Circulating microRNA signature of genotype-by-age interactions in the long-lived Ames dwarf mouse

Abstract

Recent evidence demonstrates that serum levels of specific miRNAs significantly change with age. The ability of circulating sncRNAs to act as signaling molecules and regulate a broad spectrum of cellular functions implicates them as key players in the aging process. To discover circulating sncRNAs that impact aging in the long-lived Ames dwarf mice, we conducted deep sequencing of small RNAs extracted from serum of young and old mice. Our analysis showed genotype-specific changes in the circulating levels of 21 miRNAs during aging [genotype-by-age interaction (GbA)]. Genotype-by-age miRNAs showed four distinct expression patterns and significant overtargeting of transcripts involved in age-related processes. Functional enrichment analysis of putative and validated miRNA targets highlighted cellular processes such as tumor suppression, anti-inflammatory response, and modulation of Wnt, insulin, mTOR, and MAPK signaling pathways, among others. The comparative analysis of circulating GbA miRNAs in Ames mice with circulating miRNAs modulated by calorie restriction (CR) in another long-lived mouse suggests CR-like and CR-independent mechanisms contributing to longevity in the Ames mouse. In conclusion, we showed for the first time a signature of circulating miRNAs modulated by age in the long-lived Ames mouse.

Keywords: aging; circulating miRNAs; dwarf mouse; sequencing; sncRNAs; tRNA halves.

Figure 1

Length distribution and annotation of small RNAs circulating in mice serum. Two major small RNA peaks were detected in the serum from the studied mice: at 20–24 nt, consistent with the size of miRNAs, and at 30–33 nt consisted of reads mapping to tRNA genes (a). A total of 76% and 24% of the total reads mapped to the mouse small noncoding RNAs were derived from tRNAs and miRNAs, respectively (b).

Figure 2

Gene‐by‐age interaction miRNA–mRNA network analysis. Five subnetworks of miRNA‐overtargeted transcripts enriched for biological processes and molecular functions relevant to aging are presented. (a) Calcium modulating Wnt receptor signaling pathway; (b) SNARE/syntaxin binding; (c) cell projection morphogenesis; (d) positive regulation of transcription; (e) genes containing ankyrin repeats. Noticeable is a complex pattern of hubs and interhub interactions that underscore a relevant cross talk among pathways. The genotype‐by‐age (GbA) miRNA–mRNA interaction network was constructed in Cytoscape 3.0.2 software as described in Materials and methods. Red/blue boxes: positive/negative GbA miRNAs. Cyan ovals: mRNAs (predicted to be downregulated).

Figure 3

Comparative analysis of circulating genotype‐by‐age (GbA) miRNAs in mice. (a) Venn diagram of common miRNA families between this work and the study of Dhahbi et al. (2013c). (b) GbA miRNAs in N and df/df mice exhibited four different types of expression patterns (left and middle panel). Many miRNAs circulating in the long‐lived B6C3F1 mouse (within common GbA miRNA families) are increased with age, and this effect can be antagonized by calorie restriction (CR; right panel).