miRNA relative expression estimates and their involvement in immunity and/or viral replication. (A) Expression of miRNAs were determined by TaqMan MicroRNA qRT-PCR. Fold-change expression of miRNAs that replicated in the 70% of patients in all 3 classes were visualized as box-and-whiskers plots. Three miRNAs were up-regulated, and 5 miRNAs were down-regulated. (B) Only miRNAs (N = 114), which replicated in more than 70% of patients in at least one class and varied by at least 1 log ₁₀ from healthy controls, were displayed in a heat map generated by Java TreeView 1.60 software. The heat map shows the fold-changes in miRNA expression in CD4⁺ T cells of HIV-1–infected (6 éLTNP and 8 naive) and exposed (4 MEU) patients compared with a pool of 6 healthy donors. Each colored block represents the expression of 1 miRNA (labeled on the right) in the indicated sample. PCR expression signals are converted into color (red, high signal; green, low signal). Color intensities are proportional to the variation of expression as indicated in the scale bar: values ranged from log₁₀(−4) to log₁₀(+4). Data represent 3 independent experiments. miRNAs and samples of patients were analyzed by average-linkage hierarchical clustering.²⁵  The clustering, performed using average linkage and Pearson correlation, shows that éLTNP and naive subjects have similar expression profiles of miRNAs. The miRNAs, differently expressed in at least 2 groups, are marked in red. The miR-17/92 cluster was composed of miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a-1. (C) Heat map shows miRNAs for which statistical analyses (ANOVA followed by Bonferroni posthoc test) indicated significant differences in expression changes among the 3 classes. Only miRNAs with transcript levels replicated in more than 70% of the patients of each class were considered for statistical analysis. The hierarchical clustering analysis was performed on tested miRNAs and samples of patients. (D) Expression profile analysis of 377 miRNAs implicated in either viral replication or the immune response by literature analysis cluster together naive and éLTNP patients. The analysis of miRNAs, which share both functions, results in naive and MEU patient clusters. The identified miRNAs are divided according to their reported function through Venn analysis. The overlapping area shows miRNAs involved in both fields. Into the Venn diagram the average (avg) of miRNA relative expression of each subject class is displayed using a heat map representation. The hierarchical clustering dendrogram shows the similarity of miRNA patterns between groups of patients. This analysis performed was average linkage and Pearson correlation. miRNAs expression with more than 30% missing values are in gray.