CDR1as regulated by hnRNPM maintains stemness of periodontal ligament stem cells via miR‐7/KLF4

Abstract CDR1as is a well‐identified circular RNA with regulatory roles in a variety of physiological processes. However, the effects of CDR1as on stemness of periodontal ligament stem cells (PDLSCs) and the underlying mechanisms remain unclear. In this study, we detect CDR1as in human PDLSCs, and subsequently demonstrate that CDR1as maintains PDLSC stemness. Knockdown of CDR1as decreases the expression levels of stemness‐related genes and impairs the cell's multi‐differentiation and cell migration abilities, while overexpression of CDR1as increases the expression levels of stemness‐related genes and enhances these abilities. Furthermore, our results indicate that the RNA‐binding protein hnRNPM directly interacts with CDR1as and regulates its expression in PDLSCs. In addition, we show that CDR1as promotes the expression of stemness‐related genes in PDLSCs by inhibiting miR‐7‐mediated suppression of KLF4 expression. Collectively, our results demonstrate that CDR1as participates in the molecular circuitry that regulates PDLSC stemness.

tissue-specific and highly conserved expression characteristics, circRNAs are considered to be biomarkers, or targets for diagnosis and treatment of diseases. 14 Recently, circRNAs have been shown to participate in the maintenance of pluripotency of human embryonic stem cells, 15 self-renewal of intestinal stem cells, 16 differentiation of osteoblasts and osteoclasts, 17,18 and the primary stage of rat liver regeneration. 19 Therefore, we have been suggested that circRNAs might regulate the pluripotency and differentiation of PDLSCs.
Previously, we have demonstrated that circRNAs are widely involved in PDLSC osteogenic differentiation through high-throughput sequencing, and eight differentially expressed circRNAs were selected for qRT-PCR validation. 20 Among them, the circRNA CDR1as, an antisense transcript of cerebellar degeneration-associated protein 1 (CDR1), was highly expressed in PDLSCs and up-regulated during PDLSC osteogenic differentiation. 20 Acting as a miR-7 'sponge', CDR1as was associated with human diseases and reported to exert biological functions, including activation of stem cell differentiation. 21 However, the effects and mechanisms of CDR1as on the stemness of PDLSCs remain unclear.
In this study, we comprehensively explore the functional roles of CDR1as and its regulatory effects on PDLSC stemness properties, including proliferation, migration, osteogenic and adipogenic differentiation, and the expression of pluripotency-associated genes.
Furthermore, we present findings indicating that the RNA-binding protein (RBP) hnRNPM directly interacts with CDR1as and regulates its expression in PDLSCs. In addition, we demonstrate that CDR1as promotes the expression of stemness-related genes in PDLSCs by inhibiting miR-7-mediated suppression of KLF4 expression.

| MATERIAL S AND ME THODS
All protocols for handling of human tissues were approved by the Research Ethics Committee of Stomatology Hospital of Shandong University, China (GR201710). Informed consent was obtained from all donors.

| Osteogenic induction, ALP and Alizarin Red staining
For osteogenic differentiation, PDLSCs were cultured with osteogenic inductive medium supplemented with 10 nM dexamethasone, 10 mM β-glycerophosphate and 50 mg/L vitamin C (Sigma-Aldrich, St. Louis, MO, USA). After 7 days, PDLSCs were fixed with 70% alcohol, and ALP staining was performed as described previously. 20 After 21 days, PDLSCs were fixed with 4% paraformaldehyde and stained with 2% Alizarin Red (pH = 4.2) (Sigma-Aldrich). To determine the relative amount of mineralized matrix, 10% w/v cetylpyridinium chloride (CPC) (Sigma-Aldrich) was added to the stained plates, and samples were quantified by spectrophotometric absorbance at 562 nm.

| Adipogenic induction and Oil Red O staining
For adipogenic differentiation, PDLSCs were cultured in normal medium and adipogenic inductive medium supplemented with 1 μM dexamethasone, 10 mg/L insulin, 0.5 mM 3-isobutyl-1-methylxanthine and 0.2 mM indomethacin (Sigma-Aldrich). After 14 days, PDLSCs were fixed with 4% paraformaldehyde and stained with Oil Red O (Sigma-Aldrich). To determine the relative amount of oil deposition, isopropyl alcohol was added to the stained plates, and samples were quantified by spectrophotometric absorbance at 510 nm.

| Identification of CDR1as
Total RNA was extracted with TRIzol (Takara, Tokyo, Japan) and reverse transcribed to cDNA. PDLSC gDNA was extracted using a TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China). Divergent and convergent primers were designed to specifically target the circular junction site and the linear region of CDR1as for PCR amplification of cDNA and gDNA (Table 1). These amplification products were separated by agarose gel electrophoresis. The sequence of the divergent primer amplification product using cDNA was verified by Sanger sequencing.
For CDR1as overexpression, lentiviral constructs (ov-CDR1as) were generated based on the whole CDR1as sequence. The same lentiviral vectors, yet containing non-specific RNA oligonucleotides, denoted as sh-NC and ov-NC, were used as a negative control. For KLF4 and hnRNPM knockdown, four siRNA oligonucleotides complementary to different regions of human KLF4 and hnRNPM were designed and synthesized by Oligobio (Beijing, China). For the negative control, PDLSCs were transfected with a non-specific RNA oligonucleotide.
The shRNA and siRNA sequences are presented in Table 2.

