Circular RNA SIPA1L1 promotes osteogenesis via regulating the miR-617/Smad3 axis in dental pulp stem cells

Circular RNA SIPA1L1 promotes osteogenesis via regulating the miR-617/Smad3 axis in dental pulp stem cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Circular RNA SIPA1L1 promotes osteogenesis via regulating the miR-617/Smad3 axis in dental pulp stem cells Xingyun Ge, Zehan Li, Zhou Zhou, Yibo Xia, Minxia Bian, Jinhua Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-32816/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Aug, 2020 Read the published version in Stem Cell Research & Therapy → Version 2 posted 7 You are reading this latest preprint version Show more versions Abstract Background: Bone regeneration is preferred for bone loss caused by tumors, bone defects, fractures, etc. Recently, mesenchymal stem cells are considered as optimistic tools for bone defect therapy. Dental pulp stem cells (DPSCs) are a promising candidate for regenerative medicine and bone regeneration. Our previous study showed that up-regulated circSIPA1L1 during osteogenesis of DPSCs is of significance. In this paper, the potential role of circSIPA1L1 in osteogenesis of DPSCs and its underlying mechanisms are explored. Methods: The circular structure of circSIPA1L1 was identified by Sanger sequencing and PCR. Regulatory effects of circSIPA1L1 and miR-617 on mineral deposition in DPSCs were assessed by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and in vivo bone formation assay were conducted to verify the biological influences of circSIPA1L1 on DPSCs. Western blot was performed to detect the protein expression of Smad3. Localization of circSIPA1L1 and miR-617 was confirmed by FISH. Dual-luciferase reporter assay and rescue experiments were conducted to investigate the role of the circSIPA1L1/miR-617/Smad3 regulatory axis in osteogenesis of DPSCs. Results : Sanger sequencing and back-to-back primer experiments confirmed the closed loop structure of circSIPA1L1. CircSIPA1L1 could promote the committed differentiation of DPSCs. MiR-617 was predicted to be the target binding circSIPA1L1 through MiRDB, miRTarBase, and TargetScan database analyses, which was further confirmed by dual-luciferase reporter assay. FISH results showed that circSIPA1L1 and miR-617 colocalize in the cytoplasm of DPSCs. MiR-617 exerted an inhibitory effect on osteogenesis of DPSCs. Knockdown of circSIPA1L1 or upregulation of miR-617 down-regulated phosphorylated Smad3. In addition, rescue experiments showed that knockdown of miR-617 reversed the inhibitory effect of circSIPA1L1 on osteogenesis of DPSCs. Conclusion: CircRNASIPA1L1 promotes osteogenesis of DPSCs by adsorbing miR-617 and further targeting Smad3. Stem Cell & Developmental Cell Biology Circular RNA SIPA1L1 miR-617 Smad3 Dental pulp stem cells osteogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Large bone defects caused by massive injuries, diseases or deformities can be repaired by autologous bone grafts. Nevertheless, the number of bone grafts is limited and the delicate 3D shape cannot be outlined. Therefore, effective bone regeneration for clinical needs is urgently required. Artificial bone engineering, which creates functional bone tissues using stem cells (the most optimal autologous cells) as artificial environment or scaffold, contributes to bone defect repair 1, 2 . In recent years, cell therapy, especially mesenchymal stem cells (MSCs), has shown good application prospects in the treatment of bone defects 3 . As an important member of the MSC family, the biological function of bone marrow mesenchymal stem cells has been widely recognized 4 . Due to its strong multi-lineage differentiation potentials and regenerative properties, great progress has been achieved in bone tissue engineering 5 . Sources of stem cells are diverse, including peripheral blood, bone marrows, cord blood, placenta, and teeth 6 . Notably, dental MSCs are easily available 7 . The endodontium is a special gelatinous soft connective tissues containing nerves, blood vessels and connective tissue, which protects teeth from inflammation and infection 8, 9 . Dental pulp stem cells (DPSCs) are derived from dental pulp tissues, which are featured by high proliferative potential, clonogenicity, self-renewal capacity, and multi-lineage differentiation. As an important mesenchymal stem cell derived from dental pulp, DPSCs can be collected from young permanent teeth in a non-invasive way. In addition to the advantages of a wide range of sources and convenient collection, DPSCs also have the advantages of low immunogenicity and no moral controversy 10 . Many studies have shown that DPSCs can differentiate into neurogenic, osteogenic, dentinal and myogenic cell lineages under different induction conditions 11 . In vivo studies have shown that DPSCs are capable of producing lamellar bones and differentiating into periodontal tissues. Therefore, DPSCs can be utilized for bone regeneration 12 . Moreover, clarifying osteogenesis of DPSCs is conducive to the development of regenerative medicine and bone disease treatment. As an important type of ncRNAs, the head-to-tail closed loop structure of circular RNAs (circRNAs) from 3’ end to 5’ tail results in their pronounced stability than traditional linear RNAs 13 . CircRNAs are extensively expressed in thousands of human genes, and sometime they exhibit higher expressions than corresponding homologous linear isoforms 14 . CircRNAs mainly exert transcriptional and post-transcriptional regulations on protein sponges 15, 16 , translation 17 and miRNA sponges 18 . The well-known competing endogenouse RNA (ceRNA) theory of circRNAs has been well concerned. In the nucleotide sequence of circRNA, some circRNAs contain multiple miRNA binding sites capable of binding miRNA, preventing them from binding to their mRNA target genes (sponge effect), thereby inhibiting the function of miRNA. CiRS-7, also known as CDR1as, has been clearly demonstrated as a typical example of miRNA sponge. CircRNA CDR1as contains over 70 miRNA-7 (miR-7) conserved binding sites that strongly inhibit miR-7 activity. After the CDR1as study was published in 2013, various other circRNAs have been shown to act as miRNA sponges. Using bioinformatics tools, miRNA binding sites can be predicted in circRNA sequences. Therefore, for the same miRNA, a circular RNA containing many miRNA binding sites is relatively easy to be found as a miRNA sponge 18, 19, 20 . For example, circRNA-ciRS-7 contains over 70 binding sites of miR-7, which are highly conserved and greatly inhibits its activity. However, potential functions of circRNAs in stem cell osteogenesis are rarely reported. We have previously identified differentially expressed circRNAs during osteogenesis of teeth-derived stem cells by RNA sequencing 21 , suggesting vital functions of circRNAs in osteogenesis. In the preliminary work, we found that the expression of circSIPA1L1 in the mineralization induction group was about 8 times that of the control group. CircSIPA1L1 is produced by a transcript encoding circSIPA1L1 on human chromosome 14 (NM_015556). Through miRDB, miRTarBase, and TargetScan database analysis, binding sequences in 3’UTR of miR-617 and circSIPA1L1 have been predicted 21 . As is well known, microRNAs (miRNAs) are ncRNAs with short chains (19-25 nt), which inhibiting target gene translation through complementary base pairing 22 . MiRNAs are extensively involved in bone homeostasis through mediating certain cytokines and transcription factors, thereafter affecting bone formation, remodeling, defect repair, and bone diseases 23 . Dysfunctional miRNAs in osteoporosis have been proven to exert a certain therapeutic potential. Bone destruction and strength are greatly improved in osteoporosis mice intravenously administrated with chemically synthesized miR-106b-5p, miR-17-5p or miR-451 24 . This study illustrated the influence of circSIPA1L1 on osteogenesis of DPSCs. Our findings uncovered that knockdown of circSIPA1L1 or overexpression of miR-617 remarkably inhibited osteogenesis of DPSCs. Mechanically, circSIPA1L1 sponged miR-617 to up-regulate phosphorylated Smad3. Our results provide potential therapeutic strategies for bone regeneration through targeting osteogenesis of DPSCs. Materials And Methods DPSCs extraction and cell culture Primarily healthy third molars were collected from young people aged 18-25 years after informed consent at Oral and Maxillofacial Surgery of the Jiangsu Provincial Stomatological Hospital. The collection process obeyed the ethical approval of Nanjing Medical University. Tooth was removed and then placed in PBS buffer containing 100 U / mL penicillin. After washing with a sterile saline solution, the surface adhered gingival tissues and blood clots were removed. Dental pulp was gently harvested in fresh culture medium And digested in 4 mg / mL trypsin (Gibco, Life Technologies, Grand Island, NY) containing 3 mg / mL collagenase type I (Gibco, Life Technologies) at 37°C. 30 min later, isolated cells were inoculated in 6-cm culture dishes with α-MEM (Gibco, Life Technologies) containing 10% fetal bovine serum (FBS, Gibco, Life Technologies), 100 μg/mL streptomycin and 100 U / mL penicillin in a 5% CO 2 incubator at 37°C. On the third day, solution was changed and medium was replaced every two days since after. Cell passage at a ratio of 1:3 was conducted at 70-80% confluence, and third - fifth generation cells were utilized for subsequent experiments. Isolated DPSCs were induced for osteogenesis at 50–60% confluence in osteogenesis medium (OM，Human Dental Pulp Stem Cell Osteogenic Differentiation Basal Medium, Cyagen Biosciences Inc, USA): standard GM containing 100 μM ascorbic acid, 2mM 2‐glycerophosphate, and 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA). OM was replaced every 2 days. DPSCs characterization STRO-1 is a protein-tagged gene of MSCs, which is the first isolated monoclonal antibody to identify MSCs. Cultured cells (3d) were subjected to immunofluorescence staining with an antibody of STRO-1 (1:200, Novus Biologicals, Littleton, CO, USA), followed by determination of positive expression of STRO-1. Meanwhile, cells were incubated with CD34-FITC, CD45-PerCP, CD90-PE, CD105-APC and CD73-PE (Miltenyi, Bergisch Gladbach, Germany), and subjected to FCM analysis (BD Biosciences, CA, USA). Tri-lineage differentiation of DPSCs Mineralized nodule formation of DPSCs was determined by ARS staining, as described previously 25 . Osteogenesis ability of third generation DPSCs was examined by induction in OM for 14 days according to the instructions. Then, DPSCs were reacted in 4% paraformaldehyde for 15 minutes and dyed with ARS (pH=4.2, Sigma, Aldrich) for 10 min. ARS