LncRNA‐SULT1C2A regulates Foxo4 in congenital scoliosis by targeting rno‐miR‐466c‐5p through PI3K‐ATK signalling

Abstract Congenital scoliosis (CS) is the result of anomalous vertebrae development, but the pathogenesis of CS remains unclear. Long non‐coding RNAs (lncRNAs) have been implicated in embryo development, but their role in CS remains unknown. In this study, we investigated the role and mechanisms of a specific lncRNA, SULT1C2A, in somitogenesis in a rat model of vitamin A deficiency (VAD)‐induced CS. Bioinformatics analysis and quantitative real‐time PCR (qRT‐PCR) indicated that SULT1C2A expression was down‐regulated in VAD group, accompanied by increased expression of rno‐miR‐466c‐5p but decreased expression of Foxo4 and somitogenesis‐related genes such as Pax1, Nkx3‐2 and Sox9 on gestational day (GD) 9. Luciferase reporter and small interfering RNA (siRNA) assays showed that SULT1C2A functioned as a competing endogenous RNA to inhibit rno‐miR‐466c‐5p expression by direct binding, and rno‐miR‐466c‐5p inhibited Foxo4 expression by binding to its 3′ untranslated region (UTR). The spatiotemporal expression of SULT1C2A, rno‐miR‐466c‐5p and Foxo4 axis was dynamically altered on GDs 3, 8, 11, 15 and 21 as detected by qRT‐PCR and northern blot analyses, with parallel changes in Protein kinase B (AKT) phosphorylation and PI3K expression. Taken together, our findings indicate that SULT1C2A enhanced Foxo4 expression by negatively modulating rno‐miR‐466c‐5p expression via the PI3K‐ATK signalling pathway in the rat model of VAD‐CS. Thus, SULT1C2A may be a potential target for treating CS.

possibly because RAs control the development of the segmental structure of the vertebrate body during embryogenesis. [8][9][10] Retinoic acid is a metabolite of vitamin A (retinol) that mediates the critical functions of vitamin A in growth and development. However, the mechanisms underlying VAD-induced CS remain unclear.
MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that inhibit or degrade mRNAs by binding to the 3′ untranslated region (UTR) of mRNAs. Long non-coding RNAs (lncRNAs) are non-protein coding transcripts of >200 nucleotides that modulate many physiological and pathological biological functions via epigenetic regulation and interaction with miRNAs, proteins or mRNAs. 11 Long non-coding RNAs can act as competing endogenous RNAs (ceRNAs) to inhibit miRNA activity by sequestering miRNA, thereby reducing the expression of its target genes. [12][13][14] Recently, lncRNAs were found to play an important role in embryo development. 15,16 However, the role of lncRNAs in somitogenesis remains largely unclear.
Zhang et al reported that miRNA-145-5p (miR-145) and β-catenin mRNA (CTNNB1) are overexpressed in adolescent idiopathic scoliosis (AIS) bone tissues and primary osteoblasts, and such aberrant miRNA expression has been shown to influence osteocyte function. 17 A lncRNA known as lncAIS, for its involvement in the pathogenesis of AIS, has been reported to suppress the osteogenic differentiation of mesenchymal stem cells (MSCs). Specifically, ln-cAIS impacts the function of NF90 and HOXD8 and hinders RUNX2 transcription. 18 Previously, we performed sequencing data analysis to build ceRNA networks in embryonic tissues of rats with VAD-induced CS and found that, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, a SULT1C2A-rno-miR-466c-5p-Foxo4 axis as well as the Phosphoinositide 3 kinases (PI3K)-AKT pathway appear to be involved in CS pathogenesis based on bioinformatics prediction and sequence analysis. 19 In the present study, we aimed to confirm the expression of the lncRNA SULT1C2A in a rat model of VAD-induced CS and to explore the molecular mechanism of the SULT1C2A-rno-miR-466c-5p-Foxo4 axis in CS. This study of the effect of ceRNA dysregulation within the pathogenesis of VAD-CS provides insight into the mechanisms of somitogenesis and suggests that SULT1C2A may be a potential target for treating CS. The rat model of VAD-induced CS was created as described previously. 7 Briefly, female rats (n = 96) were randomly assigned to either the VAD group (n = 48), which received a modified AIN-93G diet without any vitamin A source (Research Diets, New Brunswick, NJ) or the control group (CON, n = 48), which received an AIN-93G diet with adequate vitamin A (4 retinol equivalents (RE)/g diet). Vitamin A deficiency was detected and confirmed in the VAD group after the rats were fed the VAD diet for more than 2 weeks as described previously. 7,20 The female rats were then mated with normal male rats at 6-10 pm and continuously received the same diet during gestation. 21

| Tissue collection
Embryos were collected from six to eight pregnant rats of the CON and VAD groups on gestational days (GDs) 3, 8, 9, 11, 15 and 21.

