MicroRNA and Long Non-coding RNA Regulation in Skeletal Muscle From Growth to Old Age Shows Striking Dysregulation of the Callipyge Locus

Abstract

MicroRNAs (miRNAs) undergo high levels of regulation in skeletal muscle development and control skeletal muscle mass, function and metabolism over the lifespan. More recently, the role of long non-coding RNAs (lncRNAs) in skeletal muscle regulation has started to emerge. Following up on our recent study describing the expression pattern and putative roles of 768 miRNAs in the quadriceps muscle of mice at early life stages, we used a high-throughput miRNA qPCR-based array to assess the expression of the same miRNAs in 28-month old male mouse quadriceps muscle. In addition, we report the expression patterns of lncRNAs playing a putative role in muscle development and adaptation from growth to old age. Twelve miRNAs were significantly downregulated in 28-month old muscle when compared with 12-week old muscle. Ten of them clustered at the Dlk1-Dio3 locus, known as 'Callipyge,' which is associated with muscle development and hypertrophy. This collective downregulation was paralleled by decreases in the expression levels of the maternally expressed imprinted LncRNA coding genes Meg3 and Rian stemming from the same chromosomal region. In contrast, the paternally expressed imprinted Dlk1-Dio3 locus members Rtl1, Dio3, and Dlk1 and the muscle related lncRNAs lncMyoD1, Neat_v1, Neat_v2, and Malat1 underwent significant changes during growth, but their expression levels were not altered past the age of 12 weeks, suggesting roles limited to hyperplasia and early hypertrophy. In conclusion, collective muscle miRNA expression gradually decreases over the lifespan and a cluster of miRNAs and maternally expressed lncRNAs stemming from the Callipyge locus is significantly dysregulated in aging muscle. The Dlk1-Dio3 locus therefore represents a potential new mechanism for age-related muscle decline.

Keywords: aging; lncRNAs; miRNAs; sarcopenia; skeletal muscle.

FIGURE 1

Fold-change of miRNA expression levels in young (12-week) compared with old (28-month) quadriceps muscles of C57Bl/6J male mice. Log₁₀ (fold-change of miRNA expression with age). Positive values denote an increase in miRNA expression with age. Negative values denote a decrease in miRNA expression with age. Red dots represent the miRNAs that were returned significant after Bonferroni adjustment.

FIGURE 2

Expression levels of individual miRNAs in young (12-week) compared with old (28-month) muscles. Individual miRNA expression levels are shown for 12 miRNAs that were significantly different in 28-month, when compared with 12-week, old mouse muscle. The data are reported as Mean ± SEM. ^∗p < 0.05; ^∗∗p < 0.01; and ^∗∗∗p < 0.001.

FIGURE 3

Genomic context of the differentially expressed mRNAs, miRNAs, and lncRNAs in young (12-week) and old (28-month) muscle. We used the basic annotation of GENCODE mouse vM16 to annotate protein-coding genes (mRNAs) (purple), long non-coding genes (lncRNAs) (green), and miRNAs (blue), visible on the top panel. Differential expression between 12-week old and 28-month old mice for a given gene is visible as a vertical bar in the middle panel (log fold-change of expression). All transcripts had decreased expression with age. There was no difference in mRNA expression for the genes Dlk1, Rtl1, and Dio3 between young (12-week) and old (28-month) muscles. This graph was created using an in-house R script. FC, fold change.

FIGURE 4

MyomiRs expression levels in young (12-week) compared with old (28-month) muscles. Individual miRNA expression levels are shown for mmu-miR-1-3p, mmu-miR-133a-3p, and mmu-miR-206 in 28-month, when compared with 12-week, old mouse muscle. The data are reported as Mean ± SEM.

FIGURE 5

Expression levels of miRNAs, lncRNAs, and mRNAs from the Callipyge locus over the lifespan (5 ages). (A) Individual miRNA expression levels of mmu-miR-136-5p, mmu-miR-299-5p, mmu-miR-335-5p, mmu-miR-337, and mmu-miR-376c over the lifespan. The data are reported as Mean ± SEM. ^∗∗∗, main effect of time, p < 0.001. The grayed data have been previously published in Lamon et al. (2017). (B) Individual expression levels of Meg3, Mirg, Rian, Rtl1, Dio3, and Dlk1 over the lifespan. The data are reported as Mean ± SEM. ^∗∗∗, main effect of time, p < 0.001. Post hoc tests: ^∗p < 0.05 and ^∗∗p < 0.01.