| Cell proliferation assay
The proliferation of PDLSCs was tested by EdU At 24, 48 and 72 hour, the CCK8 reagent (10 μL) was added to each well, and cells were incubated at 37°C for 2 hour. The optical density at 450 nm was measured using a spectrophotometer.
The primer sequences are listed in Table 1. The primer of miR-7 (Set Catalog #606) was purchased from Takara. Data were analysed using the 2 −△△CT method, with GAPDH and U6 as internal standards.

| Antibodies
The antibodies used for Western blotting were the following: anti-

| RNA-protein pull-down
In the RNA pull-down assay, PDLSCs were lysed in co-IP buffer, and samples were incubated with biotinylated oligo-DNA probes against putative binding sites in CDR1as. A biotinylated random oligo probe was used as a negative control. Streptavidin-coated magnetic beads were added to each binding reaction to pull down CDR1as and RBPs.
The beads were then washed, and binding proteins were analysed by mass spectrometry to identify CDR1as binding proteins.

| Dual-luciferase reporter assay
In the dual-luciferase reporter assay, 40 ng of luciferase reporter plasmid was transfected into 293T cells together with 100 nM miR-7 mimic using Lipofectamine 3000. After transfection for 24 hour, the Renilla and firefly luciferase activities were measured separately using the Dual-Luciferase Reporter Assay System (Promega, Beijing, China) following the manufacturer's instructions. The light intensity from Renilla luciferase was normalized to that of firefly luciferase.

| Statistical analysis
Quantitative data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using a Student's t test with SPSS 17.0 software. P <.05 was considered statistically significant.

| Identification of PDLSCs and CDR1as
Primary cultured fibroblast-like PDLSCs grew radially around the tissue explants and spread ( Figure 1A). Subcultured PDLSCs were fairly uniform and exhibited strong proliferation capacity at passage number 3 ( Figure 1A). The ability of PDLSCs to form cell clusters was shown by the formation of single colonies ( Figure 1B). Cultured PDLSCs were positive for mesenchymal stem cell markers CD44 and CD90 and negative for leukocyte markers CD34 and CD45 ( Figure 1C). After osteogenic induction, the amount of mineralized matrix was increased in PDLSC cultures ( Figure 1D). Additionally, oil deposition was increased in adipo-induced PDLSCs ( Figure 1E).
These results suggest that established PDLSCs maintained selfrenewal capacity and pluripotency.
The expression of CDR1as was validated by RT-PCR followed by agarose gel electrophoresis. As a result, amplification products of convergent primers were detected using both cDNA and gDNA as template, while amplification products of divergent primers were only detected using cDNA ( Figure 1F). The amplification product of divergent primers was Sanger sequenced ( Figure 1G).

| Knockdown of CDR1as impairs PDLSC stemness
In the sh-NC, sh-CDR1as #1 and sh-CDR1as #2 groups, approximately 80% of PDLSCs were green fluorescent, and thus successfully transfected (Figure 2A). Compared with the sh-NC group, the expression of CDR1as in the sh-CDR1as #1 group was reduced by approximately 80%. However, the expression of CDR1as in sh-CDR1as #2 was not significantly reduced ( Figure 2A). Therefore, sh-CDR1as #1 was selected for subsequent experiments. The expression levels of stemness-associated genes (SOX2, OCT4 and Nanog) were significantly down-regulated in the sh-CDR1as #1 group ( Figure 2B). Next, we measured the multi-differentiation, proliferation and migration abilities of PDLSCs. The amount of mineralized matrix in osteogenic-induced PDLSC cultures was reduced in the sh-CDR1as #1 group ( Figure 2C). The expression of ALP and Runx2 were down-regulated in the sh-CDR1as #1 group ( Figure 2D). Moreover, oil deposition in adipogenic-induced PDLSCs was significantly decreased in the sh-CDR1as #1 group ( Figure 2E). The proliferation ability of PDLSCs was not significantly changed ( Figure 3A−C). In addition, knockdown of CDR1as significantly impaired the migration ability of PDLSCs ( Figure 3D and E). These results suggest that knockdown of CDR1as impairs PDLSC stemness.

| The expression of CDR1as in PDLSCs is regulated by hnRNPM
As reported, RBPs might interact with circRNA and control its expression level. 22,23 To examine whether RBPs regulate the expression of CDR1as, we carried out an RNA pull-down assay. The biotinylated CDR1as probe (biotin-CDR1as) was designed to pull down CDR1as and RBPs. Biotinylated random oligo (biotin-NC) was used as a negative control. CDR1as levels in the biotin-CDR1as elution were much higher than in the biotin-NC elution ( Figure 6A). Next, we electrophoresed and silver stained the pull-down products. Several bands were only found in the biotin-CDR1as group ( Figure 6B).
After cutting out these bands, we identified 68 proteins that bind to CDR1as by mass spectrometry (Table S1). Among them, hetero- levels of CDR1as were significantly lower in si-hnRNPM groups #1-4 than in the si-NC group ( Figure 6F). Taken together, these results suggest that hnRNPM promotes the expression of CDR1as in PDLSCs.

