hsa-miR-105 modulates TLR2 expression in oral keratinocytes

Objective: MicroRNAs regulate innate immune responses in human macrophages, monocytic cell lines and psoriatic lesions. We sought to examine the miRNA changes in human gingival keratinocytes following challenge with Porphyromonas gingivalis.

Methods: MiRNA microarray was performed after challenging epithelial cells with heat inactivated P. gingivalis and LPS. Upregulated miRNAs were subjected to an miRBase search to identify potential targets. The microarray result was verified with TaqMan miRNA real time PCR. An expression plasmid containing miR-105 sequence was transiently transfected into epithelial cells with or without anti miR-105 oligonucleotide. Toll like receptor–2 (TLR2) expression was monitored by real time PCR, western blot, immunostaining. Expression vectors were constructed using the binding site for miR-105 on TLR2 3'UTR with/without mutation which were then tagged to Luciferase reporter gene. Transient expression of Luciferase was detected by anti-Luciferase antibody.

Results: Microarray data indicated that miR-105 regulates TLR2 signaling in oral keratinocytes. Using a library of human gingival epithelial cells we discerned robust differences in the inflammatory cytokine production of P. gingivalis-challenged keratinocytes: these cells could be categorized cells into ‘normal' and ‘depressed' responses to specific TLR2/TLR4 agonists. MiR-105 was markedly downregulated in cells with the ‘normal' response (using IL-6 as the primary response variable), upon challenge with TLR2/TLR4 agonists and significantly up-regulated in the response deficient cells. Characterization by si-RNA and protein expression revealed that miR-105 regulates TLR2 signaling and cytokine response by reducing cell surface TLR-2 protein.

Conclusion: Thus we propose that miR-105 modulates TLR2 at the post transcriptional level with a resultant reduction in TLR2 protein expression and inflammatory cytokine response. Supported by grant DE017384 from NIDCR