was diluted in 10% cetylpyridinium chloride (CPC) to calculate the number of calcified nodules. OD value was determined at 570 nm. DPSCs were incubated in adipogenic differentiation medium (adult fat adipose‐derived stem cell adipogenic differentiation medium, Cyagen Biosciences Inc, USA). When the cell fusion reached 80%, the adipogenic induction group was added with 2 ml OriCell adipogenic differentiation medium A solution. After 3 days, OriCell adipogenic differentiation medium B solution was replaced. After 24 h, change the A solution to culture. After 25 days, Oil Red O staining was conducted to assess adipogenic differentiation in fixed DPSCs. Three-dimensional pellet culture of DPSCs (2.5×10 5 cells) in a 15 ml sterile tube was conducted for 25-day chondrogenic differentiation. Induction medium was changed every two days with the lids of the tube loosened. Pellets fixed and embedded in OCT compounds in 5 µm thickness (Sakura Finetek Co., Ltd., Tokyo, Japan) were dyed with Alcian Blue. Cell transfection Three circSIPA1L1 siRNAs (100 nM) were designed by Ribobio (Ribobio, China) and their sequences were as follows: siRNA-1: CTGGATGAACAAGGGAGAA; siRNA-2: ATGAACAAGGGAGAAAGCA; siRNA-3: AGGGAGAAAGCATGGGATT (Figure 1E),the si-NC group were transfected with randomized sequence of siRNA, the transfection efficacy was tested by RT-PCR and at last, circSIPA1L1 siRNA-1 and siRNA-3 were selected (Figure 1F).Meanwhile, miR-617 mimic (50 nM), miR-617 mimic NC (50 nM), miR-617 inhibitor (100 nM) and miR-617 inhibitor NC (100 nM) were purchased from Ribobio as well. To overexpress the circSIPA1L1, we designed an overexpression plasmid of circSIPA1L1 and the NC group were transfected with an empty vector, after transfection using Lipofectamine 2000 (Invitrogen, USA) for 48-96 h, the transfection efficacy was tested by RT-PCR Complete medium was replaced at 6 h. Flow cytometry DPSCs were cultured for three days, then collected by trypsin (Beyotime, Haimen, China) and fixed in alcohol overnight at 4 °C in dark. After PBS wash, samples were subjected to FACScan flow cytometer (BD Biosciences, San Jose, CA) and independently analyzed for three times. Cell proliferation assay Proliferative potential of DPSCs was determined by the Cell Counting Kit-8 (CCK-8) (Dojindo, Tokyo, Japan) assay and EdU incorporation assay. For CCK-8 assay, 3 × 10 3 DPSCs were inoculated in each well of a 96-well plate. After 24 hours of culture, the medium were replaced with osteogenesis medium. After 1, 3, 5, 7, and 9 days of culture, DPSCs were treated with CCK‐8 regents at 37°C for 2 h and the optical density (OD) at 450 nm was measured by a microplate reader. For EdU incorporation assay, 5 × 10 3 DPSCs were treated with 50 mM 5 ethynyl-20-deoxyuridine (EdU, Ribobio) at 37 °C for 6 h. After 30-min fixation in 4% paraformaldehyde (PFA), DPSCs was treated with 2 mg / ml glycine for 10 min, 0.5% Triton X-100 and 1 × Apollo reaction mixture for 30 min. Subsequently, DPSCs were treated with 1×Hoechst-33342 solution in dark for 30 min at room temperature (RT) and images were captured by fluorescence microscopy. ALP staining At day 7 of osteogenesis, DPSCs were fixed in 4% PFA for 15 min and washed with PBS for three times. ALP staining was performed using the NBT / BCIP staining kit (Beyotime, China) and images were captured using a microscope (Olympus, Japan). Western blot RIPA lysis buffer (Beyotime, China) was used to isolate cellular protein, which was further loaded onto 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, MA, USA). After 2-h blockage in 5% skim milk, the membrane was incubated with diluted OSX, RUNX2, ALP (Abcam, UK), Smad3 and GAPDH (Cell Signaling Technology) at 1:1000 overnight at 4 °C. After TBST wash for three times, the membrane was reacted with the corresponding secondary antibody for 1 h at RT. Grey value was analyzed by ImageJ software. Reverse transcription polymerase chain reaction (RT-PCR) RNAs extracted from DPSCs using TRIzol (Invitrogen, USA) underwent reverse transcription by PrimeScript RT kit (TaKaRa, Otsu, Japan). RT-PCR was performed on an ABI 7300 real-time PCR system with Universal ChamQTM SYBR Green quantitative PCR Master Mix (Vazyme, Nanjing, China). GAPDH and U6 were the internal references for mRNA and miRNA, respectively. Bulge-Loop miRNA qPCR Primer kit (RiboBio) was used for measuring miRNA-617 expression. Primer sequences for ALP, OSX, RUNX2 and GAPDH were depicted in Table 1. Expression levels were calculated by the 2 −ΔΔCt method as previously reported 25 . Immunofluorescence staining After PBS washing for three times, DPSCs were subjected to 30-min incubation in 4% paraformaldehyde, 15-min incubation in 0.1% Triton X-100 (Beyotime) and 2-h blockage in normal goat serum (DCS / BioGenex, Hamburg, Germany) at RT. After treatment with primary and T fluorescent dye-labeled designated secondary antibody at appointed time points, nuclei were counterstained with DAPI (Beyotime). Immunofluorescence images were observed under a fluorescent inverted microscope (Olympus, Shanghai, China). Dual-luciferase reporter assay Dual-luciferase reporter assay was conducted as described previously 25 . In brief, HEK293T cells seeded in 24-well plates (5×10 5 cells/well) were co-transfected with Firefly Luciferase reporter vector (800 ng), Renilla Luciferase reporter vector (5 ng wild-type or mutant-type, GeneChem, Shanghai, China) and 50 nM miR-617 mimics or negative control using Lipofectamine 2000. Luciferase activity measured by the Dual-Luciferase Reporter Assay System (Promega) was finally calculated as Firefly luciferase activity normalized to that of Renilla. Animal procedures Animal procedures followed institutional guidelines and got approval of the Ethics Committee of Nanjing Medical University. Fifteen 5-week homozygous nude mice were provided by the Animal Center of Nanjing Medical University. Mice were habituated for 1 week with 3-4 per cage. They were randomly assigned into three groups (n=5 per group) and subcutaneously transplanted with DPSCs transfected with si-NC, si-circSIPA1L1-1 or si-circSIPA1L1-3, respectively. Specifically, transfected DPSCs underwent osteogenesis for 2 weeks, followed by treatment with Bio-Oss collagen (Geistlich, Germany) scaffold for 12 h at 37 °C. Make two longitudinal incisions on the back of the nude mouse, and bluntly separate to form dorsal subcutaneous pocket, where two implants were inserted. Eight weeks later, the implant was removed and fixed in 4% PFA. Histology Sections were decalcified in 10% EDTA (pH 7.4) for 4 weeks with EDTA solution replacement every other day, dehydrated and paraffin embedded. Subsequently, sections were sagittaly sectioned, deparaffinized and visualized by hematoxylin and eosin (H&E) or Masson's trichrome staining. Images were captured using a microscope. Statistical processing Statistical Package for Social Sciences (SPSS) software 16.0 was used for statistical analyses. One-way analysis of variance (ANOVA) and Student's t -test were used for comparing differences. A two-tailed P < 0.05 considered as statistically significant. Data were expressed as mean ± SD of from at least three independent experiments. Results Phenotype identification of DPSCs The morphology of primary generation DPSCs were fibroblast- or spindle-like (Figure S1A). To identify the phenotype and qualification of extracted DPSCs, the multipotency, including chondrogenic, adipogenic and osteogenic differentiation, was tested 26 . Flow cytometry results demonstrated that the isolated DPSCs were negative for hematopoietic markers (CD34, CD45) (Figure S1B), but positive for MSC markers (CD29, CD90, CD73 and CD105) (Figure S1C). Tri‐lineage differentiation of DPSCs was firstly confirmed (Figure S1D). Meanwhile, immunofluorescence staining results showed that DPSCs were positive for the MSC surface molecule STRO-1 (Figure S1D). The above results all verified the stem cell characteristics of isolated DPSCs. Identification of the circular structure To identify the circular structure of circSIPA1L1, the SIPA1L1-expressing plasmid was designed (Figure 1A). Head-to-tail splicing of circSIPA1L1, and its genome size and sequences were confirmed by Sanger sequencing (Figure 1A). Moreover, divergent and convergent primers were used and it is found that circSIPA1L1, but not linear SIPA1L1 was resistant to RNase R digestion (Figure 1B). To exclude the possibility that head-to-tail splicing product of circSIPA1L1 comes from genomic rearrangement or trans-splicing, its cDNA and gDNA of 293T cells either with RNase R or not were detected. Supplemental expression level of reverse splicing or canonical form of SIPA1L1 was shown (Figure 1C). Subsequently, FISH identified that circSIPA1L1 was mainly distributed in the cytoplasm of DPSCs with 18S and U6 as the internal control (Figure 1D). We hypothesized that circSIPA1L1 regulates the biological characteristics of DPSCs via the ceRNA mechanism. In summary, circSIPA1L1 was identified as a stable circRNA and deserved further exploration. CircSIPA1L1 is upregulated and miR-617 is downregulated during osteogenesis of DPSCs Dynamically expressed circSIPA1L1 and miR-617 during osteogenesis in DPSCs were detected. Three circSIPA1L1 small interfering RNAs (siRNAs) specifically targeting the backsplice junction sequences at different binding sites in circSIPA1L1 were designed (Figure 1E). Small interfering RNA transfection efficiency was detected by RT-PCR. The results showed that si-circSIPA1L1-1 and si-circSIPA1L1-3 could effectively knock down the expression of circSIPA1L1(Figure 1F) . Meanwhile, the expression of circSIPA1L1 between NC and circSIPA1L1 group were detected by RT-PCR, the results showed that circSIPA1L1 could effectively increase the expression of circSIPA1L1(Figure 1G). CircSIPA1L1 was time-dependently upregulated and miR-617 was downregulated in osteogenic DPSCs. Moreover, mRNA levels of osteogenesis markers ALP , OSX and RUNX2 were remarkably upregulated during the process of osteogenesis (Figure 1H), demonstrating the successful induction of osteogenesis. CircSIPA1L1 have no effect on DPSCs proliferation To elucidate the role of circSIPA1L1 in DPSCs proliferation, CCK-8, flow cytometry and EdU assay were conducted. FCM analysis did not show significant differences in the proliferation index (PI = G2M ± S) between the NC group (9.15%) and the circSIPA1L1 group (8.15%, P > 0.05, Figure S2A). Similarly, no significant difference was found in the proliferation index between the si-NC group (4.72%), the si-circ-SIPA1L1-1 group (5.23%), and the si-circ-SIPA1L1-3 group (5.32%, P >0.05, Figure S2A). In addition, the results of the EdU assay showed no significant difference between the NC group and the circSIPA1L1 group ( P > 0.05 , Figure S2B, C) or between the si-NC, si-circ-SIPA1L1-1 and si-circ-SIPA1L1-3 groups (Figure S2B, D). The CCK-8 assay showed no significant difference in proliferation rates between the NC group and the circSIPA1L1 group (Figure S2E) or between the si-NC, si-circ-SIPA1L1-1 and si-circ-SIPA1L1-3 groups from 0 days to 9 days ( P > 0.05) (Figure S2F). Taken together, the data demonstrated that circSIPA1L1 does not affect the proliferation of DPSCs. CircSIPA1L1 stimulates DPSCs osteogenesis To further analyze the effect of circSIPA1L1 on osteogenic differentiation of DPSCs, protein and mRNA levels of ALP, OSX and RUNX2 were detected by Western blot and RT-PCR in osteogenic DPSCs. Western blot results showed that protein expression of ALP, OSX and RUNX2 were up-regulated in the overexpression group of circSIPA1L1 (Figure 2A). The results of RT-PCR indicated that circSIPA1L1 overexpression increased ALP , OSX and RUNX2 (Figure 2E).Whereas the expression of protein level was down-regulated when circSIPA1L1 was knocked down in DPSCs (Figure. 