Circ-FURIN Expression Enhances Osteoblast Differentiation In Dental Pulp Stem Cells Via SOX11 Signaling Pathway Sponging miR-125

Background: Many studies have found that circRNA plays a part in osteoblast differentiation. However, its mechanism remains unknown. Methods: High-throughput sequencing was used to identield the different expression of circRNA during osteogenic dental pulp stem cells (DPSCs) differentiation. Luciferase report analysis and RT-qPCR were used to clarify the expression and regulation relationship among circ-FURIN, miR-125 and SOX11. The heterotopic bone formation experiment was further used to conrm the osteoblast differentiation of DPSC with different expression of circ-FURIN, miR-125 and SOX11. Results: Study indicated that circ-FURIN expression remarkably increased during osteoblast differentiation, yet circ-FURIN knockdown suppressed it. Bioinformatics and luciferase results discovered that miR-125 is the downstream target of circ-FURIN. Furthermore, circ-FURIN upregulation decreased miR-125 expression. MiR-125 upregulation restored the promotion effect of circ-FURIN on osteogenic DPSC differentiation. Luciferase report analysis veried that SOX11 is miR-125 downstream target. miR-125 overexpression suppressed osteogenic DPSC differentiation through targeting SOX11. SOX11 overexpression restored miR-125 inhibitory effect on osteogenic DPSC differentiation. In vivo experiments with heterotopic bone model suggested that circ-FURIN overexpression has crucial function to enhance heterotopic bone formation. Conclusions: In summary, circ-FURIN enhances osteoblast DPSC differentiation via the SOX11 signaling pathway by sponging miR-125. These ndings suggest a novel therapeutic target for osteoporosis treatment.


Introduction
Dental pulp stem cells (DPSCs), the rapidly proliferating mesenchymal stem cells (MSCs), are ready to differentiate into neuronal, osteoblastic, hepatocytic, and myocytic lineages. DPSC multilineage capacity suggests that these cells might have a wider therapeutic potential compared to lineage-restricted adult stem cell populations, including MSCs [1,2]. DPSCs are fairly accessible and prevail in the whole life.
Interactions between dental papilla and epithelial cells during tooth formation enhance tooth morphogenesis by stimulating mesenchymal cell subpopulations to differentiate into odontoblasts, which then build primary dentin [3]. DPSCs can be used for stem cell therapies, which provide therapeutic capabilities for corneal, cardiovascular, neurologic, oro-facial, diabetic, renal, hepatic, muscular dystrophy, and autoimmune conditions. DPSCs have been increasingly used in areas of regenerative medicine, tissue engineering, and bone disorder therapies [4,5].
Being an indispensable class of ncRNAs, head-to-tail closed loop structure regarding circular RNAs (circRNAs) from 3′ to 5′ tail causes a pronounced stability compared to traditional linear RNAs [6].
The present study demonstrated circRNA in uence on DPSC osteogenesis. High-throughput sequencing discovered that circ-FURIN expression is increased during osteoblast DPSC differentiation. circ-FURIN expression enhances osteoblast differentiation regarding human DPSCs through SOX11 signaling pathway via sponging miR-125. These results indicate underlying therapeutic approaches for bone and periodontal tissue regeneration as well as reveal novel functions potential osteogenic differentiation.

Ethics statement
Animal Care Committee at the Ninth People's Hospital in Shanghai Jiao Tong University School of Medicine (Shanghai, China) approved animal experiments.
Human DPSC isolation and identi cation Cells were isolated from dental pulp. Brie y, we removed tissue and immersed it in a digestive solution (4.0 mg/mL dispase and 3.0 mg/mL: type I collagenase) for 1 h at 37°C. It was ltered using 70-μm cell strainers to get an hDPSC suspension. The cells were plated in T25 asks and cultured them in culture medium DMEM/F12 with fetal bovine serum of 10% and 1% penicillin/streptomycin at 37°C and 5% CO 2 .
For cell phenotypic analysis using surface proteins, we harvested DPSCs using 5 mM ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS). We then incubated cells with FITC-conjugated antibodies against human CD44, CD45, CD34, CD29, CD90, CD73, and CD105. Isotype antibodies that matched would serve as controls (Becton Dickinson, San Jose, CA). We washed cells with cold PBS containing 2% fetal calf serum. We acquired 1000 labeled cells and analyzed them through an immuno uorescence microscope (Becton Dickinson).
Strand-speci c high-throughput RNA-Seq library construction Total RNA was retrieved from osteogenic DPSC differentiation after 0 or 14 days through TRIzol reagent (Invitrogen, Carlsbad, CA, USA) by using ~3 μg of RNA from individual samples with VAHTS Total RNAseq (H/M/R) Library Prep Kit from Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) to erase ribosomal RNA and retain other mRNA's, like non-coding RNAs. RNAs were exposed to 40 U of RNase R (Epicenter) at 37°C for 3 h after TRIzol puri cation. KAPA Stranded RNA-Seq Library Prep Kits (Roche, Basel, Switzerland), which were subjected to deep sequencing via Illumina HiSeq 4000 at Aksomics,

