Circulating miRNAs as diagnostic biomarkers for adolescent idiopathic scoliosis

Abstract

The aetiology of adolescent idiopathic scoliosis (AIS) has been linked to many factors, such as asymmetric growth, neuromuscular condition, bone strength and genetic background. Recently, epigenetic factors have been proposed as contributors of AIS physiopathology, but information about the molecular mechanisms and pathways involved is scarce. Regarding epigenetic factors, microRNAs (miRNAs) are molecules that contribute to gene expression modulation by regulating important cellular pathways. We herein used Next-Generation Sequencing to discover a series of circulating miRNAs detected in the blood samples of AIS patients, which yielded a unique miRNA biomarker signature that diagnoses AIS with high sensitivity and specificity. We propose that these miRNAs participate in the epigenetic control of signalling pathways by regulating osteoblast and osteoclast differentiation, thus modulating the genetic background of AIS patients. Our study yielded two relevant results: 1) evidence for the deregulated miRNAs that participate in osteoblast/osteoclast differentiation mechanisms in AIS; 2) this miRNA-signature can be potentially used as a clinical tool for molecular AIS diagnosis. Using miRNAs as biomarkers for AIS diagnostics is especially relevant since miRNAs can serve for early diagnoses and for evaluating the positive effects of applied therapies to therefore reduce the need of high-risk surgical interventions.

Conflict of interest statement

J.L.G.G., S.M.M., D.H., T.B. and F.V.P.C. are inventors of this method with application reference: PCT/EP2016/063935. J.L.G.G. and S.M.M. are currently the C.E.O. and C.S.O. of EpiDisease S.L., respectively. EpiDisease is a Spin-Off of the Center for Biomedical Network Research on Rare Diseases (Spanish Institute of Health Instituto de Salud Carlos III), the Biomedical Research Center INCLIVA and the University of Valencia. P.R.B., L.P.C., E.M.G.L., D.G.R., J.S.I.C. and P.B.H. report no conflicts of interest.

Figures

Figure 1

Heatmap with the hierarchical clustering of differentially expressed miRNAs in AIS. Expression levels of the miRNAs selected by the random forest analysis and the Robinson and Smyth test. Raw count values were log-transformed and samples were ordered according to their corresponding group: Controls (C) or AIS patients (P).

Figure 2

Relative expression levels of the miRNAs with different representations found in the plasma of the AIS patients versus the control healthy subjects. Box plot of the relative expression levels of the miRNAs analysed by RT-qPCR, normalised to miR-191 as an endogenous control and calculated using the 2-∆∆Ct method. (a) miR-122 (Fold Change, FC > 2.65; p < 0.05; (b) miR-27a (FC > 1.87; p < 0.005); (c) miR-223 (FC > 1.5; p < 0.0001); (d) miR-1306 (FC > 1.03; p = 0.38); and (e) miR-671 (FC > 0.81; p = 0.53). Samples were ordered according to their corresponding group: controls (C) or AIS patients (P). An independent samples t-test was applied to analyse the biospecimens from the 17 healthy subjects (6 males and 11 females) and the 29 AIS patients (5 males and 24 females). A p < 0.05 was considered to indicate a significant difference.

Figure 3

Receiver operating characteristic curve analysis of the 4-miRNA signature validated by RT-qPCR for diagnosing AIS. The model uses a panel of 4-miRNA signature composed of miR-122, miR-27a, miR-223 and miR-1306, and achieved an AUC value of 0.95 (CI: 0.89-1). At a fixed specificity of 90%, the model achieved a sensitivity of 85.7%.

Figure 4

Schematic representation of the gene targets of miRNA identified by NGS in the AIS patients and their role in osteoblast and osteoclast differentiation depicted from KEGG and DIANA miRPath v3. The continuous blunt end line indicates the gene interactions with the overexpressed miRNA. The dotted grey blunt end line indicates the gene interactions with the down-regulated miRNA. Osteoblast progenitor formation is mediated by Smad cascade signalling through the activation of the TGFβ and Wnt signalling pathways. Smad become phosphorylated and migrates into the nucleus, where it induces Runx2, Dlx5 and Sp7/Osterix transcription to induce the differentiation of osteoblasts. In these intricate mechanisms, microRNAs can modulate key genes to control osteoblast and osteoclast differentiation. miR-671–5p blocks BMPR2 and Smad2/3, which are key regulators on this pathway. So the down-regulation of this miR-671 may contribute to the osteoblast differentiation pathway. mir-27a also blocks BMPRI/II and thus produces the inhibition of this pathway, which is also affected by the miR-122 interaction with BMP4. However, the dual role of miR-27a can activate osteoblast differentiation by the activation of Wnt signalling through the inhibition of APC. In such circumstances APC do not form a complex with glycogen synthase kinase 3 (GSK3B), so the levels of non-phosphorylated β-catenin increase and migrate to the nucleus, where it associates with members of the TCF/LEF transcription factors by controlling the gene transcription of RUNX2. However, osteoblast differentiation may undergo the upstream regulation of Wnt by miR-122, which alters this pathway. Finally, miR-223 may contribute to affect osteoclast -specific genes transcription and the maturation of functional osteoclasts by inhibiting NFIA, the inhibitor of osteoclast differentiation, which results in increased osteoclast activation.