2B), and the results of RT-PCR indicated that the level of ALP , OSX and RUNX2 were decreased in circSIPA1L1 knockdown of DPSCs (Figure 2H). After 7 days of osteogenesis, ALP staining showed decreased ALP activity after knockdown of circSIPA1L1 and obviously upregulated by circSIPA1L1 overexpression (Figure 2C). After 14 days of induction, alizarin red staining showed reduced matrix mineralization in DPSCs with circSIPA1L1 knockdown whereas circSIPA1L1 overexpression obtained the opposite effects (Figure 2C, F, I). Identically, positive expressions of ALP and OSX were downregulated by circSIPA1L1 knockdown in DPSCs as immunofluorescence revealed (Figure 2D). These results indicated that circSIPA1L1 stimulated DPSCs osteogenesis. DPSCs stably down expressing circSIPA1L1 and controls were loaded on Bio-Oss Collagen scaffolds, and implanted in the subcutaneous tissues of nude mice for 8 weeks growth. Both H&E and Masson staining showed less bone-like structures and collagen deposit in DPSCs of the circSIPA1L1-downexpressing group than control group (Figure 2G). CircSIPA1L1 sponges miR-617 CircRNAs are able to regulate downstream gene expressions and functions by sponging corresponding miRNAs. Transfection efficiency of miR-617 mimics and inhibitor was verified by RT-PCR (Figure 3A). It is shown that circSIPA1L1 expression was negatively regulated by miR-617 (Figure 3B). To further validate their interaction, FISH analysis was conducted in DPSCs, and the results revealed that miR-617 colocalized with circSIPA1L1 in the cytoplasm (Figure 3C, D). Through analyses on miRDB, miRTarBase, and TargetScan database, a binding site in 3’UTR of miR-617 and circSIPA1L1 was discovered (Figure 3E). Subsequently, dual-luciferase reporter assay was conducted to test the interaction between circSIPA1L1 and miR-617. 293T cells were co-transfected with miR-617 mimics/negative control and wild-type/mutant-type circSIPA1L1, respectively. Overexpression of miR-617 markedly quenched luciferase activity in wild-type circSIPA1L1 compared with controls, verifying the direction interaction between circSIPA1L1 and miR-617 (Figure 3F). These observations indicated that circSIPA1L1 and miR-617 coexisted in the cytoplasm, and circSIPA1L1 acted as a miRNA sponge for miR-617 in DPSCs. MiR-617 inhibits DPSCs osteogenesis Next, the potential influence of miR-617 on DPSCs osteogenesis was explored. Western blot revealed that the protein levels of ALP, OSX and RUNX2 increased in the miR-617 knockdown group and the opposite effect was observed in the miR-617 overexpression group (Figure 4A, B).RT-PCR analysis confirmed that the expression of osteogenic related genes ALP , OSX and RUNX2 were significantly lower in the miR-617 overexpressing group than in the control group, while knockdown of miR-617 increased the gene expression of these osteogenic markers (Figure 4C). After 7 days of osteogenesis, ALP staining showed that miR-617 negatively regulated ALP activity in DPSCs (Figure 4D). After 14 days of osteogenesis, alizarin red staining showed that the formation of mineralized nodules in DPSCs was negatively mediated by miR-617 as well (Figure 4E, F). Immunofluorescence staining analysis showed that positive expressions of ALP and OSX were upregulated in DPSCs with miR-617 knockdown group, which were downregulated in those overexpressing miR-617 group (Figure 4G). In conclusion, the above findings demonstrated that miR-617 was a negative regulator in DPSCs osteogenesis. MiR-617 directly targets Smad3 Similarly, downstream genes binding miR-617 were predicted using miRDB, miRTarBase, miRWalk and TargetScan algorithms (Figure 5A). A total of 10,461 potential target genes of miR-617 were obtained (see supplement file1). GO and KEGG pathway analysis indicated that these target genes were mainly involved in intracellular activities (Figure 5B, C). Interestingly, Smad3 was a shared gene predicted in miRDB, miRWalk and TargetScan databases. As an intracellular protein, Smad3 induces nuclear transportation of extracellular transforming growth factor β ligands, thereafter activating transcription of downstream genes. Binding sequences in 3’UTR of Smad3 and miR-617 were shown (Figure 5D), and the complementary regions between these different species were also highly conserved. CircSIPA1L1/ miR-617 /Smad3 axis is responsible for DPSCs osteogenesis To test the interaction between miR-617 and Smad3, pciCHECK2-Smad3 and psiCHECK2-mut-Smad3 were constructed. Dual-luciferase reporter assay uncovered decreased luciferase activity after co-transfection of miR-617 mimics and pciCHECK2-Smad3, confirming the direct interaction between miR-617 and Smad3 (Figure 5D). Interestingly, Smad3 level was positively regulated by circSIPA1L1, but negatively regulated by miR-617. CircSIPA1L1/miR-617 induces osteogenic differentiation of DPSCs by targeting Smad3. Western blot assay showed higher protein levels of Smad3 in NC group and miR-617 inhibitor than miR-617 mimic group and miR-617 inhibitor NC group respectively. Meanwhile, Si-NC group showed higher protein levels of Smad3 than Si-circSIPA1L1-3 group and Si-circSIPA1L1-1 group respectively (Figure 6A-C). Immunofluorescence assay revealed a similar result (Figure 6D). MiR-617 reversed regulatory effect of circSIPA1L1 on DPSCs osteogenesis Rescue experiments were conducted to clarify the involvement of miR-617/Smad3 in circSIPA1L1-mediated osteogenesis. Western blot results showed that downregulated RUNX2, ALP, OSX and Smad3 in osteogenic DPSCs with circSIPA1L1 knockdown were partially reversed by co-silence of miR-617 (Figure 6E, F). Discussion In recent years, critical functions of circRNAs in human diseases have been highlighted, which may provide a theoretical basis for developing novel treatments 19, 27, 28 . Serving as ceRNAs, circRNAs can sponge miRNAs, proteins and trans-acting elements, thus influencing gene transcription, expressions and functions. In this paper, we focused on the role of circSIPA1L1 in the bone regeneration and its potential mechanism. Owing to the multilineage differentiation potential, DPSCs are considered as candidates in bone regeneration. Our finding showed that circSIPA1L1 was dynamically upregulated during DPSCs osteogenesis, while miR-617 showed the opposite trend. CircSIPA1L1 was unable to influence proliferative potential of osteogenic DPSCs. However, it indeed stimulated osteogenesis of DPSCs as ALP and ARS staining indicated. As an early marker of calcification, ALP is linked to osteoblast activity and osteogenesis specificity 29 . Its expression and activity were markedly enhanced in the early stage of mineralization. RUNX2 is a transcriptional regulator responsible for early-stage osteogenesis, which directly affects gene expressions associated with intracranial secretion and bone tissue enrichment. Studies have shown that RUNX2 knockout mice performed complete lack of bone formation 30 . OSX is a critical downstream gene of RUNX2 that is involved in bone formation and osteoblast differentiation. It is reported that MSCs isolated from OSX-deficient mice cannot be differentiated into osteoblasts 31 . Here, osteogenesis markers (e.g. ALP, RUNX2, OSX) were found to be positively regulated by circSIPA1L1, further confirming our findings. In addition, a subcutaneous transplantation model in nude mice by cell scaffold material was established. histological examination results were consistent with the in vitro conclusions. Taken together, these findings indicated that circSIPA1L1 stimulated DPSCs osteogenesis. Recently, ceRNA hypothesis proposed a vital regulatory loop, that is, circRNA-miRNA-mRNA axis 32 . For instance, circNRIP1 aggravates gastric cancer progression by sponging microRNA-149-5p via the AKT1/mTOR pathway 33 . In addition, circHIPK3 promotes the proliferative and differentiation potentials of chicken myoblasts by sponging miR-30a-3p 34 . Vital functions of miRNAs in stem cell regulation have been well concerned. Many miRNAs have been identified to participate in osteoblast differentiation processes 35 . For example, miR-21, miR-26a and miR-196 are involved in MSCs osteogenesis 36, 37, 38 . To explore the potential contributing mechanisms of circSIPA1L1 in DPSCs osteogenesis, bioinformatics analysis was conducted to seek potential targets binding circSIPA1L1. Our results demonstrated that miR-617 was the target gene binding circSIPA1L1 through dual-luciferase reporter assay, which negatively mediated DPSCs osteogenesis. Notably, miR-617 was capable of abolishing regulatory effect of circSIPA1L1 on DPSCs osteogenesis. In the cytoplasm, ceRNAs can affect mRNA stability and translational regulation under the circumstances that two interacted genes should be colocalized 39, 40 . FISH results illustrated that circSIPA1L1 and miR-617 were co-localized in the cytoplasm of DPSCs. In a similar way, Smad3 was discovered to be downstream gene of miR-617, which is an important component of TGF-β signaling. Smad3 (mothers against decapentaplegic homolog 3) is a crucial regulator of TGF-β/ Smads pathway 41 . It is able to mediate the synthesis and degradation of extracellular matrix, as well as cell phenotypes 42, 43 . After TGF-β and RUNX2 induction, Smads are activated and accumulated to contribute to skeleton formation 44 . It is reported that miR-708 can effectively abolish the inhibitory effect of Dex on osteoblast differentiation by upregulating Smad3 45 . In this paper, Smad3 was proven to be the downstream gene binding miR-617. Its level was positively regulated by circSIPA1L1 and negatively regulated by miR-617. Therefore, we hypothesized a circSIPA1L1/miR-617/Smad3 axis responsible for mediating DPSCs