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. 2020 Oct 28;7:583243. doi: 10.3389/fmed.2020.583243

In silico Analysis Excavates A Novel Competing Endogenous RNA Subnetwork in Adolescent Idiopathic Scoliosis

Hui-Min Li 1,†, Yi Liu 2,†, Jing-Yu Ding 1,†, Renjie Zhang 1,†, Xiao-Ying Liu 3,*, Cai-Liang Shen 1,*
PMCID: PMC7655901  PMID: 33195333

Abstract

Background and Objective: Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional deformity of the spine. Mesenchymal stem cells (MSCs) regulate bone mass homeostasis in AIS, which might be related to the pathogenesis of AIS. However, the mRNA–miRNA–lncRNA network linked to the regulation of the genetic pathogenesis of MSCs remains unknown.

Methods: We conducted an exhaustive literature search of PubMed, EMBASE, and the Gene Expression Omnibus database to find differentially expressed genes (DEGs), differentially expressed miRNAs (DE miRNAs), and differentially expressed lncRNAs (DE lncRNAs). Functional enrichment analysis was performed through Enrichr database. Protein–protein interaction (PPI) network was constructed using STRING database, and hub genes were identified by CytoHubba. Potential regulatory miRNAs and lncRNAs of mRNAs were predicted by miRTarBase and RNA22, respectively.

Results: We identified 551 upregulated and 476 downregulated genes, 42 upregulated and 12 downregulated miRNAs, and 345 upregulated and 313 downregulated lncRNAs as DEGs, DE miRNAs, and DE lncRNAs, respectively. Functional enrichment analysis revealed that they were significantly enriched in protein deglutamylation and regulation of endoplasmic reticulum unfolded protein response. According to node degree, one upregulated hub gene and eight downregulated hub genes were identified. After drawing the Venn diagrams and matching to Cytoscape, an mRNA–miRNA–lncRNA network linked to the pathogenesis of MSCs in AIS was constructed.

Conclusion: We established a novel triple regulatory network of mRNA–miRNA–lncRNA ceRNA, among which all RNAs may be utilized as the pathogenesis biomarker of MSCs in AIS.

Keywords: adolescent idiopathic scoliosis, bioinformatics, Competing endogenous RNA (CeRNA), hub genes, lncRNAs, miRNAs

Introduction

Adolescent idiopathic scoliosis (AIS) is characterized by a complex 3D deformity of the spine, but its etiology and pathogenesis remain unclear (1–3). Abnormal skeletal growth and low bone mass are factors that may be associated with disturbed bone metabolism existing in patients with AIS (4–6). Recent studies have suggested that mesenchymal stem cells (MSCs) play a crucial role in bone mass homeostasis in AIS; however, the regulation of the genetic pathogenesis of MSCs remains yet to be elucidated (7, 8).

MicroRNAs (miRNAs) are a group of endogenous small single-stranded non-coding RNAs with a length of ~21–25 nucleotides (9). miRNAs can negatively regulate gene expression by binding to the 3′-untranslated region of messenger RNA (mRNA), leading to direct mRNA degradation or protein translation inhibition. Thus, miRNAs are involved in the regulation of many biological processes, such as proliferation, apoptosis, cell cycle, differentiation, and DNA repair (10). Competing endogenous RNA (ceRNA) hypothesis indicates that cross-talk between non-coding RNA (ncRNA) and mRNA is achieved by competitively binding to shared miRNAs (11, 12). Long non-coding RNAs (lncRNAs) are a class of ncRNA, which can decrease miRNA abundance as miRNA sponges and increase the expression of downstream target mRNAs of miRNAs (13, 14). Increasing evidence indicates that miRNAs and lncRNAs have an effect on the osteoblastic differentiation of MSCs (15–17). However, current knowledge of mRNA–miRNA–lncRNA in MSCs of AIS is limited. The aim of the present study is to explore and construct a key mRNA–miRNA–lncRNA ceRNA triple subnetwork in AIS based on comprehensive bioinformatics analysis.

