MicroRNA-Mediated Modulation of the Colony-Stimulating Factor 1 Pathway in Microglial Activation and Neuropathic...

Neuropathic pain typically develops after nerve injury and is largely driven by microglial activation in the central nervous system (CNS), with colony-stimulating factor 1 (CSF1) serving as a key initiator. We analyzed mRNA and microRNA expression profiles in CSF1-stimulated primary microglia using next-generation sequencing, followed by integrative bioinformatic analyses.

Rat microglia were cultured in microglial medium at 37°C with 5% CO₂ and treated with CSF1 (20 ng/mL) for 16 h to assess early transcriptional and miRNA-mRNA responses. Total RNA was extracted using TRIzol and quantified using a Nanodrop and Bioanalyzer. RNA libraries were prepared with the SureSelect kit and sequenced on an Illumina platform. Differentially expressed mRNAs were defined as those with a fold change ≥ 2 and an FDR-adjusted p value < 0.05, whereas differentially expressed miRNAs were identified using a fold change ≥ 1.5 for both upregulated and downregulated miRNAs with a p value < 0.05. The miRNA-mRNA interactions were assessed using miRWalk, and KEGG/GO analyses identified pathways related to inflammation, immune signaling, and extracellular matrix (ECM) remodeling.

CSF1 stimulation altered the expression of 176 miRNAs and 429 mRNAs, including 86 upregulated and 90 downregulated miRNAs. Inverse correlation analysis revealed 68 downregulated miRNAs linked to 119 upregulated mRNAs, and 69 upregulated miRNAs linked to 204 downregulated mRNAs, indicating complex regulatory interactions. The transcriptional program induced by CSF1 was characterized by increased expression of proinflammatory and proliferative genes, such as Fos, Cxcl2, and Ephb3, and suppressed expression of ECM- and glycosaminoglycan (GAG)-related genes, including Gpc6 and Prelp. Several miRNAs, including rno-miR-652-5p, rno-miR-672-5p, rno-miR-455-3p, rno-miR-145-5p, rno-miR-222-3p, rno-miR-702-5p, rno-miR-877, rno-miR-664-2-5p, and rno-miR-702-3p, emerged as potential upstream regulators of these changes. Pathway enrichment analysis highlighted the activation of TNF, IL-17, and mitogen-activated protein kinase (MAPK) signaling, coupled with the suppression of ECM remodeling.

These findings identify candidate miRNA-mRNA regulatory networks associated with CSF1-driven microglial responses and provide new insights into transcriptomic changes relevant to neuroimmune mechanisms in neuropathic pain.