osteogenesis. Overexpression of inflammation-induced miR-223-3p triggers odontoblast differentiation of DPSCs by targeting Smad3 46 . The synergistic activity of Smads following Runx2 activation is of significance in bone formation. The Smad pathway mediates differentiation of mesenchyma progenitors through converging RUNX2 47 . Taken together, we believed that circSIPA1L1/miR-617/Smad3 axis stimulated DPSCs osteogenesis. Conclusion CircSIPA1L1 is dynamically upregulated under mineralization-inducing conditions. CircSIPA1L1/miR-617/Smad3 axis is responsible for stimulating DPSCs osteogenesis, which can be utilized as bone regeneration targets. Abbreviations CircSIPA1L1: Circular RNA SIPA1L1;DPSCs: Dental pulp stem cells; miRNA: MicroRNA;ARS: Alizarin red Staining; RT-PCR: Reverse transcription polymerase chain reaction; ALP: Alkaline phosphatase; RUNX2: Runt-related transcription factor 2; MSCs: Mesenchymal stem cells; ceRNA: Competing endogenous RNA; 3′UTR: 3′ untranslated region; NC: Negative control Declarations Acknowledgements Not applicable. Authors’ contributions Xingyun Ge and Zehan Li conceived and designed the study, collected and assembled data and wrote the manuscript. These two authors contributed equally to this work. Zhou Zhou performed data analysis and interpretation, Yibo Xia and MinxiaBian reviewed the data. Jinhua Yu conceived and designed the study, provided financial support and study material, performed data analysis and interpretation, approved the final version of the manuscript. All authors read and approved the manuscript. Funding This study has been partially funded by a grant from the National Natural Science Foundation of China (81873707, 81900962), Medical Talent Project of Jiangsu Province (ZDRCA2016086). And Science and Technology Development Project of Jiangsu Province (BE2017731) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX19_1147). Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. 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Supplementary Files supplementfile1.xls Supplementfigure.docx Cite Share Download PDF Status: Published Journal Publication published 24 Aug, 2020 Read the published version in Stem Cell Research & Therapy → Version 2 posted Editorial decision: Accept 31 Jul, 2020 Review # 1 received at journal 19 Jul, 2020 Reviewer # 1 agreed at journal 18 Jul, 2020 Reviewers invited by journal 15 Jul, 2020 Editor assigned by journal 13 Jul, 2020 Submission checks completed at journal 12 Jul, 2020 Editor invited by journal 12 Jul, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-32816","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":844393,"identity":"16ad4534-3e59-47fa-9458-3c1be09a9c60","order_by":0,"name":"Xingyun Ge","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xingyun","middleName":"","lastName":"Ge","suffix":""},{"id":844394,"identity":"1414ea44-c886-4f35-88cb-ddf2be1554ff","order_by":1,"name":"Zehan Li","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zehan","middleName":"","lastName":"Li","suffix":""},{"id":844395,"identity":"15a555ad-a3bb-4704-840b-4cea831af27e","order_by":2,"name":"Zhou Zhou","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhou","middleName":"","lastName":"Zhou","suffix":""},{"id":844396,"identity":"39a88e7c-c142-466d-9021-011ccacb93e2","order_by":3,"name":"Yibo Xia","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yibo","middleName":"","lastName":"Xia","suffix":""},{"id":844397,"identity":"39988904-da29-4f50-8a0f-281da9046f15","order_by":4,"name":"Minxia Bian","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Minxia","middleName":"","lastName":"Bian","suffix":""},{"id":844398,"identity":"5a0b1d2c-2f87-4742-99d5-47d276d61d72","order_by":5,"name":"Jinhua Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYHCChAOMDQwM/EAGiAdiE6lFsoEELRBlBgcQbPzA4EbCw8OFOw7LG99ueLqZh8FGdsMB5mcPCGhJODzzzGHDbXcOpN3mYUgz3nCAzdyAoBbetsOM224kgLQcTtxwgIdNghgt9ptngLX8J15L4gYJsJYDhLVInnkA0pKePAPosJtzDJKNZx5mM8Orhe94TvJn3jZr2/4ZOWk33lTYyfYdb36GV4vCAZ4EKBPEAAUVMz71QCDfwH4AyoQzRsEoGAWjYBSgAgAKL1VYlDy4XgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4874-9910","institution":"","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jinhua","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2020-06-01 04:19:20","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-32816/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-32816/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-020-01877-3","type":"published","date":"2020-08-24T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1591344,"identity":"c5f68910-3e31-49af-a861-5964176a0f1f","added_by":"auto","created_at":"2020-07-16 20:55:09","extension":"tif","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5186970,"visible":true,"origin":"","legend":"Identification of the circular structure. A. Head-to-tail splicing of circSIPA1L1, and its genome size and sequences tested by Sanger sequencing. a. Schematic representation of SIPA1L1-expressing plasmid. b. Agarose gel electrophoresis of RT-PCR products from HEK293T cells transfected with SIPA1L1-expressing plasmid or empty vector. c. Sanger sequencing of RT-PCR products from 293T cells, the arrows indicate fusion sites. B. We further confirmed that circSIPA1L1 was resistant to RNase R. rather than linear-SIPA1L1 could resist digestion by RNase R. C. The existence of circSIPA1L1 was validated in 293T cell lines by RT-PCR. Divergent primers amplified circSIPA1L1 in cDNA but not genomic DNA (gDNA). GAPDH was used as negative control. D. FISH assay showed the localization of circSIPA1L1 in the cytoplasm. 18S and U6 ware the internal control. E. Three circSIPA1L1 small interfering RNAs (siRNAs) specifically targeting the backsplice junction sequences at different binding sites in circSIPA1L1 were designed. F. Small interfering RNA silencing efficiency was detected by RT-PCR. The results showed that si-circSIPA1L1-1 and si-circSIPA1L1-3 could effectively knock down the expression of circSIPA1L1(*P \u003c 0.05, **P \u003c 0.01). G. The expression of circSIPA1L1 between NC and circSIPA1L1 group were detected by RT-PCR. H. Dynamic expressions of ALP, RUNX2 and OSX during DPSCs osteogenesis at day 0, 3 and 7. Dynamic expressions of circSIPA1L1 and miR-617 during DPSCs osteogenesis at day 0, 3 and 7. RNA level was normalized to that at day 0. GAPDH and U6 were the internal controls, respectively. *P \u003c 0.05, **P \u003c 0.01.","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-32816/v2/Figure1.tif"},{"id":1591345,"identity":"b9831050-3187-40f2-aeaf-cf5cd5508bc7","added_by":"auto","created_at":"2020-07-16 20:55:10","extension":"tif","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15736460,"visible":true,"origin":"","legend":"CircSIPA1L1 stimulates DPSCs osteogenesis. A. Western blot results revealed that the protein levels of OSX, RUNX2 and ALP significantly increased in circSIPA1L1 group compared with the NC group. B. Western blot assay showed higher protein levels of ALP, RUNX2, and OSX in Si-NC group than Si-circSIPA1L1-3 group and Si-circSIPA1L1-1 group respectively. GAPDH was the internal control. C. Images of alkaline phosphatase (ALP) staining in the different groups, Si-NC treatment led to the highest ALP activity. Cells were cultured for 7 days. After 14 days of co-culture, the formation of mineralized nodules in DPSCs in circSIPA1L1 group generated more calcified nodules than NC group, meanwhile, Si-NC group have more calcified nodules than Si-circSIPA1L1-3 group and Si-circSIPA1L1-1 group. \nD. Immunofluorescence staining showed positive expressions of ALP and OSX in DPSCs transfected with si-NC, si-circSIPA1L1-1 or si-circSIPA1L1-3, respectively. *P \u003c 0.05, **P \u003c 0.01. E. The mRNA levels of OSX, RUNX2 and ALP in DPSCs measured by RT-PCR following 3-day osteogenesis. The results showed higher levels of ALP, RUNX2, and OSX in circSIPA1L1 group than NC group. F. Histograms showed quantification of Alizarin red staining by spectrophotometry of NC group and circSIPA1L1 group. G. H\u0026E staining and Masson staining Si-NC, si-circSIPA1L1-1 and si-circSIPA1L1-3 groups. H\u0026E and Masson staining showed less bone-like structures and collagen deposit in DPSCs of the circSIPA1L1-downexpressing group than control group. bone/dentin-like tissues(arrow), S around the scaffold, Scale Bar=100μm. H. The results of RT-PCR showed that the expression of ALP, RUNX2, and OSX were decreased in Si-circSIPA1L1-3 group and Si-circSIPA1L1-1 group compared with the Si-NC group. n = 3. **2-ΔΔCt \u003e2, P \u003c0.01; *1 \u003c 2-ΔΔCt \u003c2, P \u003c0.05. I. The quantification of Alizarin red staining by spectrophotometry revealed that si-circSIPA1L1-1 or si-circSIPA1L1-3 showed less calcified nodules than si-NC group.\n","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-32816/v2/Figure2.tif"},{"id":1591346,"identity":"7da1f8c9-3cf0-43ab-9942-fa4a77ebded8","added_by":"auto","created_at":"2020-07-16 20:55:10","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":729735,"visible":true,"origin":"","legend":"CircSIPA1L1 sponges miR-617.A. Transfection efficacy of miR-617 mimics and inhibitor. B. Relative circ-SIPA1L1 expression level in DPSCs transfected with miR-617 mimics or inhibitor (**P \u003c 0.01). C, D. FISH showed co-localization of circSIPA1L1 and miR-617 in the cytoplasm of DPSCs cells(arrow). U6 and 18S were the internal control. E. Binding sequences in 3’UTR of circSIPA1L1 and miR-617 predicted online. F. Luciferase activity in 293T cells co-transfected with miR-617 mimics/NC and wild-type/mutant-type circSIPA1L1, respectively. *P\u003c 0.05, **P\u003c 0.01.","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-32816/v2/Fig3.jpg"},{"id":1591347,"identity":"66740438-22e0-40ae-845a-736cbdb8bb16","added_by":"auto","created_at":"2020-07-16 20:55:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1095080,"visible":true,"origin":"","legend":"MiR-617 inhibits DPSCs osteogenesis. A. Western blot assay showed higher protein levels of ALP, RUNX2, and OSX in NC group and miR-617 inhibitor group than mimics group and miR-617 inhibitor NC group respectively. GAPDH was the internal control. B. Grayscale analyses. *P \u003c 0.05 or **P \u003c 0.01.C. RT‐PCR showed higher levels of ALP, RUNX2, and OSX in NC group and inhibitor than mimics group and iNC group respectively. D. ALP staining in DPSCs following 7-day osteogenesis with overexpression or knockdown of miR-617. E. Histograms showed quantification of Alizarin red staining by spectrophotometry. F. After 14 days of co-culture, Upper: alizarin red staining showed that miR-617 mimics group generated more calcified nodules than control group. MiR-617 inhibitor group generated more calcified nodules than iNC group. Lower: Mineralized nodules in different groups under the inverted microscope. (OM: osteogenic medium). G. Immunofluorescence showed positive expressions of ALP and OSX in DPSCs with overexpression or knockdown of miR-617. *P \u003c 0.05, **P \u003c 0.01.","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-32816/v2/Figure4.jpg"},{"id":1591348,"identity":"fe2e44f3-2946-4f39-a9e4-db02b09cb355","added_by":"auto","created_at":"2020-07-16 20:55:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":611412,"visible":true,"origin":"","legend":"MiR-617 directly targets Smad3.A. The Venn plots showed predicted downstream genes binding miR-617. B, C. Go annotation (B) and KEGG pathway analysis (C) showed the top 25 target genes and their enriched pathways. GO: A field directly related to reproduction.