*2.3. Identification of miRNA-Targeted mRNAs*

MiRNAs regulate expression of specific genes via hybridization to mRNAs to promote their degradation in order to inhibit their translation or both [33]. A volcano plot revealed the overall distribution of the exosomal miRNAs we identified in this study (Figure 3A). To study the possible functional roles of the differentially expressed miRNAs, their potential mRNA targets were analyzed with Targetscan. Among the 68 miRNAs, only 19 were associated with 766 of the 2844 mRNAs that were differentially expressed with age (Figure 3B), suggesting that these miRNAs contribute to the age-dependent regulation of specific mRNAs. Among them, 5 mRNAs were down-regulated and 14 were up-regulated in serum exosomes from old rats compared with those from young rats (Figure 3C). MiRNA-483-3p and miRNA-489-3p were detected only in exosomes from young rats, and miRNA-187-3p, miRNA-202-3p, miRNA-450b-5p, miRNA-501-3p, miRNA-511-5p, and miRNA-598-3p were detected only in exosomes from old rats (Table 1). Figure 3D presents results of a cluster analysis of differentially expressed miRNAs for each sample.

**Figure 3.** Profiles for differentially expressed miRNAs of serum exosomes from young and old rats. (**A**) Volcano plot showing the differentially expressed miRNAs (fold change > 1.5 and *p* < 0.05). (**B**) Venn diagram showing the differentially expressed mRNAs. (**C**) Venn diagram of the differentially expressed miRNAs. (**D**) Heat map of hierarchical clustering of 19 miRNAs that were identified in serum exosomes from young and old rats (*n* = 6 each group).