| Co-expression network construction
A co-expression network was constructed to identify interactions among mRNAs and lncRNAs as described previously. 19 A weighted gene co-expression network analysis (WGCNA) was performed to identify the associations between mRNAs and lncRNAs according to the calculated Pearson correlation coefficients. The gene encoding lncRNA SULT1C2A (length 1828 bp) is located on chromosome 9 (4621424-4624425 [+] strand) in intron 4 of Sult1c2a.

| Cell culture
HEK-293T and H9C2 cells were maintained in high glucose DMEM (Gibco, New York, NY) added 10% foetal bovine serum (Gibco). All cells were cultured in a 5% CO 2 humidified atmosphere at 37°C. The medium was exchanged daily, and cells were passaged every 3 days using 0.25% trypsin.

| Luciferase reporter assay
The target genes of SULT1C2A were predicted using a bioinformatics prediction program (miRcode and TargetScan program). PCR was performed to obtain the wild-type SULT1C2A sequence (SULT1C2A- Renilla luciferase activity was normalized to firefly luciferase activity. ized overnight at 65°C. The membranes were then exposed to the phosphor imager screen for at least 12 hours.

| SULT1C2A expression is down-regulated in embryonic tissues of rats with VAD on GD 9
Gene co-expression networks were used to identify potential interactions among different transcripts in embryos from rats with VAD, and co-expression modules with a high rate of protein-coding RNAs were selected. SULT1C2A (NONRATG027649.1) was associated with many somitogenesis-related genes, such as Foxo4, Bhlha15, Trim55 and Fads6. Thus, we identified a novel lncRNA-miRNA-mRNA regulatory network, the SULT1C2A-rno-miR-466c-5p-Foxo4 axis in VAD.
We then measured the expression of SULT1C2A, rno-miR-466c-5p and Foxo4 in embryonic tissues of the VAD and CON groups on GD 9.
The Foxo4 and SULT1C2A expression levels were significantly lower in the VAD group than the CON group ( Figure 1A-C).

| Somitogenesis is reduced in embryonic tissues of the VAD group on GD 9
We next measured the expression of somitogenesis-associated genes in embryonic tissues from rats with VAD at GD 9. The mRNA levels of Pax1, Nkx3-2 and Sox9 were significantly lower in the embryos of the VAD group compared with the CON group ( Figure 1D-F).
Furthermore, a siRNA against SULT1C2A was designed for transfection of H9C2 cells. According to the qRT-PCR results, the SULT1C2A and Foxo4 levels were effectively reduced in the H9C2 cells transfected with the siRNA Lnc compared with levels in the blank and NC groups ( Figure 2G,H).
Luciferase reporter assays showed that rno-miR-466c-5p mimics reduced the luciferase activity in cells transfected with pmirGLO-Foxo4-wt, but not with pmirGLO-Foxo4-mt ( Figure 2F). The qRT-PCR results revealed that the rno-miR-466c-5p level was effectively increased in H9C2 cells treated with the siRNA Lnc compared with that in the NC group ( Figure 2I). In addition, transfection with rno-miR-466c-5p mimics or the siRNA for SULT1C2A or cotransfection F I G U R E 1 Expression of long non-coding RNA SULT1C2A, miR-466c-5p and Foxo4 in rat embryos on GD 9. (A,C) quantitative real-time PCR results showing that relative expression levels of SULT1C2A and Foxo4 expression were significantly lower in the vitamin A deficiency (VAD) group than in the control (CON) group. B, The relative expression level of miR-466c-5p was significantly increased in the VAD group compared with the CON group. (D-F) The relative expression levels of Pax1, Nkx3-2 and Sox9 were lower in rat embryos of the VAD group compared with those of the CON group. (*P < 0.05, **P < 0.001, n = 50 for each group). All experiments were independently repeated three times of both led to down-regulated expression of pmirGLO-Foxo4-wt, but this was not observed for pmirGLO-Foxo4-mt ( Figure 2J). These findings suggest that rno-miR-466c-5p regulates Foxo4 expression by directly binding to the 3′-UTR and also revealed that SULT1C2A functions as a ceRNA, interacting with rno-miR-466c-5p.
Compared with that in the CON group, Foxo4 expression in the VAD group was significantly down-regulated on GDs 8 and 11 ( Figure 3C).

| PI3K-AKT expression is altered in embryos of rats with VAD
From the STRING database, the protein interaction and correlation score between Foxo4 and AKT1 was 0.984, 22 and as a substrate of the PI3K-AKT pathway, we selected this interaction as the target. The AKT, p-AKT and PI3K (p85 regulatory subunit) expression levels were measured via western blot analysis. Compared with the CON group, AKT expression was generally up-regulated in the VAD group, but p-AKT expression was significantly reduced on GDs 3, 8 and 11 and then increased on GD 21 in the VAD group ( Figure 5A). p85 expression was significantly reduced on GD 11 but increased on GDs 3 and 8 in the VAD group ( Figure 5B). Together, these data indicate that GD 11 represents a key period of VAD during somitogenesis, which is consistent with the results of qRT-PCT and northern blotting.

