Abstract
IntroductionThe risk of tendon injury and disease increases significantly with increasing age. The aim of the study was to characterise transcriptional changes in human Achilles tendon during the ageing process in order to identify molecular signatures that might contribute to age-related degeneration.MethodsRNA for gene expression analysis using RNA-Seq and quantitative real-time polymerase chain reaction analysis was isolated from young and old macroscopically normal human Achilles tendon. RNA sequence libraries were prepared following ribosomal RNA depletion, and sequencing was undertaken by using the Illumina HiSeq 2000 platform. Expression levels among genes were compared by using fragments per kilobase of exon per million fragments mapped. Differentially expressed genes were defined by using Benjamini-Hochberg false discovery rate approach (P <0.05, expression ratios 1.4 log2 fold change). Alternative splicing of exon variants were also examined by using Cufflinks. The functional significance of genes that showed differential expression between young and old tendon was determined by using ingenuity pathway analysis.ResultsIn total, the expression of 325 transcribed elements, including protein-coding transcripts and non-coding transcripts (small non-coding RNAs, pseudogenes, long non-coding RNAs and a single microRNA), was significantly different in old compared with young tendon (±1.4 log2 fold change, P <0.05). Of these, 191 were at higher levels in older tendon and 134 were at lower levels in older tendon. The top networks for genes differentially expressed with tendon age were from cellular function, cellular growth, and cellular cycling pathways. Notable differential transcriptome changes were also observed in alternative splicing patterns. Several of the top gene ontology terms identified in downregulated isoforms in old tendon related to collagen and post-translational modification of collagen.ConclusionsThis study demonstrates dynamic alterations in RNA with age at numerous genomic levels, indicating changes in the regulation of transcriptional networks. The results suggest that ageing is not primarily associated with loss of ability to synthesise matrix proteins and matrix-degrading enzymes. In addition, we have identified non-coding RNA genes and differentially expressed transcript isoforms of known matrix components with ageing which require further investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0544-2) contains supplementary material, which is available to authorized users.
Highlights
The risk of tendon injury and disease increases significantly with increasing age
Sample collection and preparation All human Achilles tendons used in this study—RNA-Seq and quantitative real-time polymerase chain reaction—were harvested from limbs amputated during surgical procedures to treat sarcomas at the Royal National Orthopaedic Hospital, Stanmore
Identification of differentially expressed genes and isoforms A principal component analysis (PCA) plot of log2 gene expression data indicated that the effect of age on gene expression was distinct as data were clustered in two groups (Figure 1A)
Summary
The risk of tendon injury and disease increases significantly with increasing age. The aim of the study was to characterise transcriptional changes in human Achilles tendon during the ageing process in order to identify molecular signatures that might contribute to age-related degeneration. Similar changes have been observed in macroscopically abnormal equine flexor tendon [7], another common site of age-associated tendon injury. Histological abnormalities are more often observed in older individuals [6], the relationship with ageing and the apparent change in cell function is not clear. It has been suggested that early degenerative changes in tendon result from an accumulation of micro-damage within the extracellular matrix (ECM) due to an imbalance between anabolic and catabolic pathways [10]. Another study found that flexor tendon explants from older horses were more susceptible to fatigue damage following cyclical loading in vitro than explants from young horses and that this was a cellmediated process involving the matrix metallo-proteinases (MMPs) [12]
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