\nD. Binding sequences in 3’UTR of miR-617 and Smad3 predicted online. The red letters represent the binding sequence of miR-617.Luciferase activity in 293T cells co-transfected with miR-617 mimics/NC and wild-type/mutant-type smad3, respectively.","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-32816/v2/Fig5.jpg"},{"id":1591349,"identity":"fe475b1b-0e72-4f74-b2d1-c8a74c17c606","added_by":"auto","created_at":"2020-07-16 20:55:10","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":860659,"visible":true,"origin":"","legend":"CircSIPA1L1/miR-617/Smad3 axis is responsible for DPSCs osteogenesis. A. Western blot assay showed higher protein levels of Smad3 in miR-NC group and miR-617 inhibitor than miR-617 mimic group and miR-617 inhibitor NC group respectively. Meanwhile, si-circSIPA1L1-downexpressing group showed lower protein levels of Smad3 than control group. B,C. Grayscale analyses.**P\u003c 0.01 or ***P\u003c 0.001.CImmunofluorescence assay revealed upregulated Smad3 in NC group and miR-617 inhibitor than miR-617 mimics group and miR-617 inhibitor NC group respectively. D. Immunofluorescence assay revealed upregulated Smad3 in Si-NC group compared with Si-circSIPA1L1-3 group and Si-circSIPA1L1-1 group. E. Protein levels of RUNX2, ALP, OSX and Smad3 in co-transfected DPSCs. *P\u003c 0.05, **P\u003c 0.01. F. Results of western blotting was analyzed with ImageJ software and data were presented as ratio of target protein to GAPDH in the form of grayscale value. (*P \u003c 0.05, **P \u003c 0.01).","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-32816/v2/Fig6.jpg"},{"id":13551625,"identity":"f92ecfcd-bf08-4e23-9ad9-46ae509d7b27","added_by":"auto","created_at":"2021-09-17 02:29:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9286835,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-32816/v2/1cfa26ec-ea2e-47ec-81dd-028c459417ce.pdf"},{"id":1591351,"identity":"0b65b73b-80d5-4a15-8f71-9e4342e96112","added_by":"auto","created_at":"2020-07-16 20:55:11","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":413450,"visible":true,"origin":"","legend":"","description":"","filename":"supplementfile1.xls","url":"https://assets-eu.researchsquare.com/files/rs-32816/v2/supplementfile1.xls"},{"id":1591352,"identity":"29c40deb-981c-4eb3-b815-8b1d2178dce1","added_by":"auto","created_at":"2020-07-16 20:55:11","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2187743,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-32816/v2/Supplementfigure.docx"}],"financialInterests":"","formattedTitle":"Circular RNA SIPA1L1 promotes osteogenesis via regulating the miR-617/Smad3 axis in dental pulp stem cells","fulltext":[{"header":"Background ","content":"\u003cp\u003eLarge bone defects caused by massive injuries, diseases or deformities can be repaired by autologous bone grafts. Nevertheless, the number of bone grafts is limited and the delicate 3D shape cannot be outlined. Therefore, effective bone regeneration for clinical needs is urgently required. Artificial bone engineering, which creates functional bone tissues using stem cells (the most optimal autologous cells) as artificial environment or scaffold, contributes to bone defect repair \u003csup\u003e1, 2\u003c/sup\u003e. In recent years, cell therapy, especially mesenchymal stem cells (MSCs), has shown good application prospects in the treatment of bone defects \u003csup\u003e3\u003c/sup\u003e. As an important member of the MSC family, the biological function of bone marrow mesenchymal stem cells has been widely recognized \u003csup\u003e4\u003c/sup\u003e. Due to its strong multi-lineage differentiation potentials and regenerative properties, great progress has been achieved in bone tissue engineering \u003csup\u003e5\u003c/sup\u003e. Sources of stem cells are diverse, including peripheral blood, bone marrows, cord blood, placenta, and teeth \u003csup\u003e6\u003c/sup\u003e. Notably, dental MSCs are easily available \u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe endodontium is a special gelatinous soft connective tissues containing nerves, blood vessels and connective tissue, which protects teeth from inflammation and infection \u003csup\u003e8, 9\u003c/sup\u003e. Dental pulp stem cells (DPSCs) are derived from dental pulp tissues, which are featured by high proliferative potential, clonogenicity, self-renewal capacity, and multi-lineage differentiation. As an important mesenchymal stem cell derived from dental pulp, DPSCs can be collected from young permanent teeth in a non-invasive way. In addition to the advantages of a wide range of sources and convenient collection, DPSCs also have the advantages of low immunogenicity and no moral controversy\u003csup\u003e10\u003c/sup\u003e. Many studies have shown that DPSCs can differentiate into neurogenic, osteogenic, dentinal and myogenic cell lineages under different induction conditions \u003csup\u003e11\u003c/sup\u003e. \u003cem\u003eIn vivo\u003c/em\u003e studies have shown that DPSCs are capable of producing lamellar bones and differentiating into periodontal tissues. Therefore, DPSCs can be utilized for bone regeneration \u003csup\u003e12\u003c/sup\u003e. Moreover, clarifying osteogenesis of DPSCs is conducive to the development of regenerative medicine and bone disease treatment.\u003c/p\u003e\n\u003cp\u003eAs an important type of ncRNAs, the head-to-tail closed loop structure of circular RNAs (circRNAs) from 3\u0026rsquo; end to 5\u0026rsquo; tail results in their pronounced stability than traditional linear RNAs \u003csup\u003e13\u003c/sup\u003e. CircRNAs are extensively expressed in thousands of human genes, and sometime they exhibit higher expressions than corresponding homologous linear isoforms \u003csup\u003e14\u003c/sup\u003e. CircRNAs mainly exert transcriptional and post-transcriptional regulations on protein sponges \u003csup\u003e15, 16\u003c/sup\u003e, translation \u003csup\u003e17\u003c/sup\u003e and miRNA sponges \u003csup\u003e18\u003c/sup\u003e. The well-known competing endogenouse RNA (ceRNA) theory of circRNAs has been well concerned. In the nucleotide sequence of circRNA, some circRNAs contain multiple miRNA binding sites capable of binding miRNA, preventing them from binding to their mRNA target genes (sponge effect), thereby inhibiting the function of miRNA. CiRS-7, also known as CDR1as, has been clearly demonstrated as a typical example of miRNA sponge. CircRNA CDR1as contains over 70 miRNA-7 (miR-7) conserved binding sites that strongly inhibit miR-7 activity. After the CDR1as study was published in 2013, various other circRNAs have been shown to act as miRNA sponges. Using bioinformatics tools, miRNA binding sites can be predicted in circRNA sequences. Therefore, for the same miRNA, a circular RNA containing many miRNA binding sites is relatively easy to be found as a miRNA sponge\u003csup\u003e18, 19, 20\u003c/sup\u003e. For example, circRNA-ciRS-7 contains over 70 binding sites of miR-7, which are highly conserved and greatly inhibits its activity. However, potential functions of circRNAs in stem cell osteogenesis are rarely reported. We have previously identified differentially expressed circRNAs during osteogenesis of teeth-derived stem cells by RNA sequencing \u003csup\u003e21\u003c/sup\u003e, suggesting vital functions of circRNAs in osteogenesis. In the preliminary work, we found that the expression of circSIPA1L1 in the mineralization induction group was about 8 times that of the control group. CircSIPA1L1 is produced by a transcript encoding circSIPA1L1 on human chromosome 14 (NM_015556). Through miRDB, miRTarBase, and TargetScan database analysis, binding sequences in 3\u0026rsquo;UTR of miR-617 and circSIPA1L1 have been predicted \u003csup\u003e21\u003c/sup\u003e. As is well known, microRNAs (miRNAs) are ncRNAs with short chains (19-25 nt), which inhibiting target gene translation through complementary base pairing \u003csup\u003e22\u003c/sup\u003e. MiRNAs are extensively involved in bone homeostasis through mediating certain cytokines and transcription factors, thereafter affecting bone formation, remodeling, defect repair, and bone diseases \u003csup\u003e23\u003c/sup\u003e. Dysfunctional miRNAs in osteoporosis have been proven to exert a certain therapeutic potential. Bone destruction and strength are greatly improved in osteoporosis mice intravenously administrated with chemically synthesized miR-106b-5p, miR-17-5p or miR-451 \u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis study illustrated the influence of circSIPA1L1 on osteogenesis of DPSCs. Our findings uncovered that knockdown of circSIPA1L1 or overexpression of miR-617 remarkably inhibited osteogenesis of DPSCs. Mechanically, circSIPA1L1 sponged miR-617 to up-regulate phosphorylated Smad3. Our results provide potential therapeutic strategies for bone regeneration through targeting osteogenesis of DPSCs.