In this study, we acquired differentially expressed genes (DEGs), differentially expressed miRNAs (DE miRNAs), and differentially expressed lncRNAs (DE lncRNAs) by mining previously published datasets and studies. The DEGs were analyzed by gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Then, a protein–protein interaction (PPI) network was established to identify hub genes, and potential regulatory miRNAs were predicted. The key DE miRNAs were identified by drawing Venn diagrams, and the functions of the key DE miRNAs were also determined. Subsequently, potential regulatory lncRNAs were predicted, and the key DE lncRNAs were identified using Venn diagrams. A novel mRNA–miRNA–lncRNA regulatory network associated with the pathogenesis of patients with AIS was successfully established.

Materials and Methods

Search Literature and Microarray Assay

We conducted an exhaustive literature search of PubMed, EMBASE, and the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) for studies that compared the gene or miRNA or lncRNA expression in bone marrow MSCs from patients with AIS and those from healthy individuals. Studies were selected and excluded based on the following criteria: (1) The study population consisted of patients with AIS who underwent a comprehensive clinical and radiological examination to rule out other causes of scoliosis and to determine the diagnosis of AIS. (2) In the non-AIS patients' group, each of the subjects had a straight spine and normal forward bending test on physical examination, determined not to have any associated medical conditions or spinal deformity. (3) Bone marrow aspirates were obtained from both groups. (4) Studies containing more than five AIS samples and five normal samples were included, whereas studies based on animal models were excluded. The titles and abstracts of these studies were screened, and information on the studies of interest was further evaluated. The search terms included “adolescent idiopathic scoliosis,” “AIS,” “mesenchymal stem cells,” and “MSCs.” We did not limit the languages or publication date.

Differential Expression Analysis

DEGs and DE miRNAs were obtained through literature search of previous studies (8, 18). Gene reannotation was performed, and “Limma” R package was used for differential expression analysis of lncRNAs. |FoldChange| > 1 and p < 0.05 were set as the cut-off criteria.

Functional Analysis

The GO and KEGG pathways of DEGs were analyzed based on the Enrichr database (http://amp.pharm.mssm.edu/Enrichr/) by performing functional annotation (biological process, BP, molecular function, MF, and cellular component, CC) and pathway enrichment analysis. p < 0.05 was considered statistically significant (19, 20). Then, the top 10 enriched GO terms and KEGG pathways were downloaded from the webpage. The functions of key DE miRNAs were analyzed through TAM 2.0 tools (21, 22) by using the hypergeometric test to evaluate the overexpression of each miRNA set (257 sets) in the “miRNA list.”

PPI Network

The potential PPI relationship of DEGs was mapped to the Search Tool for the Retrieval of Interacting Genes (STRING) database (http://string-db.org/), which is designed to analyze PPI information (23). PPI node pairs with a medium combined score ≥0.4 were selected for further analysis. The PPI network was then visualized using the Cytoscape software (version 3.7.2, www.cytoscape.org/). Nodes with a high degree of connectivity are essential in maintaining the stability of the entire network (24). CytoHubba is a plugin in Cytoscape that calculates the degree of each protein node. In our study, the top 30 hub genes were visualized in Cytoscape, and the top 10 hub genes were chosen for subsequent analysis.

Prediction and Validation of miRNA and Construction of the mRNA–miRNA Network

The miRTarbase database, which collects experimentally validated microRNA-target interactions by a reporter assay, Western blot analysis, qPCR, microarray, and next-generation sequencing experiments, was used to predict the potential miRNAs binding to hub genes (25). Then, the common DE miRNAs between predicted and previous DE miRNAs were identified as the key DE miRNAs by drawing Venn diagrams. The network of key genes and miRNA was constructed through the Cytoscape software.

Prediction and Validation of lncRNA and Construction of the mRNA–miRNA–lncRNA Network

The RNA22 tool was used to predict the potential lncRNAs binding to previously identified commonly DE miRNAs (26). Then, the commonly DE lncRNAs between predicted and previously DE lncRNAs were identified as the key DE lncRNAs by drawing Venn diagrams. The ceRNA network was constructed through the Cytoscape software.

Results

Study Search and Identification of DEGs, DE miRNAs, and DE lncRNAs

A summary of the study selection process is shown in Figure 1A. A total of 102 relevant studies were inspected via electronic search. A total of 27 duplicate studies were excluded. After the titles and abstracts were assessed, 51 studies that did not meet the eligibility criteria were eliminated. After the full text of the remaining 24 studies was verified, only three studies were included (8, 17, 18). The gene dataset was performed on the Affymetrix Gene Chip Human Transcript 2.0 arrays containing ten human AIS samples and five non-AIS samples and as did the miRNA dataset. The lncRNA dataset GSE110359 was also analyzed on the Affymetrix Gene Chip Human Transcript 2.0 arrays containing 12 human AIS samples and five non-AIS samples. Gene, miRNA, lncRNA datasets of the AIS group and the control group were from three different cohorts.

Figure 1.

Figure 1

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(A) Flow diagram of study selection. (B) Volcano plot of the DE lncRNAs. The black dots represent lncRNAs that are not differentially expressed, and the red dots and green dots represent the upregulated and downregulated lncRNAs, respectively.

A total of 1,027 genes (551 upregulated and 476 downregulated genes) and 54 miRNAs (42 upregulated and 12 downregulated miRNAs) were extracted from the included studies (8, 18) and identified as DEGs and DE miRNAs. Based on differential expression analysis, 658 lncRNAs (345 upregulated and 313 downregulated lncRNAs) were identified as DE lncRNAs, and the volcano plots of these DE lncRNAs is shown in Figure 1B.

GO and KEGG Analyses of DEGs

As shown in Figures 2A1–A3, the upregulated genes between AIS and normal samples are significantly enriched in protein deglutamylation, negative regulation of mitotic cell cycle, and positive regulation of nuclear-transcribed mRNA poly (A) tail shortening in the BP category, cullin-RING ubiquitin ligase complex, SWI/SNF complex, and mitotic spindle in the CC category and in transcription coactivator activity, syntaxin binding, and metallocarboxypeptidase activity in the MF category. KEGG enrichment analysis for these DEGs revealed that the thyroid hormone signaling pathway, p53 signaling pathway, and pancreatic cancer are significantly enriched pathways (Figure 3A).

Figure 2.

Figure 2

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GO functional annotation for the significant DEGs. (A1) The top 10 enriched BP of the upregulated significant DEGs. (A2) The top 10 enriched CC of the upregulated significant DEGs. (A3) The top 10 enriched MF of the upregulated significant DEGs. (B1) The top 10 enriched BP of the downregulated significant DEGs. (B2) The top 10 enriched CC of the downregulated significant DEGs. (B3) The top 10 enriched MF of the downregulated significant DEGs. Ranking by combined score. Red color indicates p < 0.05 in BP; Pink color indicates p < 0.05 in CC; Jacinth color indicates p < 0.05 in MF.

Figure 3.

Figure 3

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KEGG pathway enrichment analysis for the significant DEGs. (A) Upregulated significant DEGs. (B) Downregulated significant DEGs. Ranking by combined score. Blue color indicates p < 0.05; Gray color indicates p ≥ 0.05.

As shown in Figures 2B1–B3, downregulated genes between AIS and normal samples were significantly enriched in regulation of endoplasmic reticulum unfolded protein response, glycolytic process, and oligosaccharide-lipid intermediate biosynthetic process in the BP category; endoplasmic reticulum lumen, integral component of endoplasmic reticulum membrane, and clathrin-coated vesicle membrane in the CC category; and MAP kinase phosphatase activity, dolichyl-diphosphooligosaccharide-protein glycotransferase activity, and D-glucose transmembrane transporter activity in the MF category. KEGG enrichment analysis for these DEGs revealed that N-glycan biosynthesis, antigen processing and presentation, and complement and coagulation cascades were significantly enriched pathways (Figure 3B).

PPI Network Construction and Hub Gene Identification

Protein interactions among the DEGs were predicted with STRING tools. A total of 403 nodes and 537 edges were involved in the PPI network (PPI enrichment p < 7.47e−08) of upregulated DEGs, as shown in Figure 4A. The top 30 upregulated genes are shown in Figure 4B and Table 1. A total of 437 nodes and 739 edges were involved in the PPI network (PPI enrichment p < 1.0e−16) of downregulated DEGs, as presented in Figure 4C, and the top 30 downregulated genes are shown in Figure 4D and Table 1. The top 10 upregulated hub genes included CREBBP, WDTC1, KAT2B, PRKACG, TP53BP1, UBXN7, SMARCC2, NCOA1, SMAD3, and NCOA2. The top ten downregulated hub genes included HSPA5, CYCS, PDIA3, KDR, PGK1, PDIA6, SERPINE1, PPIB, CKAP4, and TGOLN2. Those 20 hub genes were chosen for the following analyses.

Figure 4.

Figure 4

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(A) PPI network of the significant upregulated DEGs. (B) The top 30 hub genes of the significant upregulated DEGs. (C) The PPI network of the significant downregulated DEGs. (D) The top 30 hub genes of the significant downregulated DEGs.

Table 1.

The top 30 hub genes in PPI networks.

Upregulated gene Degree* Downregulated gene Degree*
CREBBP 33 HSPA5 31
WDTC1 23 CYCS 27
KAT2B 20 PDIA3 19
PRKACG 18 KDR 16
TP53BP1 16 PGK1 16
UBXN7 16 PDIA6 16
SMARCC2 16 SERPINE1 16
NCOA1 15 PPIB 16
SMAD3 13 CKAP4 15
NCOA2 12 TGOLN2 15
NF1 11 SEC61A1 15
PRKG1 11 RPN1 15
H2AFJ 11 CDKN2A 14
COMMD3 10 RPN2 14
POLR2G 10 HK2 13
ERBB2 10 TF 13
FBXL7 9 STT3A 13
FBXL20 9 LDHA 12
FBXO32 9 SLC2A3 12
YAP1 9 LAMB1 12
DDX17 9 SLC2A1 11
KMT2A 9 AGTR1 11
RARG 9 A2M 11
AGO1 9 TPM3 11
DDB2 9 NIP7 11
CHD3 9 RPF2 11
PRKAR2B 9 LAMC1 11
SMARCA2 9 GOLPH3 10
HECW2 8 CA9 10
CUL4B 8 FGF23 10
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*

Degree indicates connectivity.

Prediction and Validation of Potential Key miRNAs of Key Genes

The potential miRNAs binding to the 20 key genes were predicted through the miRTarBase, and a total of 241 miRNAs were identified for upregulated DEGs. After Venn diagrams were constructed with DE miRNAs, one downregulated miRNA that could potentially regulate one key gene (WDTC1) expression was identified as presented in Figure 5A. Potential miRNAs binding to the nine key upregulated genes (CREBBP, KAT2B, PRKACG, TP53BP1, UBXN7, SMARCC2, NCOA1, SMAD3, and NCOA2) were not observed. For downregulated hub genes, a total of 439 miRNAs were identified. After the construction of Venn diagrams with DE miRNAs, we identified 18 upregulated miRNAs that could potentially regulate eight key gene (HSPA5, CYCS, KDR, PGK1, PDIA6, PPIB, CKAP4, and TGOLN2) expression as presented in Figure 5B. Potential miRNAs binding to two key downregulated genes (PDIA3 and SERPINE1) were not observed.

Figure 5.

Figure 5

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(A) Identification of key downregulated miRNAs by combining prediction and experiment analyses. (B) Identification of key unregulated miRNAs by combining prediction and experiment analyses. (C) Construction of the miRNA–gene network.

Construction of the Key mRNA–miRNA Network in AIS

Based on the classical inverse relationship between miRNA and target gene, the 18 potential upregulated miRNAs binding to eight downregulated genes (HSPA5, CYCS, KDR, PGK1, PDIA6, PPIB, CKAP4, and TGOLN2) were identified, and only one potential downregulated miRNA binding to an upregulated gene (WDTC1) was identified. The network (miR-93-3p/miR-16-5p/miR-548x-3p - HSPA5, miR-16-5p/miR-17-5p/miR-93-5p/miR-106a-5p/miR-197-3p/miR-106b-5p/miR-21-3p/miR-93-3p/miR-3918 - CYCS, miR-16-5p - KDR, miR-324-5p/miR-18a-3p/miR-125a-3p/miR-548x-3p - PGK1, miR-15a-5p/miR-16-5p/miR-181b-5p/miR-548ac - PDIA6, miR-92a-3p - PPIB, miR-615-3p - CKAP4, miR-16-5p/miR-17-5p/miR-93-5p/miR-106b-5p - TGOLN2, miR-4419b - WDTC1) of key mRNA and miRNA is shown in Figure 5C.

Functional Analysis of Key miRNAs

Functional enrichment analysis for these miRNAs revealed that aging, immune system, and immune response are significantly enriched pathways (Figure 6).

Figure 6.

Figure 6

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Functional enrichment analysis for the key miRNAs. Ranking by –log 10 (p-value).

Prediction and Validation of Potential Key lncRNAs Binding to Key miRNAs

Given that no direct database can accommodate queries on the pairing relationship between lncRNAs and miRNAs in large quantities, RNA22 was used to predict the potential lncRNAs binding to the 19 key miRNAs, and a total of 6,293 lncRNAs were identified for downregulated miRNAs. After Venn diagrams with previous DE lncRNAs were constructed, 15 lncRNAs that could potentially regulate one key downregulated miRNA (miR-4419b) expression was identified as presented in Figure 7A. A total of 12,065 lncRNAs were identified for upregulated miRNAs. After Venn diagrams with previous DE lncRNAs were constructed, 41 lncRNAs that could potentially regulate the expression of 17 key miRNAs (miR-15a-5p, miR-16-5p, miR-17-5p, miR-92a-3p, miR-93-5p, miR-106a-5p, miR-197-3p, miR-181b-5p, miR-106b-5p, miR-324-5p, miR-18a-3p, miR-615-3p, miR-21-3p, miR-93-3p, miR-125a-3p, miR-3918, and miR-548x-3p) were identified as shown in Figure 7B. Potential lncRNAs binding to one key upregulated miRNA (miR-548ac) were not observed.

Figure 7.

Figure 7

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Screening the key lncRNAs in AIS. (A) Identification of key upregulated miRNAs by combining prediction and experiment analyses. (B) Identification of key downregulated miRNAs by combining prediction and experiment analyses.

Construction of Key mRNA–miRNA–lncRNA Triple Subnetwork in AIS

Based on the aforementioned analysis, an mRNA–miRNA–lncRNA network was constructed using Cytoscape. The triple subnetwork of upregulated and downregulated hub genes is shown in Figures 8, 9. The detailed information of subnetworks is listed in Supplementary Tables 1, 2. Each of the 18 miRNAs (miR-15a-5p, miR-16-5p, miR-17-5p, miR-92a-3p, miR-93-5p, miR-106a-5p, miR-197-3p, miR-181b-5p, miR-106b-5p, miR-324-5p, miR-18a-3p, miR-615-3p, miR-21-3p, miR-93-3p, miR-125a-3p, miR-3918, miR-548x-3p, and miR-4419b) was regulated by lncRNAs, which then regulated the target genes of miRNAs. The differential expression level of lncRNA RAP2C-AS1 was the highest in the network regulating miR-4419b, and other certain lncRNAs (HCG18 and TSPEAR-AS2) and miRNAs (miR-16-5p, miR-93-3p, and miR-93-5p) may play important roles in the ceRNA network as they regulate the majority of the downregulated hub genes. Therefore, the RAP2C-AS1 - miR-4419b - WDTC1, TSPEAR-AS2 - miR-16-5p - CYCS/KDR/PDIA6/TGOLN2/HSPA5, TSPEAR-AS2/HCG18 - miR-93-3p - CYCS/HSPA5, TSPEAR-AS2/HCG18 - miR-93-5p - CYCS/ TGOLN2, TSPEAR-AS2/HCG18 - miR-615-3p - CKAP4, and TSPEAR-AS2/HCG18 - miR-125a-3p - PGK1 axes may play critical roles in the network (Figure 10).

Figure 8.

Figure 8

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Novel upregulated key mRNA's competing endogenous RNA (ceRNA) triple regulatory network. The green rectangle in the network represents downregulated miRNAs. The red ellipse in the network represents upregulated hub genes. The red V in the network represents upregulated lncRNAs.

Figure 9.

Figure 9

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Novel downregulated key mRNAs' ceRNA triple regulatory network. The red rectangle in the network represents upregulated miRNAs. The green ellipse in the network represents downregulated hub genes. The green V in the network represents downregulated lncRNAs.

Figure 10.

Figure 10

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Critical ceRNA triple regulatory network. The rectangle in the network represents miRNAs. The ellipse in the network represents hub genes. The diamond in the network represents lncRNAs. Green represents downregulation, while red represents upregulation.

Discussion

This study was designed to investigate the specific ceRNA network based on the “mRNA–miRNA–lncRNA” order pattern. A total of 1,027 significant DEGs, consisting of 551 upregulated and 476 downregulated DEGs were extracted from the included studies. These genes are involved in multiple biological processes, including protein deglutamylation, small GTPase-mediated signal transduction, phosphatidylinositol 3-kinase signaling, RNA polymerase II transcription coactivator activity, MAP kinase phosphatase activity, negative regulation of the mitotic cell cycle, regulation of endoplasmic reticulum-unfolded protein response, glycolytic process, and positive regulation of the nuclear-transcribed mRNA poly (A) tail shortening. Although Zhuang et al. (8) used a different functional annotation database, they found that small GTPase-mediated signal transduction, phosphatidylinositol 3-kinase signaling, RNA polymerase II transcription coactivator activity, and MAP kinase phosphatase activity were enriched in GO analysis of DEGs. Elevated levels of the small GTPase Cdc42 could induce senescence in male rat MSCs (27). Domingues et al. (28) found that Cofilin-1 levels were elevated in human mesenchymal stem/stromal cells cultured on soft substrates, which could promote Cofilin-1-dependent increased RNA transcription and faster RNA polymerase II-mediated transcription elongation. It has been reported that miR-16 regulated MgCl2-induced promotion of osteogenic differentiation of MSCs by targeting FGF2-mediated activation of the ERK/MAPK pathway (29). Meanwhile, the pathway enrichment analysis of DEGs and key miRNAs revealed the presence of multiple enriched pathways, including the thyroid hormone signaling pathway, p53 signaling pathway, aging, regulation of Akt pathway, and apoptosis. Yoon et al. (30) have found that NRF2 could determine the self-renewal and osteogenic differentiation potential of human MSCs via the P53-SIRT1 axis. Other teams reported that PI3K/Akt signaling is the main contributor to MSC proliferation in response to PDGFRβ activation and Erk activation by PDGFRβ signaling, potently inhibiting the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression (31). Through the activation of the AKT/glycogen synthase kinase signaling pathway and suppression of apoptosis, PBX homeobox 1 enhances hair follicle MSC proliferation and reprogramming. Thus, these significant DEGs may be involved in the molecular pathogenesis of AIS initiation and development.

To analyze the relationships and functions of significant DEGs, we obtained PPI networks from STRING database and identified one upregulated hub gene (WDTC1) and eight downregulated hub genes (HSPA5, CYCS, KDR, PGK1, PDIA6, PPIB, CKAP4, and TGOLN2) that may be related to the pathogenesis of AIS. Most of these key genes have been well-investigated in MSCs. For example, WDTC1 is a conserved and single-copy gene in humans (32) and studies have linked the function of WDTC1 to the negative regulation of adipogenesis (33, 34). Besides, Liang et al. have reported that the adipogenic ability of MSCs from AIS girls was lower than that of controls (35). HSPA5 encodes heat shock protein 70 (HSP70), which was involved in proliferation, differentiation, and apoptosis of MSCs (36, 37). CYCS positively regulates MSC apoptosis (16) and is involved in neuronal developmental disorders, amyotrophic lateral sclerosis, and neuron death (38). KDR, also known as VEGF receptor 2, is a critical regulator of MSC osteogenesis following the activation of the ERK/RUNX2 signaling pathway (39). PGK1 overexpression may induce osteoblastic differentiation of bone marrow stromal cells and inhibit osteoclastogenesis (40). These publications partially support the accuracy of potential core genes found in our bioinformatics analyses, while the function of these core genes in MSCs of AIS requires further research.