| CDR1as regulates stemness of PDLSCs via miR-7 and KLF4
It is known that CDR1as has approximately 70 conserved miR-7 binding sites and performs biological functions by acting as a miR-7 sponge. 18 To determine whether CDR1as regulates the stemness of PDLSCs through interactions with miR-7, we first showed by qRT-PCR that the relative expression level of miR-7 was up-regulated in the sh-CDR1as #1 group ( Figure 7A). The expression levels of CDR1as and miR-7 are negatively correlated. To further uncover the downstream molecules by which CDR1as and miR-7 affect PDLSC stemness maintenance, we analysed potential target genes of miR-7 using TargetScan7. Moreover, KLF4 expression was down-regulated in the sh-CDR1as #1 group ( Figure 7A). Notably, we found that the

| D ISCUSS I ON
A critical requirement in periodontal tissue regeneration is the stemness of a PDLSC-enriched population that retains the features of its original constituent cells. 24 Stemness of PDLSCs is closely related to age, oestrogen levels, hypoxia and the inflammatory microenvironment, strongly limiting their usefulness in tissue engineering. [25][26][27][28] Maintaining stemness of PDLSCs might provide a new strategy for repair of injured periodontal tissue and in situ regeneration. With the ongoing development in bioinformatics, many non-coding RNAs have been found to participate in stemness regulation. 29 Recently, circRNA, another type of endogenous non-coding RNA, has been demonstrated to control plasticity of mesenchymal stem cells. 30 In our previous study, we indicated that CDR1as is highly expressed in PDLSCs and is up-regulated during PDLSC osteogenic differentiation. 20 In this study, we comprehensively explored the functional role of CDR1as in regulating PDLSC stemness properties.
Our study suggests that knockdown of CDR1as decreases the  35 Our previous study indicated that a variety of circRNAs is differentially expressed upon PDLSC osteogenic differentiation and might play important regulatory roles. 20 In this study, we suggest that CDR1as could enhance the osteogenic and adipogenic differentiation of PDLSCs.
Cell migration is another stemness property of stem cells.
Studies have demonstrated that signalling molecules like substance P, stromal-derived factor 1a and stem cell factor participate in the regulation of this process. 36  (SCAP). 38 Similarly, hypoxia had no effect on SCAP proliferation, but it evoked the up-regulation of genes specific for osteogenic differentiation, neuronal differentiation and angiogenesis. 39 In summary, our study has explored the functional roles of CDR1as and its regulatory effects on PDLSC stemness properties, including proliferation, migration, differentiation and the expression of In this study, PDLSCs were used at passage number 3 to maintain stemness. Telomerase activity of stem cells could be regulated by transcription factors. For instance, SOX2 could enhance multipotential and self-renewal of neural progenitor cells by maintaining telomerase activity. 41 However, the effects of CDR1as on telomerase activity and stemness of long-term culture PDLSCs remain unknown, which will be considered in our future studies.
A previous study indicated that the expression of circRNA has cell type-specific features. 42 In our previous study, we identified the differential expression of CDR1as between normal and osteodifferentiated PDLSCs. 20

ACK N OWLED G EM ENTS
This study was supported by grants from the National Natural Science Foundation of China (81771030), the Construction Engineering Special Fund of 'Taishan Scholars' (tsqn20161068) and Open Foundation of Shandong Provincial Key Laboratory of Oral Tissue Regeneration (SDKQ201902).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

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 from the corresponding author upon reasonable request. F I G U R E 7 CDR1as regulates stemness of PDLSCs via miR-7 and KLF4. A. The expression levels of miR-7 and KLF4 in the sh-CDR1as#1 and sh-NC groups were analysed. B. Schematic illustration showing the differences between the two luciferase reporters, including one containing the complete KLF4 3′-UTR sequence (KLF4-wt) and one containing the KLF4 3′-UTR sequence, with mutated sequences in the two miR-7 binding sites (KLF4-mut). The reporter assay showed the luciferase activity of KLF4-wt and KLF4-mut in 293T cells cotransfected with miR-7 mimics. C. KLF4 mRNA and protein expression levels in PDLSCs transfected with si-NC and four siRNAs targeting different regions of KLF4, as analysed by qRT-PCR and Western blot. D. mRNA and protein expression levels of stemness-associated genes (SOX2, OCT4 and Nanog) in PDLSCs transfected with si-NC and siRNA targeting KLF4, as measured by qRT-PCR and Western blot. Quantitative qRT-PCR data are presented as mean ± SD of three experiments. **P < .01, by Student's t test