Figure 2.

microRNAs differentially expressed in smokers regulate the PI3K-AKT-FOXO signaling pathway. Isolated CD8⁺ T cells from human peripheral blood were activated with 30 nM PMA and 500 nM ionomycin for 2 h. (A) Fold change of miR expression in smokers compared to non-smokers. MiRs with complete data are included; those exceeding an average microarray signal intensity of 80 are presented in black. MiRs included in further analyses are highlighted in color orange and blue for up- or downregulated, respectively. (B) Kegg pathways that involve the miRs that were significantly down- or upregulated in smokers. Pathways of T cell effector/memory differentiation, PI3K-AKT signaling or nicotine signaling are indicated by color as indicated with color key. (C) A graphic map of the Kegg pathways indicated with bold in (B). MiRs predicted to inhibit expression of proteins involved in these pathways are placed adjacent to the protein. (D) Heatmap and clustering of miRs differentially expressed in smokers. Clustering was based on Ward's minimum clustering method and distances were calculated with Spearman correlations. (E) A scoring system was constructed to differentiate between naïve-memory and effector phenotype. A frequency of CD27⁺CD8⁺ cells above the median of all samples, or a higher than median expression of IL7R, FOXO1, or TCF7 mRNA were rewarded with one point each. Samples with at least two points were considered to have a naïve-memory phenotype. P-values between the two groups were calculated with the Mann-Whitney U-test. The smoking status of each individual is indicated below. (F) Volcano plot of the fold change of miRs in samples with naïve-memory vs. effector phenotype according to the scoring system in (E). S, smoker; NS, non-smoker. An X indicates missing data.