Emerging roles of non‐coding RNAs in scoliosis

Abstract Scoliosis, a complex three‐dimensional deformity of the spine with the Cobb angle (a measure of the spinal lateral curvature) >10 degree, encompasses a spectrum of pathologies, including congenital, idiopathic, syndromic and neuromuscular aetiologies. The pathogenesis is multifactorial involving both environmental and genetic factors but the exact cellular and molecular mechanisms of disease development remain largely unknown. Emerging evidence showed that non‐coding RNAs (ncRNAs), namely microRNAs, long ncRNAs and circular RNAs, are deregulated in many orthopaedic diseases, including scoliosis. Importantly, these deregulated ncRNAs functionally participate in the initiation and progression of scoliosis. Here, we review recent progress in ncRNA research on scoliosis.

scoliosis so as to identify novel molecular markers for early identification of patients at risk and the development of mechanism-driven therapeutics.
Non-coding RNAs (ncRNAs) are an important class of regulatory, non-protein-coding transcripts, which encompass three major subclasses, namely long ncRNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs). [17][18][19][20] While the mechanism by which miR-NAs regulate gene expression is relatively straightforward (ie, guiding the RNA-induced silencing complex to target mRNAs through base-pairing to induce their degradation and/or inhibit their translation), 21 lncRNAs and circRNAs could regulate gene expression at multiple levels (eg, DNA methylation, histone modification, recruitment of transcriptional factors, sponging of miRNAs and regulation of mRNA stability for lncRNAs; sponging of miRNAs, transcription regulation, modulation of alternative splicing, direct interactions with RNA-binding proteins and protein translation via rolling circle amplification for circRNAs). 22,23 ncRNAs are essential for the regulation and execution of key cellular processes, including proliferation, apoptosis, autophagy, differentiation, metabolism, migration, differentiation and invasion. [24][25][26][27][28][29][30] It therefore comes as no surprise that ncRNAs are deregulated in most, if not all, kinds of diseases, including neoplastic, inflammatory and metabolic diseases. [31][32][33][34][35] From a clinical perspective, the frequent alteration of ncRNA abundance in the body fluid, such as saliva, blood and urine, during diseases make this class of molecules promising candidates for development into biomarkers for early diagnosis and prognostication. [36][37][38] A growing body of evidence now suggests that deregulated expression of ncRNAs is a key to the development of orthopaedic diseases, including osteosarcoma, osteoporosis, osteoarthritis and intervertebral disc degeneration. 25,28,[39][40][41] Emerging evidence also suggests that ncRNAs are deregulated in scoliosis and functionally participate in its pathogenesis. 42,43 Here, we first summarize recent ncRNA profiling studies in scoliosis followed by discussing specific ncRNAs that are functionally important to the development of scoliosis. The potential clinical utilities of ncRNAs as molecular biomarkers and therapeutic targets for the management of scoliosis are also addressed.

| N CRNA E XPRE SS I ON PROFILING IN SCOLIOS IS
Profiling the expression of ncRNAs with whole-transcriptome sequencing, microarray or PCR array followed by confirmation with reverse transcription (RT)-quantitative PCR is the most common approach to identify and validate deregulated ncRNAs in specific disease states. 17,44,45 In this regard, multiple groups of investigators have embarked on profiling studies to depict the transcriptome-wide expression landscape of ncRNAs in scoliosis (Table 1).
Liu and colleagues performed Agilent mRNA and lncRNA human Array V3.0 microarray to identify the expression patterns of mRNA and lncRNA in peripheral blood of healthy children and AIS cases. 46 Their data demonstrated that a total of 546 mRNAs and 139 lncRNAs were differentially expressed between healthy control and AIS cases, among which NR024075 and ENST00000440778.1 were the most  Efforts have also been put forth to profile circulating miRNAs in patients with scoliosis. García-Giménez and colleagues profiled circulating miRNAs by RNA sequencing in 17 AIS patients and 10 healthy controls, followed by validation with RT-qPCR in 30 AIS patients and 13 healthy controls. 47 Their data suggested that circulating miRNAs of AIS patients exhibited differential abundance profiles as compared with healthy subjects. miR-122-5p, miR-27a-5p and miR-223-5p were found to be more abundant in patients with These profiling studies, mostly performed with peripheral blood of patients, collectively suggested that ncRNAs are deregulated in scoliosis. It is expected that the use of other tissue types, such as bone, paravertebral muscle and cartilage, collected during surgery for transcriptome-wide profiling will depict a much clearer picture of ncRNA deregulation in a cell-type-specific manner that will guide the choice of cell and animal models for subsequent functional studies.

| H19/miR-675-5p
Imbalance between two sides of paravertebral muscle is impli- imprinting control region was responsible the differential expression of H19. This study highlighted the potential involvement of the CCTF-H19/miR-675-5p-ADIPOQ axis in AIS development.

| LncAIS
Abnormal osteogenic differentiation of mesenchymal stem cells

| SULT1C2A and miR-466c-5p
Our team has shown that embryonic expression of the lncRNA SULT1C2A was decreased whereas miR-466c-5p was upregulated in the rat model of vitamin A deficiency-induced congenital scoliosis. 43 Somitogenesis-related genes, such as Foxo4, Sox9, Pax1 and Nkx3-2, were downregulated on gestational day 9 upon maternal vitamin A deficiency. In this regard, reduced expression of SULT1C2A was found

| miR-15a
Li and colleagues studied the morphological changes, histone meth-

| CON CLUS I ON AND FUTURE PER S PEC TIVE S
The cellular and molecular pathogenesis of scoliosis is complex, involves multiple aetiologies and depends on varying contributions of biomechanical, neuromuscular, developmental and biochemical abnormalities. 8,29 Among different subtypes of scoliosis, the ncRNA expression landscape has been relatively well depicted in AIS. Nevertheless, only few of the identified ncRNAs (ie miR-145, miR-675-5p, LncAIS, SULT1C2A, miR-466c-5p, miR-15a) have been functionally verified ( Figure 1 and Table 2). These ncRNAs were found to primarily affect processes that are pertinent to the development In terms of treatment, it would be tantalizing to determine whether scoliosis progression could be prevented through restoring the expression of downregulated ncRNAs or silencing of the aberrantly upregulated ncRNAs. Nevertheless, the best way to deliver ncRNA-directed therapeutics into specific tissue or cell types remain poorly defined. It is hopeful that, with more translational research in this area, ncRNAs will one day become part of our armamentarium to fight this potentially devastating disease.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest.