MicroRNA-Mediated Modulation of the Colony-Stimulating Factor 1 Pathway in Microglial Activation and Neuropathic Pain: A Next-Generation Sequencing-Based Transcriptomic Study

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 a Bioanalyzer. RNA libraries were prepared with the SureSelect kit and sequenced on an Illumina platform.

Differential expression was analyzed using Cuffdiff after excluding low-abundance transcripts; genes with a fold change ≥ 2 and an FDR-adjusted p value < 0.05 were considered significantly differentially expressed. Potential miRNA-mRNA regulatory interactions were identified using miRWalk, and GO and KEGG pathway enrichment analyses were used to identify pathways related to inflammation, immune signaling, and extracellular matrix remodeling.

Results: 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 with 119 upregulated mRNAs, and 69 upregulated miRNAs with 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 extracellular matrix (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. Conclusion: 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.