Circular RNAs in nucleus pulposus cell function and intervertebral disc degeneration

Abstract Intervertebral disc degeneration (IDD) is a common cause of low back pain, which inflicts more global disability than any other condition. Although IDD was deemed to be a natural process that comes with ageing, a growing body of evidence suggested that both genetic and environmental factors could modify the development of IDD. In this connection, aberrant function of nucleus pulposus cells has been implicated in IDD pathogenesis. Circular RNAs are a novel class of endogenous non‐coding RNAs that play crucial regulatory roles in diverse cellular processes. Recently, deregulation of circRNAs in nucleus pulposus cells was found to functionally participate in IDD development. In this review, we summarize the current knowledge regarding the deregulation of circRNAs in IDD in relation to their actions on nucleus pulposus cell functions, including cell proliferation, apoptosis and extracellular matrix synthesis/degradation. The potential clinical utilities of circRNAs as therapeutic targets for the management of IDD are also discussed.


| INTRODUC TI ON
Low back pain is an extremely common condition with an estimated global point prevalence of ~10%, causing distress and economic losses due to pain, activity limitation and work absence. It also ranks highest in terms of overall disability among 291 conditions studied in the Global Burden of Disease 2010 Study. [1][2][3] Intervertebral disc degeneration (IDD) of the lumbar spine, which is characterized by progressive structural failure and advanced signs of ageing of the intervertebral disc, is strongly associated with an increased risk for low back pain. 4 Both genetic (eg polymorphisms in genes encoding collagen I, IX, XI and aggrecan) and lifestyle (eg the lack of sports activities and night shift work) factors have been linked to IDD development. 5,6 However, the exact cellular and molecular mechanisms underlying IDD remain largely elusive. Anatomically, the intervertebral disc consists of a gelatinous core known as nucleus pulposus (NP) surrounded by a lamella of fibrous cartilage termed annulus fibrosus. 7,8

Funding information
Our study was supported by National Natural Science Foundation of China (81871829).

Abstract
Intervertebral disc degeneration (IDD) is a common cause of low back pain, which inflicts more global disability than any other condition. Although IDD was deemed to be a natural process that comes with ageing, a growing body of evidence suggested that both genetic and environmental factors could modify the development of IDD.
In this connection, aberrant function of nucleus pulposus cells has been implicated in IDD pathogenesis. Circular RNAs are a novel class of endogenous non-coding RNAs that play crucial regulatory roles in diverse cellular processes. Recently, deregulation of circRNAs in nucleus pulposus cells was found to functionally participate in IDD development. In this review, we summarize the current knowledge regarding the deregulation of circRNAs in IDD in relation to their actions on nucleus pulposus cell functions, including cell proliferation, apoptosis and extracellular matrix synthesis/ degradation. The potential clinical utilities of circRNAs as therapeutic targets for the management of IDD are also discussed. integrity of intervertebral discs via producing extracellular matrix (ECM) components, such as aggrecan as well as type II and type X collagen. 9,10 A growing body of evidence now suggests that aberrant NP cell functions, including altered cell proliferation, apoptosis, ECM production/degradation and cytokine secretion, are key to IDD pathogenesis. 11,12 Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long ncRNAs (lncRNAs) and the recently discovered circular RNAs (cir-cRNAs), are important regulatory elements encoded by the genome and play critical roles in diverse cellular processes. 13,14 CircRNAs are a type of single-stranded RNAs that form covalently closed loops that primarily serve as competing endogenous RNAs (ceRNAs) to sponge miRNAs. A subset of circRNAs have also been reported to exert their biological functions via transcription regulation, modulation of alternative splicing, direct interactions with RNA-binding proteins and protein translation via rolling circle amplification. [15][16][17] Concerning transcriptional regulation, a class of circRNAs known as exon-intron circRNAs characterized by circularized exons with introns retained in between has been identified. This class of cir-cRNAs was found to exhibit nuclear localization, interacts with the U1 small nuclear ribonucleoprotein and enhances transcription of their parental genes in a cis-acting manner. 18 An accumulating number of studies have demonstrated that circRNAs play pivotal roles in cellular functions such as proliferation, differentiation, invasion, migration and metabolism, 19 and their deregulation has been documented in different types of diseases, including cancers, neurodegeneration, inflammatory/immune diseases and orthopaedic conditions, such as scoliosis, osteoporosis and osteoarthritis. [20][21][22][23][24][25] Recently, it has been reported that circRNAs are involved in the development of IDD. 26 In terms of translational value, intracellular circRNAs are insensitive to exonuclease in contrast with other classes of linear RNAs, 27 rendering circRNAs suitable for further development into tissue biomarkers owing to their high intracellular stability.
This review serves to provide an overview of the current understanding regarding the functional roles of deregulated circRNAs in modulating phenotypes that are pertinent to IDD pathogenesis, including NP cell proliferation, apoptosis, ECM synthesis/degradation and pro-inflammatory cytokine production. We also discussed the translational value of circRNAs in terms of their clinical utilities as therapeutic targets for the management of IDD.