ALP staining
We used NBT/BCIP staining kit (CoWin Biotech, Beijing, China) for ALP staining following instructions. DPSCs were seeded in 24-well plates to be cultured in osteogenic medium for 1~2 w. Cellswere xed with 4% PFA for half of an hour and incubated them in a staining reagent in dark for twenty minutes.

Mineralization assay
In order for detection of extracellular matrix calcium deposition, we seeded DPSCs in 24-well tissue plates and cultivated them for one to two weeks in osteogenic medium. Then, 0.1% Alizarin Red S (ARS, Sigma-Aldrich, Saint Louis, MO, USA) solution at pH 4.2 was used to stain nodules that calci ed followed by xing DPSCs.
In vivo heterotopic bone formation assay DPSCs were induced in osteogenic medium for one week before in vivo investigation. Cells were resuspended to be incubated with 7 mm × 5 mm × 2 mm Bio-Oss Collagen (Geistlich, GEWO GmbH, Baden-Baden, Germany) scaffolds for one hour at 37°C after centrifugation at 150 g for 5 min. Cells were subcutaneously implanted on backs of ve-week-old BALB/c homozygous nude (nu/nu) mice ( ve mice per group) as previously described [15]. The implants were harvested after eight weeks.

Hematoxylin and eosin (H&E) and Masson's trichrome staining and immunohistochemical analyses
Specimens in 10% EDTA (pH 7.4) was decalci ed for 1 min after embedding in para n and dehydration. We cut sections (5 μm) and stained them with H&E as well as Masson's trichrome. We performed immunohistochemical analysis following previously described procedure [16]. Specimens were maintained in 5% normal goat serum for half of an hour and incubated with primary antibody against OCN (Santa Cruz Biotechnology) at 4°C overnight. Our team processed sections via ABC detection kit (Vector Laboratories, Burlingame, CA) and visualized with Olympus microscope (Olympus Co., Tokyo, Japan).

Statistical analyses
Our team expressed results as mean ± standard deviation (SD). GraphPad Prism software (GraphPad, La Jolla, CA, USA) was used to compute signi cance. P-value ≤0.05 inferred statistical signi cance.

Overexpression of circ-FURIN promotes osteogenic DPSC differentiation
The recent research determined that hsa_circ_0036872 was cyclized and derived from FURIN gene exon that is located at chr15:91411884-91426687. Therefore, hsa_circ_0036872 was named circ-FURIN ( Figure

Discussion
Research have discovered that circRNA plays an indispensable role during osteogenic differentiation [18,19]. The present study isolated DPSCs and found that circRNA was abnormally expressed during osteogenesis differentiation using high-throughput sequencing. Study results also veri ed that circ-FURIN expression was increased during osteogenesis differentiation. Overexpressing circ-FURIN promoted osteogenesis DPSC differentiation, while downregulating circ-FURIN suppressed it. To determine the regulatory mechanism of circ-FURIN in osteogenesis differentiation, bioinformatics analysis and luciferase reporting experiments con rmed that miR-125 is the target of circ-FURIN.
MiR-125 is broadly expressed in various cells and tissues. It binds disparate proteins to inhibit cancer occurrence and is involved in immunity, heart protection from ischemic injury, differentiation of skeletal muscle cells, and angiogenesis inhibition [20][21][22][23][24]. Previous studies have also found that miR-125 promotes osteoclastogenesis [25], suggesting that miR-125 expression can inhibit osteogenic differentiation. It was also discovered that miR-125 expression decreases during osteogenic differentiation, circ-FURIN overexpression suppresses miR-125 expression, while miR-125 overexpression restores the promotion effect of circ-FURIN on osteogenic DPSC differentiation.
Further research advised that SOX11 is the downstream target of miR-125, which was con rmed by luciferase report analysis. Availability of data and material All data in this study was availability.

Funding
This work was supported by grants from STCSM (20ZR1431300, 18ZR1422700).