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MIR147B: A NOVEL KEY REGULATOR OF IL-1β-MEDIATED INFLAMMATION IN ASTROCYTES
basal conditions and after stimulation with IL-1β (p<0.0001, Fig 2D). After consultation of the online databases mirDB, targetScan and DIANA tools and based on previous liter- ature 26, disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), DEP domain-containing mTOR-interacting protein (DEPTOR) and interleukin 32 (IL-32) were selected as potential targets for further investigation. Of both DEPTOR and ADAM15 mRNA levels were reduced after miR147b overexpression (p<0.0001 and p=0.0004 respectively, Fig 2E), both under basal and inflammatory conditions. IL-32 showed a trend towards reduced expression (data not shown).
Altered proliferation rate after miRNA mimic transfection
Fetal astrocyte proliferation was decreased after transfection with miR146a (p=0.0022) and miR147b mimics (p=0.0022), as determined by Ki67 staining (Fig 3 A and B). Flow cytometric analysis of PI staining confirmed these results, as the percentage of cells in the S-phase was decreased after transfection with either miR146a (p=0.0043) or miR147b mimic (p=0.015, Fig 3C).
Table 2. Enriched pathway terms and associated differential expressed mRNAs as determined by mRNA-sequencing of control and IL-1β stimulated human astrocyte cultures.
Pathway
Gene count
Genes
P value
Adjusted p-valuea
hsa04668: TNF signaling pathway
8
NFKBIA, BIRC3, CCL20, LIF, CSF1, VCAM1, IL6, TNFAIP3
7.74E-07
7.97E-05
hsa04064: NF-kappa B signaling pathway
7
NFKBIA, BIRC3, RELB, VCAM1, NFKB2, CXCL8, TNFAIP3
4.05E-06
1.39E-04
hsa05162: Measles
8
NFKBIA, OAS2, OAS3, MX1, IFIH1, IL6, TNFAIP3, OAS1
3.58E-06
1.85E-04
hsa05164: Influenza A
8
NFKBIA, OAS2, OAS3, MX1, IFIH1, IL6, CXCL8, OAS1
2.11E-05
5.43E-04
hsa05160: Hepatitis C
7
NFKBIA, IRF1, OAS2, OAS3, CLDN1, CXCL8, OAS1
4.66E-05
9.59E-04
hsa04621: NOD-like receptor signaling pathway
5
NFKBIA, BIRC3, IL6, CXCL8, TNFAIP3
1.57E-04
0.00230227
hsa05134: Legionellosis
5
NFKBIA, NFKB2, IL6, CXCL8, C3
1.46E-04
0.00249934
hsa05168: Herpes simplex infection
7
NFKBIA, OAS2, OAS3, IFIH1, IL6, C3, OAS1
2.72E-04
0.00349796
hsa05133: Pertussis
5
IRF1, C1R, IL6, CXCL8, C3
5.20E-04
0.00592968
hsa05323: Rheumatoid arthritis
5
CCL20, IL11, CSF1, IL6, CXCL8
9.51E-04
0.00887256
hsa04060: Cytokine- cytokine receptor interaction
7
CCL20, LIF, CSF1, IL6, CSCL8, IL7R
9.20E-04
0.00943084
hsa04622: RIG-I-like receptor signaling pathway
4
NFKBIA, IFIH1, ISG15, CXCL8
0.00516713
0.04349202
aBenjamini-Hochberg adjusted p-value.
153
six