Background and objectives MiR-31 is upregulated in repeatedly activated Th1 and in effector/memory Th lymphocytes isolated from the synovial fluid of patients suffering from rheumatic joint diseases. Several respective miR-31 targets are downregulated and essential for the cytoskeletal rearrangement of repeatedly activated Th1 cells. This suggests that miR-31 regulates the motility and immobilises proinflammatory Th1 cells in inflamed tissues.
Materials and methods Assuming that Th cells involved in chronic inflammation have a history of repeated stimulation by autoantigens, we have in vitro generated four times activated Th1 cells. We have determined the transcriptomes and the migratory behaviour of these cells after Antagomir mediated miR-31 knockdown by using microarrays and in vitro transwell migration assays. For the identification of potential direct targets we have applied the target prediction algorithm TargetScan. In order to determine the putative function of miR-31 in proinflammatory Th1 cells a gene set enrichment (GSE) analysis of the obtained transcriptome data after miR-31 knockdown was performed.
Results Specific inhibition of miR-31 in repeatedly activated Th1 cells promoted their migration up to 30% in a transwell migration assay. In sum, we have identified 283 differentially expressed genes in repeatedly activated Th1 cells after treatment with a miR-31 specific or an unspecific Antagomir for 36, 48 and 72 h. The GSE analysis revealed an enrichment of genes, which regulate cell-intrinsic and extrinsic pathways involved in motility and tissue localization. Among the putative direct targets we found actin binding LIM Protein 1 (Ablim1), Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Epsilon (Ywhae), actin gamma 1 (Actg1), actin beta (Actb), Lamellipodin (Raph1), T-cell lymphoma invasion and metastasis-inducing protein 1 (TIAM1) and Ras homolog gene family, member A (RhoA). From these genes, we could verify the upregulation of Actg1, Actb, Raph1 and RhoA after 48 to 72 h of Antagomir-31 treatment from 1.2 to 3 fold using quantitative RT-PCR.
Conclusion We show that miR-31 restricts the motility of repeatedly activated Th1 cells in vitro most likely by repressing genes involved in cytoskeletal rearrangement. MiR-31 might represent a molecular switch important for the persistence of proinflammatory Th cells in inflamed tissues.