MiRNAs and lncRNAs are involved in regulating gene expression and function through the mechanism of ceRNA. Previous studies have shown that some miRNAs binding to the key genes that we screened were identical to our analytic results. For example, miR-17-5p and miR-106a upregulation could promote adipogenesis and inhibit osteogenesis by targeting BMP2 in the modulation of human adipose-derived MSCs (41). By targeting Smad5, miR-17-5p and miR-106b-5p could suppress the osteogenic differentiation of C2C12 cells and inhibit bone formation (42). miR-16-5p inhibits osteoclastogenesis in giant cell tumor of bone via the direct inhibition of receptor activator of nuclear factor-κB ligand- (RANKL-) (15). Downregulation of miRNA-16-5p could accelerate fracture healing by negatively regulating Bcl-2 and Cyclin-D1 expression in MC3T3-E1 cells (43). miR-93-5p suppresses the osteogenic differentiation of mouse MSCs (C3H10T1/2) by targeting Smad5 (44). In trauma-induced osteonecrosis of patients with femoral head fractures, increased miRNA-93-5p inhibits osteogenic differentiation by targeting bone morphogenetic protein-2 (45). Considering that the ceRNA hypothesis indicates that cross-talk between lncRNAs and mRNAs is achieved through competitive binding to shared miRNAs, 56 potential key lncRNAs binding to these key miRNAs were identified. However, studies on the role of lncRNA of AIS are few. Only the group of Zhuang has found a novel lncAIS (gene symbol: ENST00000453347) that could suppress osteogenic differentiation of MSCs in AIS (17). Upregulation of BDNF-AS promotes bone marrow-derived MSCs' self-proliferation but inhibits osteogenic differentiation through BDNF regulation (46); BDNF-AS expression was also upregulated in this study. A pathogenesis-associated mRNA–miRNA–lncRNA network in AIS was successfully established, and such a network has been identified in previous literature on osteogenic differentiation. For example, downregulated FAM83H-AS1 modulates the SpA-inhibited osteogenic differentiation in human bone MSCs by FAM83H-AS1/miR-541-3p/WNT3A axis (47). lncRNA MSC-AS1 could promote osteogenic differentiation of bone marrow-derived MSCs through sponging miRNA-140-5p to upregulate BMP2 (48). Collectively, although a series of bioinformatics analyses has yielded some intriguing findings in our current study, further laboratory experiments and large-scale clinical trials are needed in the future. Besides, the artificial selection of ten upregulated and downregulated genes may result in some risk of selection bias.

Conclusion

In summary, through integrated bioinformatics analysis and exhaustive literature search, we constructed a novel triple regulatory network of mRNA–miRNA-lncRNA ceRNA, in which all RNAs have significant predictive value for the pathogenesis of MSCs in AIS. We also identified that the RAP2C-AS1 - miR-4419b - WDTC1, TSPEAR-AS2 - miR-16-5p - CYCS/KDR/PDIA6/TGOLN2/HSPA5, TSPEAR-AS2/HCG18 - miR-93-3p - CYCS/HSPA5, TSPEAR-AS2/HCG18 - miR-93-5p - CYCS/ TGOLN2, TSPEAR-AS2/HCG18 - miR-615-3p – CKAP4 and TSPEAR-AS2/HCG18 - miR-125a-3p - PGK1 axes may play critical roles in the ceRNA network. In addition to the prognostic value of this mRNA–miRNA–lncRNA network for AIS, our findings provide important insights into the molecular mechanism of AIS. However, further studies are needed to validate these findings.

Data Availability Statement

Publicly available datasets were analyzed in this study. This data can be found here: lncRNA (GSE110359); gene and miRNA datasets come from the Supplementary Material in (8, 18).

Author Contributions

C-LS and X-YL designed the study. H-ML wrote the manuscript. YL and J-YD revised and polished the manuscript. H-ML, YL, J-YD, and RZ performed the statistical analysis of the data. All authors read and approved the final manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Glossary

Abbreviations

AIS

Adolescent idiopathic scoliosis

MSCs

mesenchymal stem cells

miRNA

microRNA

lncRNA

long non-coding RNA

ceRNA

competing endogenous RNA

GEO

Gene Expression Omnibus

DEGs

differentially expressed genes

GO

gene ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

PPI

protein-protein interaction

BP

biological process

MF

molecular function

CC

cellular component

STRING

Search Tool for the Retrieval of Interacting Genes.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed.2020.583243/full#supplementary-material

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Click here for additional data file. (13.8KB, DOCX)
Click here for additional data file. (27.3KB, DOCX)

Data Availability Statement

Publicly available datasets were analyzed in this study. This data can be found here: lncRNA (GSE110359); gene and miRNA datasets come from the Supplementary Material in (8, 18).

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