| D ISCUSS I ON
Congenital scoliosis is caused by abnormal vertebrae growth during embryogenesis, and vitamin A and RA are known to play important roles in normal physiological process. We previously found that VAD during pregnancy causes CS in postnatal rats. 7 In the present study, we found a dynamic correlation between SULT1C2A, rno-miR-466c-5p and We previously identified ceRNA regulatory networks of embryonic somite development in VAD-induced CS and found many ncRNA expression profiles, as well as associations between mRNAs and ncRNAs in CS. 19 Here, we further found that SULT1C2A and Foxo4 expression levels were down-regulated, while rno-miR-466c-5p expression was up-regulated on GD 9 in embryos of the VAD-induced CS rat model. Furthermore, luciferase reporter assays confirmed that SULT1C2A directly targets rno-miR-466c-5p and may function as a ceRNA. The assay also showed that rno-miR-466c-5p inhibited  27 It has been reported that spatiotemporal expression of Foxo4, which is transcriptionally regulated by PI3K signalling, is involved in the developing brain and retina in zebrafish. 28 In addition, AKT was shown to increase Foxo4 phosphorylation in response to severe dehydration. 29 However, we found that the dynamic expression of p85 and p-AKT were consistent with that of Foxo4 in embryos of the VAD group, suggesting that PI3K/AKT signalling regulates Foxo4 expression in CS.
Retinoic acid is an important modulator that promotes embryonic cell differentiation and plays an important role in vertebrate development. 30 Retinoic acid is required during embryogenesis as it regulates clustering of Hox gene expression. 31 In addition, RA contributes to the critical role of Foxc1a in early somitogenesis in zebrafish. 32 Furthermore, RA is crucial for temporal control of embryonic development, because embryonic molecular clock genes, such as limb clock hairy2, are regulated by RA. 33 The PI3K/AKT pathway was also reported to play an essential role in the process of RA-induced cell differentiation. 34 The PI3K/AKT signalling pathway was shown to also be important for the temporal control of limb development. 33 In MSCs, RA promotes osteogenic differentiation by activating the PI3K/AKT signalling pathway. 35 In addition, we found abnormal changes in PI3K and AKT phosphorylation in the VAD group in the present study, suggesting that the PI3K/AKT pathway is important for VAD-induced CS.
Previous research indicated that the somite development of rat begins from GD 9 and ends on GD 16, with the fastest stage at GD F I G U R E 6 A schematic diagram of the proposed function of the lncRNA SULT1C2A-rno-miR-466c-5p-Foxo4 axis during somitogenesis stage from GDs 9 to 11. We speculate that VAD leads to down-regulation of Foxo4, and spatiotemporally expressed lncRNA-SULT1C2A, as a ceRNA, binds to miR-466c-5p to promote Foxo4-associated somitogenesis of the rat embryo via activation of the PI3K-AKT signalling pathway 11 when approximately 21-33 somites (from lower thoracic to sacral) are developed. 36,37 We specifically observed dynamic changes in the expression of the SULT1C2A-rno-miR-466c-5p-Foxo4 axis over GDs 8 to 15, especially from GDs 9 to 11, suggesting that the spatiotemporal changes in the expression of the SULT1C2A-rno-miR-466c-5p-Foxo4 axis are important for somitogenesis in VAD-induced CS. In the VAD group, on GDs 8 and 9, lncRNA SULT1C2A and Foxo4 expression levels were decreased but miR-466c-5p expression was increased on GD 9. A critical point in this balance seemed to occur on GD 11 with up-regulation of SULT1C2A to compete with the increased miR-466c-5p expression. On GD 15, although Foxo4 expression was increasing, the level was still lower than that in the CON group. Meanwhile, the expression of p-AKT and p85 was consistent with the level of Foxo4 expression on GD 11. Therefore, we suggest that VAD during gestation initially reduces the expression of Foxo4, as the target, and subsequently up-regulates miR-466c-5p.
To maintain the relative balance of embryonic development, lncRNA SULT1C2A is up-regulated on GD 11 and functions as a sponge to antagonize miRNAs. The dynamic changes in the expression of the SULT1C2A-rno-miR-466c-5p-Foxo4 axis may be initiated on GD 9 and were most obvious on GD 11 during the early-mid stage of somitogenesis ( Figure 6). In addition, a dynamic profile of AKT phosphorylation, an important signalling pathway for somitogenesis, was found in the VAD group, and this novel finding warrants further research.
In conclusion, our results suggest that lncRNA SULT1C2A expression is increased in CS and that SULT1C2A functions as a ceRNA to spatiotemporally regulate Foxo4 expression by targeting rno-miR-466c-5p and regulating the PI3K-ATK signalling pathway.

ACK N OWLED G EM ENT
This work was supported by the National Natural Science Foundation of China (No.81330044 and 81772424).

CO N FLI C T O F I NTE R E S T
The authors confirm that there is no conflict of interest. Bi wrote the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available because of privacy or ethical restrictions.