Introduction
Methods
MOG35–55-EAE generation
Tissue collection and total RNA extraction
Next-generation sequencing
Analysis of differential expression
Ontological analysis with GOrilla/REViGo
Functional analysis with ingenuity pathway analysis
qPCR validation
qPCR statistical analysis
Results
RNASeq
Detection of EAE-related sex-specific signatures
REViGO term | -log(p value) | Dispensability | Frequency (%) |
---|---|---|---|
Extracellular region | 23.8069 | 0 | 2.38 |
Cell surface | 19.1549 | 0 | 0.24 |
Membrane | 27.0114 | 0 | 61.59 |
Vesicle | 9.279 | 0 | 1.36 |
Extracellular region part | 30.5186 | 0 | 1.31 |
Side of membrane | 20.0101 | 0 | 0.21 |
Membrane region | 12.4225 | 0.037 | 0.12 |
External side of plasma membrane | 19.3224 | 0.046 | 0.06 |
Cell part | 20.251 | 0.087 | 52.39 |
Cytoplasmic part | 14.284 | 0.214 | 12.66 |
REViGO term | -log(p value) | Dispensability | Frequency (%) |
---|---|---|---|
Membrane | 19.9136 | 0 | 61.59 |
NADH dehydrogenase complex | 12.2565 | 0 | 0.04 |
Organelle | 9.8665 | 0 | 20.79 |
Extracellular region part | 11.5017 | 0 | 1.31 |
Respiratory chain | 9.8239 | 0.035 | 0.30 |
Cell surface | 11.9872 | 0.045 | 0.24 |
Mitochondrial membrane part | 11.4377 | 0.053 | 0.38 |
Cell part | 17.4437 | 0.093 | 52.39 |
Receptor complex | 9.9747 | 0.233 | 0.12 |
Cytoplasmic part | 16.0414 | 0.261 | 12.66 |
Validation of RNASeq
DEG/DEmiR | Female | Male | Main effect | p value | 2° effect | p value | ||
---|---|---|---|---|---|---|---|---|
Fold change | Adjusted p value | Fold change | Adjusted p value | |||||
C3 | 2.48 | 0.0029 | 0.17 | < 0.0001 | Sex | < 0.0001 | Disease | 0.0384 |
C5ar1 | 2.69 | 0.0079 | 1.23 | 0.8121 | Disease | 0.0170 | ~ | ~ |
Stat1 | 3.13 | < 0.0001 | 0.33 | 0.0005 | Sex | < 0.0001 | ~ | ~ |
Trem2 | 1.80 | 0.0072 | 0.61 | 0.0375 | Sex | 0.0004 | ~ | ~ |
Tlr7 | 1.85 | 0.0182 | 0.80 | 0.6256 | Sex | 0.0174 | ~ | ~ |
Tlr8 | 2.82 | < 0.0001 | 0.60 | 0.0478 | Sex | < 0.0001 | ~ | ~ |
miR-21 | 3.78 | < 0.0001 | 4.76 | < 0.0001 | Disease | < 0.0001 | ~ | ~ |
miR-122 | 4.36 | 0.0232 | 0.22 | 0.1188 | Sex | 0.0037 | ~ | ~ |
miRSeq
Differences in transcriptional signatures by sex within EAE
Ingenuity canonical pathways | -log(p value) | Ratio | z-score |
---|---|---|---|
LXR/RXR activation | 5.06 | 0.145 | 2.673 |
Acute phase response signaling | 4.44 | 0.116 | 1.155 |
Neuroinflammation signaling pathway | 3.57 | 0.0871 | 0.229 |
Complement system | 3.54 | 0.212 | 1.342 |
FXR/RXR activation | 3.12 | 0.113 | N/A |
Phagosome formation | 2.88 | 0.107 | N/A |
Cytotoxic T lymphocyte-mediated apoptosis of target cells | 2.39 | 0.185 | 1 |
Hepatic fibrosis/hepatic stellate cell activation | 2.21 | 0.0838 | N/A |
GP6 signaling pathway | 2.14 | 0.0909 | 0.302 |
Interferon signaling | 2.12 | 0.161 | 2.236 |