:0031344) | 2.03 | 3.79E-02 |
G1 phase (GO:0051318) | 3.28 | 3.00E-02 | neuron development (GO:0048666) | 1.95 | 2.14E-02 |
mitotic G1 phase (GO:0000080) | 3.28 | 3.00E-02 | intracellular protein transport (GO:0006886) | 1.79 | 3.90E-02 |
regulation of transcription involved in G1/S transition of mitotic cell cycle (GO:0000083) | 3.27 | 1.87E-03 | neuron differentiation (GO:0030182) | 1.78 | 3.58E-02 |
response to muscle stretch (GO:0035994) | 3.22 | 2.35E-02 | generation of neurons (GO:0048699) | 1.78 | 1.80E-02 |
negative regulation of cyclin-dependent protein kinase activity (GO:1904030) | 3.18 | 6.66E-03 | neurogenesis (GO:0022008) | 1.72 | 1.79E-02 |
regulation of sister chromatid cohesion (GO:0007063) | 3.13 | 3.85E-02 | intracellular transport (GO:0046907) | 1.62 | 3.97E-02 |
negative regulation of cyclin-dependent protein serine/threonine kinase activity (GO:0045736) | 3.11 | 1.12E-02 | cellular macromolecule localization (GO:0070727) | 1.61 | 3.15E-02 |
regulation of histone H3-K9 methylation (GO:0051570) | 3.09 | 3.02E-02 | cellular protein localization (GO:0034613) | 1.60 | 3.66E-02 |
positive regulation of pri-miRNA transcription by RNA polymerase II (GO:1902895) | 3.05 | 2.47E-03 | nitrogen compound transport (GO:0071705) | 1.56 | 3.40E-02 |
Validation of predicted mRNA targets miRNAs based on mRNA sequencing data
Interictal vs Healthy | ||||
---|---|---|---|---|
Target | logFC | Node Strength | Description | Pain/inflammation related disease/processes (if applicable) |
PLCXD2 | -1.337 | 1 | phosphatidylinositol specific phospholipase C X domain containing 2 | |
TNF | 2.814 | -1 | tumor necrosis factor | |
EGR1 | 2.616 | -1 | early growth response 1 | regulates proteins involved in inflammation [56] |
EREG | 3.063 | -1 | epiregulin | temporomandibular disorder [53] |
CD83 | 2.504 | -1 | CD83 molecule | |
NFKBIA | 1.861 | -1 | NFKB inhibitor alpha | cancer pain [53] |
IER3 | 1.810 | -1 | immediate early response 3 | sarcoidosis [55] |
TNFAIP6 | 2.464 | -1 | tumor necrosis factor alpha-induced protein 6 | rheumatoid arthritis [59] |
ID1 | 1.723 | -1 | inhibitor of DNA binding 1, HLH protein | response to oxidative stress [60] |
OSR2 | 1.936 | -1 | odd-skipped related transciption factor 2 | upregulated in mirror image pain in rat CRPS model [61] |
NR4A2 | 1.752 | -1 | nuclear receptor subfamily 4 group A member 2 | |
CNTNAP3 | 2.178 | -1 | contactin associated protein-like 3 | Crohn’s Disease [65], |
FOSB | 1.524 | -1 | FosB proto-oncogene, AP-1 transcription factor subunit | |
IL6 | 1.826 | -1 | interleukin 6 | neuraxial pain, analgesia, musculoskeletal pain, cancer pain, arthritis; irritable bowel syndrome; sciatica intervertebral disc disease pain [53] |
EGR3 | 1.694 | -1 | early growth response 3 | neuropathy[56] |
DUSP1 | 1.265 | -1 | dual specificity phosphatase 1 | |
SOCS3 | 1.284 | -1 | suppressor of cytokine signalling 3 | |
SAT1 | 1.147 | -1 | spermidine/spermine N1-acetyltransferase 1 | neuroinflammation [70] |
RGS1 | 1.681 | -1 | regulator of G protein signalling 1 | undifferentiated spondylarthritis [71] |
PLAU | 1.312 | -1 | plasminogen activator, urokinase | psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease [55] |
MIPOL1 | 1.252 | -1 | mirror-image polydactyly 1 | nasopharyngeal carcinoma [72] |
JUNB | 1.059 | -1 | JunB proto-oncogene, AP-1 transcription factor subunit | psoriasis [73] |
SGK1 | 0.975 | -1 | serum/glucocorticoid regulated kinase 1 | pain developement [74] |
MAP3K7CL | 0.878 | -1 | MAP3K7 C-terminal like | non‐small cell lung cancer [75] |
CXCL8 (IL8) | 2.806 | -2 | C-X-C motif chemokine ligand 8 (interleukin 8) | cancer pain [53] |
PLAUR | 1.428 | -2 | plasminogen activator, urokinase receptor | inflammatory bowel disease [76] |
DUSP2 | 1.521 | -2 | dual specificity phosphatase 2 | endometriosis, cancer, immune and inflammatory resonses [77] |
RBKS | 1.202 | -2 | ribokinase | |
GXYLT2 | 1.888 | -3 | glucoside xylosyltransferase 2 | ulcerative colitis [78] |
SOD2 | 1.429 | -4 | superoxide dismutase 2 | migraine [55] |
SOCS1 | 1.033 | -4 | suppressor of cytokine signalling 1 | Crohn's disease, psoriasis [55] |
Ictal vs Interictal | ||||
---|---|---|---|---|
Target | logFC | Node strength | Description | Pain/inflammation related disease/processes (if applicable) |
RAB3B | -1.370 | 3 | RAB3B, member RAS oncogene family | |
LRRTM2 | -1.051 | 2 | leucine rich repeat transmembrane neuronal 2 | |
NOX5 | -1.051 | 1 | NADPH oxidase 5 | oxidative stress [82] |
FAT3 | -1.003 | 1 | FAT atypical cadherin 3 | neuropathy [55] |
CBARP | -0.955 | 1 | CACN subunit beta associated regulatory protein | Negatively regulates voltage-gated calcium channels [56] |
BEND6 | -0.890 | 1 | BEN domain containing 6 | |
RPS27A | -0.675 | 1 | ribosomal protein S27a | |
TBCA | -0.639 | 1 | tubulin folding cofactor A | brain injury, ischemia [66] |
FRG1B | -0.620 | 1 | FSHD region gene 1 family member B, pseudogene | |
ZNF730 | 1.175 | -1 | zinc finger protein 730 | transcriptional regulation [56] |
ZNF704 | 0.967 | -1 | zinc finger protein 704 | |
MKI67 | 0.826 | -1 | marker of proliferation Ki-67 | cellular proliferation [56] |