\u003c/p\u003e"},{"header":"Materials And Methods ","content":"\u003cp\u003e\u003cstrong\u003eDPSCs extraction and cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimarily healthy third molars were collected from young people aged 18-25 years after informed consent at Oral and Maxillofacial Surgery of the Jiangsu Provincial Stomatological Hospital. The collection process obeyed the ethical approval of Nanjing Medical University. Tooth was removed and then placed in PBS buffer containing 100 U / mL penicillin. After washing with a sterile saline solution, the surface adhered gingival tissues and blood clots were removed. Dental pulp was gently harvested in fresh culture medium And digested in 4 mg / mL trypsin (Gibco, Life Technologies, Grand Island, NY) containing 3 mg / mL collagenase type I (Gibco, Life Technologies) at 37\u0026deg;C. 30 min later, isolated cells were inoculated in 6-cm culture dishes with \u0026alpha;-MEM (Gibco, Life Technologies) containing 10% fetal bovine serum (FBS, Gibco, Life Technologies), 100 \u0026mu;g/mL streptomycin and 100 U / mL penicillin in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C. On the third day, solution was changed and medium was replaced every two days since after. Cell passage at a ratio of 1:3 was conducted at 70-80% confluence, and third - fifth generation cells were utilized for subsequent experiments. Isolated DPSCs were induced for osteogenesis at 50\u0026ndash;60% confluence in osteogenesis medium (OM，Human Dental Pulp Stem Cell Osteogenic Differentiation Basal Medium, Cyagen Biosciences Inc, USA):\u0026nbsp; standard GM containing 100 \u0026mu;M ascorbic acid, 2mM 2‐glycerophosphate, and 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA). OM was replaced every 2 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDPSCs characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSTRO-1 is a protein-tagged gene of MSCs, which is the first isolated monoclonal antibody to identify MSCs. Cultured cells (3d) were subjected to immunofluorescence staining with an antibody of STRO-1 (1:200, Novus Biologicals, Littleton, CO, USA), followed by determination of positive expression of STRO-1. Meanwhile, cells were incubated with CD34-FITC, CD45-PerCP, CD90-PE, CD105-APC and CD73-PE (Miltenyi, Bergisch Gladbach, Germany), and subjected to FCM analysis (BD Biosciences, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTri-lineage differentiation of DPSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMineralized nodule formation of DPSCs was determined by ARS staining, as described previously\u003csup\u003e25\u003c/sup\u003e. Osteogenesis ability of third generation DPSCs was examined by induction in OM for 14 days according to the instructions. Then, DPSCs were reacted in 4% paraformaldehyde for 15 minutes and dyed with ARS (pH=4.2, Sigma, Aldrich) for 10 min. ARS was diluted in 10% cetylpyridinium chloride (CPC) to calculate the number of calcified nodules. OD value was determined at 570 nm.\u003c/p\u003e\n\u003cp\u003eDPSCs were incubated in adipogenic differentiation medium (adult fat adipose‐derived stem cell adipogenic differentiation medium, Cyagen Biosciences Inc, USA). When the cell fusion reached 80%, the adipogenic induction group was added with 2 ml OriCell adipogenic differentiation medium A solution. After 3 days, OriCell adipogenic differentiation medium B solution was replaced. After 24 h, change the A solution to culture. After 25 days, Oil Red O staining was conducted to assess adipogenic differentiation in fixed DPSCs.\u003c/p\u003e\n\u003cp\u003eThree-dimensional pellet culture of DPSCs (2.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells) in a 15 ml sterile tube was conducted for 25-day chondrogenic differentiation. Induction medium was changed every two days with the lids of the tube loosened. Pellets fixed and embedded in OCT compounds in 5 \u0026micro;m thickness (Sakura Finetek Co., Ltd., Tokyo, Japan) were dyed with Alcian Blue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree circSIPA1L1 siRNAs (100 nM) were designed by Ribobio (Ribobio, China) and their sequences were as follows: siRNA-1: CTGGATGAACAAGGGAGAA; siRNA-2: ATGAACAAGGGAGAAAGCA; siRNA-3: AGGGAGAAAGCATGGGATT (Figure 1E),the si-NC group were transfected with randomized sequence of siRNA, the transfection efficacy was tested by RT-PCR and at last, circSIPA1L1 siRNA-1 and siRNA-3 were selected (Figure 1F).Meanwhile, miR-617 mimic (50 nM), miR-617 mimic NC (50 nM), miR-617 inhibitor (100 nM) and miR-617 inhibitor NC (100 nM) were purchased from Ribobio as well. To overexpress the circSIPA1L1, we designed an overexpression plasmid of circSIPA1L1 and the NC group were transfected with an empty vector, after transfection using Lipofectamine 2000 (Invitrogen, USA) for 48-96 h, the transfection efficacy was tested by RT-PCR Complete medium was replaced at 6 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDPSCs were cultured for three days, then collected by trypsin (Beyotime, Haimen, China) and fixed in alcohol overnight at 4 \u0026deg;C in dark. After PBS wash, samples were subjected to FACScan flow cytometer (BD Biosciences, San Jose, CA) and independently analyzed for three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProliferative potential of DPSCs was determined by the Cell Counting Kit-8 (CCK-8) (Dojindo, Tokyo, Japan) assay and EdU incorporation assay. For CCK-8 assay, 3 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e DPSCs were inoculated in each well of a 96-well plate. After 24 hours of culture, the medium were replaced with osteogenesis medium. After 1, 3, 5, 7, and 9 days of culture, DPSCs were treated with CCK‐8 regents at 37\u0026deg;C for 2 h and the optical density (OD) at 450 nm was measured by a microplate reader.\u003c/p\u003e\n\u003cp\u003eFor EdU incorporation assay, 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e DPSCs were treated with 50 mM 5 ethynyl-20-deoxyuridine (EdU, Ribobio) at 37 \u0026deg;C for 6 h. After 30-min fixation in 4% paraformaldehyde (PFA), DPSCs was treated with 2 mg / ml glycine for 10 min, 0.5% Triton X-100 and 1 \u0026times; Apollo reaction mixture for 30 min. Subsequently, DPSCs were treated with 1\u0026times;Hoechst-33342 solution in dark for 30 min at room temperature (RT) and images were captured by fluorescence microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eALP staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt day 7 of osteogenesis, DPSCs were fixed in 4% PFA for 15 min and washed with PBS for three times. ALP staining was performed using the NBT / BCIP staining kit (Beyotime, China) and images were captured using a microscope (Olympus, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRIPA lysis buffer (Beyotime, China) was used to isolate cellular protein, which was further loaded onto 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, MA, USA). After 2-h blockage in 5% skim milk, the membrane was incubated with diluted OSX, RUNX2, ALP (Abcam, UK), Smad3 and GAPDH (Cell Signaling Technology) at 1:1000 overnight at 4 \u0026deg;C. After TBST wash for three times, the membrane was reacted with the corresponding secondary antibody for 1 h at RT. Grey value was analyzed by ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse transcription polymerase chain reaction (RT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNAs extracted from DPSCs using TRIzol (Invitrogen, USA) underwent reverse transcription by PrimeScript RT kit (TaKaRa, Otsu, Japan). RT-PCR was performed on an ABI 7300 real-time PCR system with Universal ChamQTM SYBR Green quantitative PCR Master Mix (Vazyme, Nanjing, China). GAPDH and U6 were the internal references for mRNA and miRNA, respectively. Bulge-Loop miRNA qPCR Primer kit (RiboBio) was used for measuring miRNA-617 expression. Primer sequences for ALP, OSX, RUNX2 and GAPDH were depicted in Table 1. Expression levels were calculated by the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method as previously reported \u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter PBS washing for three times, DPSCs were subjected to 30-min incubation in 4% paraformaldehyde, 15-min incubation in 0.1% Triton X-100 (Beyotime) and 2-h blockage in normal goat serum (DCS / BioGenex, Hamburg, Germany) at RT. After treatment with primary and T fluorescent dye-labeled designated secondary antibody at appointed time points, nuclei were counterstained with DAPI (Beyotime). Immunofluorescence images were observed under a fluorescent inverted microscope (Olympus, Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual-luciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDual-luciferase reporter assay was conducted as described previously \u003csup\u003e25\u003c/sup\u003e. In brief, HEK293T cells seeded in 24-well plates (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) were co-transfected with Firefly Luciferase reporter vector (800 ng), Renilla Luciferase reporter vector (5 ng wild-type or mutant-type, GeneChem, Shanghai, China) and 50 nM miR-617 mimics or negative control using Lipofectamine 2000. Luciferase activity measured by the Dual-Luciferase Reporter Assay System (Promega) was finally calculated as Firefly luciferase activity normalized to that of Renilla.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal procedures \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal procedures followed institutional guidelines and got approval of the Ethics Committee of Nanjing Medical University. Fifteen 5-week homozygous nude mice were provided by the Animal Center of Nanjing Medical University. Mice were habituated for 1 week with 3-4 per cage. They were randomly assigned into three groups (n=5 per group) and subcutaneously transplanted with DPSCs transfected with si-NC, si-circSIPA1L1-1 or si-circSIPA1L1-3, respectively. Specifically, transfected DPSCs underwent osteogenesis for 2 weeks, followed by treatment with Bio-Oss collagen (Geistlich, Germany) scaffold for 12 h at 37 \u0026deg;C. Make two longitudinal incisions on the back of the nude mouse, and bluntly separate to form dorsal subcutaneous pocket, where two implants were inserted. Eight weeks later, the implant was removed and fixed in 4% PFA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSections were decalcified in 10% EDTA (pH 7.4) for 4 weeks with EDTA solution replacement every other day, dehydrated and paraffin embedded. Subsequently, sections were sagittaly sectioned, deparaffinized and visualized by hematoxylin and eosin (H\u0026amp;E) or Masson's trichrome staining. Images were captured using a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical Package for Social Sciences (SPSS) software 16.0 was used for statistical analyses. One-way analysis of variance (ANOVA) and Student's \u003cem\u003et\u003c/em\u003e-test were used for comparing differences. A two-tailed \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 considered as statistically significant. Data were expressed as mean \u0026plusmn; SD of from at least three independent experiments.