| CIRCRNA S E XPRE SS I ON PROFILING IN IDD
Circular RNAs expression profiling with transcriptome sequencing, microarray or PCR array followed by validation with reverse transcription-quantitative PCR (RT-qPCR) is the most frequently adopted approach to identify and confirm the differential expression of circRNAs in specific disease states. 20,28,29 Profiling studies of circRNAs in IDD are listed in Table 1.
Microarray-based profiling of circRNAs deregulated in IDD was first performed by Liu and colleagues in which they conducted a comprehensive profiling of mRNAs, miRNAs, lncRNAs and circRNAs in five normal discs from cadaveric donors versus five degenerative discs from patients with IDD. The microarray data were then deposited in a public database. 30 Lan and colleagues conducted an integrative analysis on these datasets with an aim to comprehensively depict the RNA landscape in human IDD. 30 Among 2894 annotated circRNAs, 636 circRNAs were found to be differentially expressed (354 upregulated and 282 downregulated) in the degenerated discs as compared with normal control discs. Notably, a single miRNA was predicted to interact with a multitude of circRNAs. The upregulation of circRNA-101852 and downregulation of circRNA-101645 were confirmed by RT-qPCR. 31 Zou and colleagues also utilized the same microarray datasets and identified a total of 76 pairs of differentially expressed circRNAs and their host genes in IDD. Pathway analysis revealed that host genes that encode the upregulated and downregulated circRNAs in IDD are involved in signalling pathways such as Wnt and integrin signalling. Significant upregulation of circ_0008305 and downregulation of circ_0041946 were confirmed in an independent set of human lumbar NP specimens by RT-qPCR. 32 Another follow-up bioinformatic study by Zhang and colleagues using these deposited datasets identified 568 mRNAs, 55 miRNAs, 765 lncRNAs and 586 circRNAs that were significantly differentially expressed in degenerative discs than in normal discs.
The authors then reconstructed the ceRNA networks in which three circRNAs, namely circ_0005139, circ_0037858 and circ_0087890, were predicted to be key regulators in IDD progression. The deferentially expressed circRNA circ_0000189 was also predicted to play a central role and have crosstalk with miR-486-5p, the ln-cRNA DANCR and 6 mRNAs-PYCR2 (Pyrroline-5-Carboxylate Reductase 2), TOB1 (Transducer of ERBB2-1), ARHGAP5 (Rho  The recent advancements in circRNA enrichment and bioinformatic workflows for next-generation sequencing have facilitated the use of this unbiased genome-wide approach for the identification of circRNAs. Bioinformatic tools used for predicting and identifying circRNAs have been extensively reviewed by Kulcheski and colleagues. 37 In this regard, thousands of loci in the human and mouse genomes have been identified to code for circRNAs, which could account for 1% as many molecules as poly(A) RNA. CircRNA isoform splice site selection, the circular to linear isoform ratios, and N 6 -methyladenosine (m6A) modification of circRNA were all found to be cell type-specific. 38,39 It is hopeful that further data mining from existing circRNA microarray datasets together with the application of novel circRNA sequencing approaches could facilitate the identification of key pathogenic circRNAs and potential therapeutic targets in IDD.

| Circ-GRB10
Guo and colleagues reanalysed Liu et al's 30   pathway was involved in IDD development. 44 Restoring the expression of circ-GRB10 or enhancing the ERBB2 signalling might thus serve a new therapeutic approach for the IDD.