Dissecting T-cell-specific miRNA/mRNA target relationships relevant for human islet autoimmunity

Molecular mechanisms triggering the onset of islet autoimmunity and progression to clinically overt type 1 diabetes (T1D) remain poorly understood. microRNAs (miRNAs) critically regulate immune homeostasis. While profiles of total miRNA abundance in T-cells have been reported previously, none have determined which miRNAs are actively engaged in mRNA regulation and which mRNAs are specifically targeted.

Therefore, we performed HITS-CLIP analysis of microRNAs and mRNA fragments present in the RNA-induced silencing complex (RISC) of human CD4+T-cells. The analysis resulted in 271 unique miRNAs present within the RISC in human CD4+T-cells and 7,829 mRNA targets. The most abundant miRNA was miR142, suggesting an essential role of this miRNA in CD4+T-cells. Gene ontology analysis of the 500 most targeted mRNAs showed a significant enrichment of biological processes associated with immune activation and regulation. To link the abundance of individual miRNAs to the activation of islet autoimmunity, we used high-throughput sequencing to analyze the miRNA abundance in activated CD4+T cells of children with and without islet autoimmunity. We could identify multiple differentially expressed miRNAs, with both down- and upregulation of up to tenfold. One of the miRNAs upregulated in children with islet autoimmunity was miR142-3p. The differential expression of miR142-3p was validated by qPCR analysis of CD4+T-cells isolated from individuals with recent onset of T1D and without T1D (4.6 +/- 0,92 vs. 3.4 +/- 1.03 10-ΔCq; P = 0.04). In addition, activated CD4+T-cells of insulin-autoantibody positive NOD mice also showed elevated levels of miR142-3p expression compared to IAA- littermates (10.58 +/- 0.69 vs. 7.8 +/- 1.34 10-ΔCq; P = 0.001).

These discoveries suggest HITS CLIP as a valuable tool to improve our understanding of the mechanisms underlying the onset of autoimmunity, facilitating the future development of innovative strategies aiming at the reduction of islet autoimmunity.