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePhenotype identification of DPSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphology of primary generation DPSCs were fibroblast- or spindle-like (Figure S1A). To identify the phenotype and qualification of extracted DPSCs, the multipotency, including chondrogenic, adipogenic and osteogenic differentiation, was tested \u003csup\u003e26\u003c/sup\u003e. Flow cytometry results demonstrated that the isolated DPSCs were negative for hematopoietic markers (CD34, CD45) (Figure S1B), but positive for MSC markers (CD29, CD90, CD73 and CD105) (Figure S1C). Tri‐lineage differentiation of DPSCs was firstly confirmed (Figure S1D). Meanwhile, immunofluorescence staining results showed that DPSCs were positive for the MSC surface molecule STRO-1 (Figure S1D). The above results all verified the stem cell characteristics of isolated DPSCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of the circular structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the circular structure of circSIPA1L1, the SIPA1L1-expressing plasmid was designed (Figure 1A). Head-to-tail splicing of circSIPA1L1, and its genome size and sequences were confirmed by Sanger sequencing (Figure 1A). Moreover, divergent and convergent primers were used and it is found that circSIPA1L1, but not linear SIPA1L1 was resistant to RNase R digestion (Figure 1B). To exclude the possibility that head-to-tail splicing product of circSIPA1L1 comes from genomic rearrangement or trans-splicing, its cDNA and gDNA of 293T cells either with RNase R or not were detected. Supplemental expression level of reverse splicing or canonical form of SIPA1L1 was shown (Figure 1C). Subsequently, FISH identified that circSIPA1L1 was mainly distributed in the cytoplasm of DPSCs with 18S and U6 as the internal control (Figure 1D). We hypothesized that circSIPA1L1 regulates the biological characteristics of DPSCs via the ceRNA mechanism. In summary, circSIPA1L1 was identified as a stable circRNA and deserved further exploration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCircSIPA1L1 is upregulated and miR-617 is downregulated during osteogenesis of DPSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDynamically expressed circSIPA1L1 and miR-617 during osteogenesis in DPSCs were detected. Three circSIPA1L1 small interfering RNAs (siRNAs) specifically targeting the backsplice junction sequences at different binding sites in circSIPA1L1 were designed (Figure 1E). Small interfering RNA transfection efficiency was detected by RT-PCR. The results showed that si-circSIPA1L1-1 and si-circSIPA1L1-3 could effectively knock down the expression of circSIPA1L1(Figure 1F)\u003cstrong\u003e.\u003c/strong\u003e Meanwhile, the expression of circSIPA1L1 between NC and circSIPA1L1 group were detected by RT-PCR, the results showed that circSIPA1L1 could effectively increase the expression of circSIPA1L1(Figure 1G).\u003c/p\u003e\n\u003cp\u003eCircSIPA1L1 was time-dependently upregulated and miR-617 was downregulated in osteogenic DPSCs. Moreover, mRNA levels of osteogenesis markers \u003cem\u003eALP\u003c/em\u003e, \u003cem\u003eOSX\u003c/em\u003e and \u003cem\u003eRUNX2\u003c/em\u003e were remarkably upregulated during the process of osteogenesis (Figure 1H), demonstrating the successful induction of osteogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCircSIPA1L1 have no effect on DPSCs proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the role of circSIPA1L1 in DPSCs proliferation, CCK-8, flow cytometry and EdU assay were conducted. FCM analysis did not show significant differences in the proliferation index (PI = G2M \u0026plusmn; S) between the NC group (9.15%) and the circSIPA1L1 group (8.15%, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, Figure S2A). Similarly, no significant difference was found in the proliferation index between the si-NC group (4.72%), the si-circ-SIPA1L1-1 group (5.23%), and the si-circ-SIPA1L1-3 group (5.32%, \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05, Figure S2A). In addition, the results of the EdU assay showed no significant difference between the NC group and the circSIPA1L1 group (\u003cem\u003eP \u0026gt; 0.05\u003c/em\u003e, Figure S2B, C) or between the si-NC, si-circ-SIPA1L1-1 and si-circ-SIPA1L1-3 groups (Figure S2B, D). The CCK-8 assay showed no significant difference in proliferation rates between the NC group and the circSIPA1L1 group (Figure S2E) or between the si-NC, si-circ-SIPA1L1-1 and si-circ-SIPA1L1-3 groups from 0 days to 9 days (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) (Figure S2F). Taken together, the data demonstrated that circSIPA1L1 does not affect the proliferation of DPSCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCircSIPA1L1 stimulates DPSCs osteogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further analyze the effect of circSIPA1L1 on osteogenic differentiation of DPSCs, protein and mRNA levels of ALP, OSX and RUNX2 were detected by Western blot and RT-PCR in osteogenic DPSCs. Western blot results showed that protein expression of ALP, OSX and RUNX2 were up-regulated in the overexpression group of circSIPA1L1 (Figure 2A). The results of RT-PCR indicated that circSIPA1L1 overexpression increased \u003cem\u003eALP\u003c/em\u003e, \u003cem\u003eOSX\u003c/em\u003e and \u003cem\u003eRUNX2\u003c/em\u003e\u0026nbsp; (Figure 2E).Whereas the expression of protein level was down-regulated when circSIPA1L1 was knocked down in DPSCs (Figure. 2B), and the results of RT-PCR indicated that the level of \u003cem\u003eALP\u003c/em\u003e, \u003cem\u003eOSX\u003c/em\u003e and \u003cem\u003eRUNX2\u003c/em\u003e were decreased in circSIPA1L1 knockdown of DPSCs (Figure 2H). After 7 days of osteogenesis, ALP staining showed decreased ALP activity after knockdown of circSIPA1L1 and obviously upregulated by circSIPA1L1 overexpression (Figure 2C). After 14 days of induction, alizarin red staining showed reduced matrix mineralization in DPSCs with circSIPA1L1 knockdown whereas circSIPA1L1 overexpression obtained the opposite effects (Figure 2C, F, I). Identically, positive expressions of ALP and OSX were downregulated by circSIPA1L1 knockdown in DPSCs as immunofluorescence revealed (Figure 2D). These results indicated that circSIPA1L1 stimulated DPSCs osteogenesis.\u003c/p\u003e\n\u003cp\u003eDPSCs stably down expressing circSIPA1L1 and controls were loaded on Bio-Oss Collagen scaffolds, and implanted in the subcutaneous tissues of nude mice for 8 weeks growth. Both H\u0026amp;E and Masson staining showed less bone-like structures and collagen deposit in DPSCs of the circSIPA1L1-downexpressing group than control group (Figure 2G).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCircSIPA1L1 sponges miR-617\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCircRNAs are able to regulate downstream gene expressions and functions by sponging corresponding miRNAs. Transfection efficiency of miR-617 mimics and inhibitor was verified by RT-PCR (Figure 3A). It is shown that circSIPA1L1 expression was negatively regulated by miR-617 (Figure 3B). To further validate their interaction, FISH analysis was conducted in DPSCs, and the results revealed that miR-617 colocalized with circSIPA1L1 in the cytoplasm (Figure 3C, D). Through analyses on miRDB, miRTarBase, and TargetScan database, a binding site in 3\u0026rsquo;UTR of miR-617 and circSIPA1L1 was discovered (Figure 3E). Subsequently, dual-luciferase reporter assay was conducted to test the interaction between circSIPA1L1 and miR-617. 293T cells were co-transfected with miR-617 mimics/negative control and wild-type/mutant-type circSIPA1L1, respectively. Overexpression of miR-617 markedly quenched luciferase activity in wild-type circSIPA1L1 compared with controls, verifying the direction interaction between circSIPA1L1 and miR-617 (Figure 3F). These observations indicated that circSIPA1L1 and miR-617 coexisted in the cytoplasm, and circSIPA1L1 acted as a miRNA sponge for miR-617 in DPSCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-617 inhibits DPSCs osteogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, the potential influence of miR-617 on DPSCs osteogenesis was explored. Western blot revealed that the protein levels of ALP, OSX and RUNX2 increased in the miR-617 knockdown group and the opposite effect was observed in the miR-617 overexpression group (Figure 4A, B).RT-PCR analysis confirmed that the expression of osteogenic related genes \u003cem\u003eALP\u003c/em\u003e,\u003cem\u003e OSX \u003c/em\u003eand \u003cem\u003eRUNX2 \u003c/em\u003ewere significantly lower in the miR-617 overexpressing group than in the control group, while knockdown of miR-617 increased the gene expression of these osteogenic markers (Figure 4C). After 7 days of osteogenesis, ALP staining showed that miR-617 negatively regulated ALP activity in DPSCs (Figure 4D). After 14 days of osteogenesis, alizarin red staining showed that the formation of mineralized nodules in DPSCs was negatively mediated by miR-617 as well (Figure 4E, F). Immunofluorescence staining analysis showed that positive expressions of ALP and OSX were upregulated in DPSCs with miR-617 knockdown group, which were downregulated in those overexpressing miR-617 group (Figure 4G). In conclusion, the above findings demonstrated that miR-617 was a negative regulator in DPSCs osteogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-617 directly targets Smad3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSimilarly, downstream genes binding miR-617 were predicted using miRDB, miRTarBase, miRWalk and TargetScan algorithms (Figure 5A). A total of 10,461 potential target genes of miR-617 were obtained (see supplement file1). GO and KEGG pathway analysis indicated that these target genes were mainly involved in intracellular activities (Figure 5B, C). Interestingly, Smad3 was a shared gene predicted in miRDB, miRWalk and TargetScan databases. As an intracellular protein, Smad3 induces nuclear transportation of extracellular transforming growth factor \u0026beta; ligands, thereafter activating transcription of downstream genes. Binding sequences in 3\u0026rsquo;UTR of Smad3 and miR-617 were shown (Figure 5D), and the complementary regions between these different species were also highly conserved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCircSIPA1L1/\u003c/strong\u003e\u003cstrong\u003emiR-617\u003c/strong\u003e\u003cstrong\u003e/Smad3 axis\u003c/strong\u003e\u003cstrong\u003e is responsible for DPSCs osteogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test the interaction between miR-617 and Smad3, pciCHECK2-Smad3 and psiCHECK2-mut-Smad3 were constructed. Dual-luciferase reporter assay uncovered decreased luciferase activity after co-transfection of miR-617 mimics and pciCHECK2-Smad3, confirming the direct interaction between miR-617 and Smad3 (Figure 5D). Interestingly, Smad3 level was positively regulated by circSIPA1L1, but negatively regulated by miR-617. CircSIPA1L1/miR-617 induces osteogenic differentiation of DPSCs by targeting Smad3. Western blot assay showed higher protein levels of Smad3 in NC group and miR-617 inhibitor than miR-617 mimic group and miR-617 inhibitor NC group respectively. Meanwhile, Si-NC group showed higher protein levels of Smad3 than Si-circSIPA1L1-3 group and Si-circSIPA1L1-1 group respectively (Figure 6A-C). Immunofluorescence assay revealed a similar result (Figure 6D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-617 reversed regulatory effect of circSIPA1L1 on DPSCs osteogenesis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRescue experiments were conducted to clarify the involvement of miR-617/Smad3 in circSIPA1L1-mediated osteogenesis. Western blot results showed that downregulated RUNX2, ALP, OSX and Smad3 in osteogenic DPSCs with circSIPA1L1 knockdown were partially reversed by co-silence of miR-617 (Figure 6E, F).