Objectives: The aim of this study was to investigate how miRNA levels are altered in oral epithelial cells after initial (t=4h) and prolonged (t=24h) challenged with periodontopathogens.
Methods: Immortalised oral keratinocyte cells OK-F6-Tert2 were challenged with either P. gingivalis or T. forsythia for 4h and 24 h at an MOI of ~ 1:100. Following RNA extraction, levels of miRNAs in challenged and unchallenged cells were determined using a qPCR-based Tiling Low Density Array (TLDA). Altered miRNAs were validated using TaqMan qPCR. Potential targets were identified using in-silico analysis and are currently being validated.
Results: Several miRNAs were downregulated after challenge with either P. gingivalis or T. forsythia. Whilst some were downregulated by both pathogens (miR-10b and miR-424) others were found to be pathogen specific; for example miR-101 was downregulated only by T. forsythia. Changes in miRNA were also time-dependent with levels of several members of the Hsa-Let family consistently downregulated after 24h but not after 4h. Of the miRNAs identified, miR-10b, miR-424, miR-101 and miR-106b, have been validated by qPCR (p<0.05). In-silico analysis suggests that potential targets include some of the interleukin receptors and several transcription factors and these are currently being experimentally validated.
Conclusion: Our data show that alterations observed in miRNA levels following bacterial challenge were pathogen-specific and time-dependent, suggesting that miRNAs play key role in the response to periodontal infection by modulating gene expression.