Viral MicroRNAs Modulate Transcriptome in Oral Keratinocytes and Alter Cytokine Levels in Myeloid Cells

MicroRNAs (miRNAs) are small, non-coding RNAs of ~18-25 nucleotides that have gained extensive attention as critical regulators in complex gene networks. Some of the key regulations involve immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and function. Many viruses encode miRNAs that directly modulate expression of genes of the innate immune system, which includes proteins involved in promoting apoptosis and recruitment of cells of the immune system. In this study, we examined the expression profiles of three known v-miRNAs (vmiRs) from HSV-1 (miR-H1), KSHV (miR-K12-3), and HCMV (miR-US4) in healthy and diseased periodontal tissues from non-obese and obese subjects and observed increased levels of these vmiRs in diseased tissues compared to respective controls. Next, we investigated the impact of miR-H1 and miR-K12-3 overexpression on the host transcriptome focusing on gingival epithelial cells that are target sites for various HHVs. Expression of more than 1300 genes were altered in human oral keratinocytes (HOK) transfected with miR-H1 and miR-K12-3. Global pathway analysis identified dysregulation of key signaling genes and pathways that may favor virus persistence. Using bioinformatic analysis, we identified hundreds of potential vmiR binding sites on genes downregualted by miR-H1 and miR-K12-3 suggesting direct regualtion of these genes by vmiRs. We also assessed the expression of six selected vmiR-deregulated genes in myeloid inflammtory cells viz., macrophages and dendritic cells. Our results show that three genes exhibit similar changes in myeloid cells as observed in HOK while the remaining genes exhibit cell specific changes. Finally, cytokine responses of vmiR expressing myeloid cells challenged with lipopolysacchride (LPS) derived from the periodontal pathogen, Aggregatibacter actinomycetemcomitans (Aa) show increased levels of TNF-α but reduced IL-12p40 indicating immunomoduatory functions of vmiRs. Overall, our resutls demonstrate clinical and functional relevance of pathogenic viral molecules viz., vmiRs capable of modulating expression of key gene and pathways in HOK and cytokine expression in myeloid cells which may favor an environment condusive to periodontal pathology.