Abstract
Summary
We compare the difference in expression profiles of miRNAs during fracture healing between adult and aged female mice. This study reveals the possibility to improve impaired fracture healing in aged females by regulating key miRNAs at early stage.
Introduction
Impaired fracture healing in aged female skeleton is still a clinical challenge (Holroyd et al., Best Pract Res Clin Endocrinol Metab, 2008, Virk, Lieberman, Arthritis Res Ther, 2012). Angiogenesis and osteogenesis are the two key stages during fracture healing, which are impaired in aged female (Naik et al., J Bone Miner Res, 2009). MicroRNAs (miRNAs) are key post-transcriptional non-coding regulators of gene expression, which has demonstrated important roles in angiogenesis and osteogenesis (Bae et al., Hum Mol Genet, 2012, Plummer et al., Cancer Res, 2013). Understanding how non-coding regulatory RNA in fracture healing changes with age will help identifying novel therapeutic targets that can be exploited to improve fracture healing in the aged females.
Materials and methods
Bilateral femur transverse fractures were created in 9 female 12-month-old mice (Aged Group) and 9 female 12-week-old mice (Adult Group). Three mice in each group were sacrificed at 0, 2 and 4 weeks post fracture, respectively. Total RNA was extracted and hybridised on Agilent 8×60K Mouse miRNA Microarray. Then, differentially expressed miRNAs were identified in adult and aged female fracture mice, respectively (2-vs-0 weeks, 4-vs-0 weeks, P-value <= 0.05 & Fold change >=2.0). With the experimentally validated interactions among miRNAs and their targets, we constructed fracture-healing-related molecular network. Thereafter, we performed topological and dynamic network analysis to find key hub miRNAs in female fracture healing. Person correlation coefficient (r) analysis was performed on the expression data of the miRNAs in all the 18 mice to identify co-expression modules in the female fracture healing progress. Meanwhile, in order to analyze the angiogenesis in the early stage and osteogenesis in the later stage of female fracture healing, we performed microCT-based angiography at 2 weeks post fracture and micro-CT examination at 4 weeks post fracture on the right femur callus samples.
Results & Discussion
Angiography showed smaller blood vessel volume in aged mice at early stage when compared to that in the adult mice. Reconstructed calluses showed lower bridging mineralization tissues within the gap in aged mice than that in the adult mice at the later stage. We found that the top hub miRNAs were differentially expressed in adult female mice but not in aged ones during fracture healing. Moreover, the differential expression of the top hub miRNAs was only observed at early stage (2 weeks) during fracture healing in adult female mice. This may help explain the difference of fracture healing between adult and aged female mice. It also indicated the molecular events controlled by the hub miRNAs in early stage could lead to the following differences between the adult and aged female mice at 4 weeks. The person correlation coefficient analysis revealed that there were five co-expression miRNA modules (r>0.8) participated in female fracture healing. The top hub miRNAs in fracture-healing-related molecular network were all included in the two largest modules. These results implied the possibility to improve the aged female fracture healing by regulating key miRNAs at early stage.
