Identification of Circulating miRNAs as Fracture-related Biomarkers
Elena Della Bella, Ursula Menzel, Andreas Naros, Eva Johanna Kubosch, Mauro Alini, Martin J. Stoddart
Abstract
Fracture non-unions affect many patients worldwide, however, known risk factors alone do not predict individual risk. The identification of novel biomarkers is crucial for early diagnosis and timely patient treatment. This study focused on the identification of microRNA (miRNA) related to the process of fracture healing. Serum of fracture patients and healthy volunteers was screened by RNA sequencing to identify differentially expressed miRNA at various times after injury. The results were correlated to miRNA in the conditioned medium of human bone marrow mesenchymal stromal cells (BMSCs) during in vitro osteogenic differentiation. hsa-miR-1246, hsa-miR-335-5p, and miR-193a-5p were identified both in vitro and in fracture patients and their functional role in direct BMSC osteogenic differentiation was assessed. The results showed no influence of the downregulation of the three miRNAs during in vitro osteogenesis. However, miR-1246 may be involved in cell proliferation and recruitment of progenitor cells. Further studies should be performed to assess the role of these miRNA in other processes relevant to fracture healing.
Introduction
Fracture non-unions affect many patients leading to functional and psychosocial disability [1]. Several risk factors have been described and include biological, surgical, and mechanical factors. However, none of them are able to determine if healing abnormalities will arise [1, 2]. As a consequence, there are no prognostic markers to monitor fracture healing, leaving no option but to wait until a non-union has occurred. Bone turnover markers have some utility but there are still uncertainties in their clinical use for monitoring fracture healing [3, 4]. The identification of new biomarkers, alone or in combination, revealing healing abnormalities at early stages are therefore crucial to improve non-union treatment or to prevent its occurrence.
miRNA are short nucleotide sequences [∼20–25 nt) that modulate gene expression at a post-transcriptional level, thereby regulating virtually all biological processes [5]. miRNAs are not only found in the intracellular space, but they are also secreted and can be easily detected in many biological fluids, including serum and plasma [6]. Endogenous circulating miRNA are usually protected from RNase-mediated degradation and have found to be dysregulated in a variety of pathological conditions.
Materials and methods
Fracture patients and samples
Whole blood was collected with full ethical approval and signed informed patient consent (EK-Freiburg 105/17 and ZH BASEC-Nr.2017-01390) from 10 healthy volunteers and 12 fracture patients (S1 Table). From fracture patients, blood was collected at day 3 (n = 10, "early"), between day 5 and 12 (n = 5, "late"), and between day 19 and 56 (n = 6, "very late") after fracture. After clotting, samples were centrifuged for 10 min at 2500g and 4°C. The serum was then filtered and frozen at –80°C.
Results
miRNA signature in osteogenic conditioned medium from human BMSCs
In day 7 medium, sequencing identified 74 downregulated and 46 upregulated miRNAs in osteogenic differentiation vs. undifferentiated controls (Fig 1A). Table 1 reports the 25 most significant miRNAs, while S3 Table includes all differentially expressed miRNAs. On day 7, miR-362-5p, miR-136-5p, miR-374a-3p, miR-3065-5p, miR-942-5p were not detected during osteogenic differentiation, while being detectable in undifferentiated cells with low TMM values (S3 Table). qPCR analysis confirmed the downregulation of hsa-miR-31-5p, hsa-miR-125a-5p, hsa-miR-335-5p, hsa-miR-23b-3p, hsa-miR-411-5p, hsa-miR-26b-5p, and hsa-miR-103a-3p, and the upregulation of hsa-miR-7704, hsa-miR-193a-5p, and hsa-miR-423-3p (Fig 1B).
Discussion
The identification of biomarkers to stratify patients’ non-union risk is crucial to allow early intervention. Blood-based biomarkers would be ideal since they can be obtained following a minimally invasive procedure and can be easily combined with the analysis of other biochemical parameters. To be robust, the marker must be present in a broad range of patients, for this reason we utilized an unmatched patient population. Despite this, we successfully identified potential markers and more clearly defined the time period when sample present the greatest number of miRNA changes. This study identified miRNAs in the serum of fracture patients or secreted during in vitro osteogenic differentiation that may play a physiological role in fracture healing. Within the patients’ serum, several miRNAs were regulated within the first 12 days post-injury, and this number was reduced from 19 days onwards. It is likely that the release of fracture-related miRNA into the bloodstream is reduced once the vasculature has recovered and maintains its integrity. miRNA changes during in vitro osteogenic differentiation were also more substantial at early timepoints. Taken together, the identification of early markers of fracture appears feasible. Three miRNAs were selected for functional validation during in vitro BMSCs osteogenic differentiation. However, the inhibition of these miRNAs at the start of differentiation did not seemingly affect this process.
Conclusions
miR-1246, miR-335-5p, and miR-193a-5p can be involved in fracture healing, though they do not have a role in direct osteogenesis of human BMSCs in vitro. However, direct ossification is not the only or the main mechanism going on during fracture healing. Therefore, it is possible that those miRNAs can be involved in other key processes such as cell recruitment, vascularization, or endochondral ossification. miR-1246 seems the most promising to be further characterized, as it is mechanosensitive, promotes proliferation and its expression can be regulated by key chemokines involved in MSC recruitment and bone formation.
Citation: Della Bella E, Menzel U, Naros A, Kubosch EJ, Alini M, Stoddart MJ (2024) Identification of circulating miRNAs as fracture-related biomarkers. PLoS ONE 19(5): e0303035. https://doi.org/10.1371/journal.pone.0303035
Editor: Carlos Alberto Antunes Viegas, Universidade de Trás-os-Montes e Alto Douro: Universidade de Tras-os-Montes e Alto Douro, PORTUGAL
Received: May 12, 2023; Accepted: April 16, 2024; Published: May 31, 2024
Copyright: © 2024 Bella et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Sequencing data deposited in NCBI’s Gene Expression Omnibus with accession number GSE217879. The minimal underlying data set is available as supplementary file.
Funding: MJS and EDB obtained funding from AO Foundation and AO Trauma. https://www.aofoundation.org/what-we-do/research-innovation/about https://www.aofoundation.org/trauma The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0303035#abstract0
