Multiple targets identified with genome wide profiling of small RNA and mRNA expression are linked to fracture healing in mice

Abstract

Long-bone fracture is a common injury and its healing process at the fracture site involves several overlapping phases, including inflammation, migration of mesenchymal progenitors into the fracture site, endochondral ossification, angiogenesis and finally bone remodelling. Increasing evidence shows that small noncoding RNAs are important regulators of chondrogenesis, osteogenesis and fracture healing. MicroRNAs are small single-stranded, non-coding RNA-molecules intervening in most physiological and biological processes, including fracture healing. Angiogenin-cleaved 5′ tRNA halves, also called as tiRNAs (stress-induced RNAs) have been shown to repress protein translation. In order to gain further understanding on the role of small noncoding RNAs in fracture healing, genome wide expression profiles of tiRNAs, miRNAs and mRNAs were followed up to 14 days after fracture in callus tissue of an in vivo mouse model with closed tibial fracture and, compared to intact bone and articular cartilage at 2 months of age. Total tiRNA expression level in cartilage was only approximately one third of that observed in control D0 bone. In callus tissue, 11 mature 5′end tiRNAs out of 191 tiRNAs were highly expressed, and seven of them were differentially expressed during fracture healing. When comparing the control tissues, 25 miRNAs characteristic to bone and 29 miRNAs characteristic to cartilage tissue homeostasis were identified. Further, a total of 54 out of 806 miRNAs and 5420 out of 18,700 mRNAs were differentially expressed (DE) in callus tissue during fracture healing and, in comparison to control bone. They were associated to gene ontology processes related to mesenchymal tissue development and differentiation. A total of 581 miRNA-mRNA interactions were identified for these 54 DE miRNAs by literature searches in PubMed, thereby linking by Spearman correlation analysis 14 downregulated and 28 upregulated miRNAs to 164 negatively correlating and 168 positively correlating miRNA-mRNA pairs with chondrogenic and osteogenic phases of fracture healing. These data indicated that tiRNAs and miRNAs were differentially expressed in fracture callus tissue, suggesting them important physiological functions during fracture healing. Hence, the data provided by this study may contribute to future clinical applications, such as potential use as biomarkers or as tools in the development of novel therapeutic approaches for fracture healing.

Signature of circulating small non-coding RNAs during early fracture healing in mice

“…The study plan and the use of animal material were approved by the National Animal Experiment Board ELLA (project license ESAVI/6129/04.10.03/2011). C57BL/6NHsd mice were supplied by Envigo (The Netherlands), and animals were maintained in the Central Animal Laboratory of the University of Turku, and they received a soya-free diet as previously reported ( Bourgery et al, 2021 ). Animal care was in accordance with the international guidelines following the 3Rs principles.…”

“…Standard closed fracture (age 70–74 days) was produced in mouse tibia by an impact device as previously described ( Bourgery et al, 2021 ; Hiltunen et al, 1993 ; Puolakkainen et al, 2017 ).…”

“…Changes in small RNA profiles during fracture healing were followed by collecting callus tissue samples (published in Bourgery et al, 2021 ) and blood samples at D0 (intact control tibial diaphysis) and at D1, D5, D7, D10, and D14 after fracture.…”

“…Small RNA libraries were prepared using TruSeq Small RNA Sample Preparation Kit (Illumina, USA) with multiplexing adapters, following the kit user guide (Rev. E), and sequenced by MiSeq V3 flow cell using Illumina MiSeq reagent kit V3 for 150 cycles and 36 bp reads with single-end chemistry as previously described ( Bourgery et al, 2021 ).…”

“…Further, an increasing number of 5′end tiRNAs have been associated with cell differentiation processes ( Krishna et al, 2019 ). More recently, we reported tsRNA expression in bone and callus tissues during fracture healing ( Bourgery et al, 2021 ). Currently, there is only limited data available concerning tsRNAs in circulation and no studies regarding fracture healing.…”