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, critical functions of circRNAs in human diseases have been highlighted, which may provide a theoretical basis for developing novel treatments \u003csup\u003e19, 27, 28\u003c/sup\u003e. Serving as ceRNAs, circRNAs can sponge miRNAs, proteins and trans-acting elements, thus influencing gene transcription, expressions and functions. In this paper, we focused on the role of circSIPA1L1 in the bone regeneration and its potential mechanism.\u003c/p\u003e\n\u003cp\u003eOwing to the multilineage differentiation potential, DPSCs are considered as candidates in bone regeneration. Our finding showed that circSIPA1L1 was dynamically upregulated during DPSCs osteogenesis, while miR-617 showed the opposite trend. CircSIPA1L1 was unable to influence proliferative potential of osteogenic DPSCs. However, it indeed stimulated osteogenesis of DPSCs as ALP and ARS staining indicated. As an early marker of calcification, ALP is linked to osteoblast activity and osteogenesis specificity \u003csup\u003e29\u003c/sup\u003e. Its expression and activity were markedly enhanced in the early stage of mineralization. RUNX2 is a transcriptional regulator responsible for early-stage osteogenesis, which directly affects gene expressions associated with intracranial secretion and bone tissue enrichment. Studies have shown that RUNX2 knockout mice performed complete lack of bone formation \u003csup\u003e30\u003c/sup\u003e. OSX is a critical downstream gene of RUNX2 that is involved in bone formation and osteoblast differentiation. It is reported that MSCs isolated from OSX-deficient mice cannot be differentiated into osteoblasts \u003csup\u003e31\u003c/sup\u003e. Here, osteogenesis markers (e.g. ALP, RUNX2, OSX) were found to be positively regulated by circSIPA1L1, further confirming our findings. In addition, a subcutaneous transplantation model in nude mice by cell scaffold material was established. histological examination results were consistent with the \u003cem\u003ein vitro \u003c/em\u003econclusions. Taken together, these findings indicated that circSIPA1L1 stimulated DPSCs osteogenesis.\u003c/p\u003e\n\u003cp\u003eRecently, ceRNA hypothesis proposed a vital regulatory loop, that is, circRNA-miRNA-mRNA axis \u003csup\u003e32\u003c/sup\u003e. For instance, circNRIP1 aggravates gastric cancer progression by sponging microRNA-149-5p \u003cem\u003evia\u003c/em\u003e the AKT1/mTOR pathway \u003csup\u003e33\u003c/sup\u003e. In addition, circHIPK3 promotes the proliferative and differentiation potentials of chicken myoblasts by sponging miR-30a-3p \u003csup\u003e34\u003c/sup\u003e. Vital functions of miRNAs in stem cell regulation have been well concerned. Many miRNAs have been identified to participate in osteoblast differentiation processes \u003csup\u003e35\u003c/sup\u003e. For example, miR-21, miR-26a and miR-196 are involved in MSCs osteogenesis \u003csup\u003e36, 37, 38\u003c/sup\u003e. To explore the potential contributing mechanisms of circSIPA1L1 in DPSCs osteogenesis, bioinformatics analysis was conducted to seek potential targets binding circSIPA1L1. Our results demonstrated that miR-617 was the target gene binding circSIPA1L1 through dual-luciferase reporter assay, which negatively mediated DPSCs osteogenesis. Notably, miR-617 was capable of abolishing regulatory effect of circSIPA1L1 on DPSCs osteogenesis. In the cytoplasm, ceRNAs can affect mRNA stability and translational regulation under the circumstances that two interacted genes should be colocalized \u003csup\u003e39, 40\u003c/sup\u003e. FISH results illustrated that circSIPA1L1 and miR-617 were co-localized in the cytoplasm of DPSCs.\u003c/p\u003e\n\u003cp\u003eIn a similar way, Smad3 was discovered to be downstream gene of miR-617, which is an important component of TGF-\u0026beta; signaling. Smad3 (mothers against decapentaplegic homolog 3) is a crucial regulator of TGF-\u0026beta;/ Smads pathway \u003csup\u003e41\u003c/sup\u003e. It is able to mediate the synthesis and degradation of extracellular matrix, as well as cell phenotypes \u003csup\u003e42, 43\u003c/sup\u003e. After TGF-\u0026beta; and RUNX2 induction, Smads are activated and accumulated to contribute to skeleton formation \u003csup\u003e44\u003c/sup\u003e. It is reported that miR-708 can effectively abolish the inhibitory effect of Dex on osteoblast differentiation by upregulating Smad3 \u003csup\u003e45\u003c/sup\u003e. In this paper, Smad3 was proven to be the downstream gene binding miR-617. Its level was positively regulated by circSIPA1L1 and negatively regulated by miR-617. Therefore, we hypothesized a circSIPA1L1/miR-617/Smad3 axis responsible for mediating DPSCs osteogenesis. Overexpression of inflammation-induced miR-223-3p triggers odontoblast differentiation of DPSCs by targeting Smad3 \u003csup\u003e46\u003c/sup\u003e. The synergistic activity of Smads following Runx2 activation is of significance in bone formation. The Smad pathway mediates differentiation of mesenchyma progenitors through converging RUNX2 \u003csup\u003e47\u003c/sup\u003e. Taken together, we believed that circSIPA1L1/miR-617/Smad3 axis stimulated DPSCs osteogenesis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCircSIPA1L1 is dynamically upregulated under mineralization-inducing conditions. CircSIPA1L1/miR-617/Smad3 axis is responsible for stimulating DPSCs osteogenesis, which can be utilized as bone regeneration targets.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCircSIPA1L1: Circular RNA SIPA1L1;DPSCs: Dental pulp stem cells; miRNA: MicroRNA;ARS: Alizarin red Staining; RT-PCR: Reverse transcription polymerase chain reaction; ALP: Alkaline phosphatase; RUNX2: Runt-related transcription factor 2; MSCs: Mesenchymal stem cells; ceRNA: Competing endogenous RNA; 3\u0026prime;UTR: 3\u0026prime; untranslated region; NC: Negative control\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXingyun Ge and Zehan Li conceived and designed the study, collected and assembled data and wrote the manuscript. These two authors contributed equally to this work. Zhou Zhou performed data analysis and interpretation, Yibo Xia and MinxiaBian reviewed the data. Jinhua Yu conceived and designed the study, provided financial support and study material, performed data analysis and interpretation, approved the final version of the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been partially funded by a grant from the National Natural Science Foundation of China (81873707, 81900962), Medical Talent Project of Jiangsu Province (ZDRCA2016086). And Science and Technology Development Project of Jiangsu Province (BE2017731) and Postgraduate Research \u0026amp; Practice Innovation Program of Jiangsu Province (KYCX19_1147).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from\u003c/p\u003e\n\u003cp\u003ethe corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudies were carried out in accordance with the Declaration of Helsinki and\u003c/p\u003e\n\u003cp\u003egot approval of the Ethical Committee of Nanjing Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eHuang GT, Gronthos S, Shi S. 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TGF-beta and BMP signaling in osteoblast differentiation and bone formation. \u003cem\u003eInt J Biol Sci\u003c/em\u003e 2012, \u003cstrong\u003e8\u003c/strong\u003e(2)\u003cstrong\u003e:\u003c/strong\u003e 272-288.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Circular RNA SIPA1L1, miR-617, Smad3, Dental pulp stem cells, osteogenesis","lastPublishedDoi":"10.21203/rs.3.rs-32816/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-32816/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eBone regeneration is preferred for bone loss caused by tumors, bone defects, fractures, etc. Recently, mesenchymal stem cells are considered as optimistic tools for bone defect therapy. Dental pulp stem cells (DPSCs) are a promising candidate for regenerative medicine and bone regeneration. Our previous study showed that up-regulated circSIPA1L1 during osteogenesis of DPSCs is of significance. In this paper, the potential role of circSIPA1L1 in osteogenesis of DPSCs and its underlying mechanisms are explored.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The circular structure of circSIPA1L1 was identified by Sanger sequencing and PCR. Regulatory effects of circSIPA1L1 and miR-617 on mineral deposition in DPSCs were assessed by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and \u003cem\u003ein vivo \u003c/em\u003ebone formation assay were conducted to verify the biological influences of circSIPA1L1 on DPSCs. Western blot was performed to detect the protein expression of Smad3. Localization of circSIPA1L1 and miR-617 was confirmed by FISH. Dual-luciferase reporter assay and rescue experiments were conducted to investigate the role of the circSIPA1L1/miR-617/Smad3 regulatory axis in osteogenesis of DPSCs.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Sanger sequencing and back-to-back primer experiments confirmed the closed loop structure of circSIPA1L1. CircSIPA1L1 could promote the committed differentiation of DPSCs. MiR-617 was predicted to be the target binding circSIPA1L1 through MiRDB, miRTarBase, and TargetScan database analyses, which was further confirmed by dual-luciferase reporter assay. FISH results showed that circSIPA1L1 and miR-617 colocalize in the cytoplasm of DPSCs. MiR-617 exerted an inhibitory effect on osteogenesis of DPSCs. Knockdown of circSIPA1L1 or upregulation of miR-617 down-regulated phosphorylated Smad3. In addition, rescue experiments showed that knockdown of miR-617 reversed the inhibitory effect of circSIPA1L1 on osteogenesis of DPSCs.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e CircRNASIPA1L1 promotes osteogenesis of DPSCs by adsorbing miR-617 and further targeting Smad3.\u003c/p\u003e","manuscriptTitle":"Circular RNA SIPA1L1 promotes osteogenesis via regulating the miR-617/Smad3 axis in dental pulp stem cells","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2020-07-16 20:55:08","doi":"10.21203/rs.3.rs-32816/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2020-07-31T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-07-19T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's 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