miRNA-124-3p.1 Inhibits the Osteogenic and Odontogenic Differentiation of SCAPs via MACF1/smad7 Axis

Background: Previous research has indicated that altered expression of micro-RNAs (miRNAs) is in connection with differentiation of stem cells from apical papillae (SCAPs). We investigated the mechanisms that miR-124-3p.1 inhibited osteogenic and odontogenic differentiation of SCAPs. Methods: SCAPs were isolated from dental apical papilla. MiR-124-3p.1 mimic and inhibitor were used for overexpression and knockdown assays. For overexpression and knockdown of microtubule actin cross ‐ linking factor 1 (MACF1), lentivirus infection and siRNA transfection were performed. Luciferase reporter assay was performed to determine the relationship between miR-124-3p.1 and MACF1. The osteogenic and odontogenic differentiation potential was analyzed by alkaline phosphatase activity analysis (ALP), alizarin red S (ARS) staining, quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR), western blot and immunouorescence (IF) staining. Results: We observed a time dependent decrease of miR ‐ 124 ‐ 3p.1 level in mineralization induction of SCAPs. Further study found that miR ‐ 124 ‐ 3p.1 exhibited an inhibitory effect on SCAPs osteo/odontogenic differentiation. Similarly, we found that the overexpression of miR ‐ 124 ‐ 3p.1 dramatically inhibited MACF1 protein level in SCAPs and knockdown of miR ‐ 124 ‐ 3p.1 signicantly increased MACF1 protein level in SCAPs. Moreover, MACF1 was veried as the targeting of miR ‐ 124 ‐ 3p.1. Meanwhile, the expression of MACF1 was related to smad7 nuclear translocation. Conclusion: Collectively, diverse data demonstrated that miR ‐ 124 ‐ 3p.1 is a regulator of MACF1/smad7, playing plays an important role in osteogenic and odontogenic differentiation of SCAPs via MACF1/smad7 axis.


Introduction
Pulpitis and periapical periodontitis are common oral diseases. Root canal treatment (RCT) is commonly used to treat these problems and relieve the pain of patients. But the traditional method is limited by the incomplete root formation with the open apex and short roots [1]. Treating immature permanent teeth with pulpitis and periapical periodontitis is a challenge in dental medicine [2]. The immature pulpless tooth with an open apex become easy to fracture, resulting in a higher incidence of tooth extraction [3]. Adult stem cells play a signi cant role in maintaining homeostasis of tissue and in the process of tissue repair and regeneration [4,5]. In fact, adult mesenchymal stem cells (MSCs) are pluripotent adult stem cells that are isolated from a variety of tissues or organs [6]. Additionally, a recent report indicates that MSCs have chondrogenic, osteogenic, or adipogenic differentiation potential. Stem cells from the tooth mesoderm derived from neural crest cells may be sources for tissue regeneration, including stem cells from apical papilla (SCAPs) [7,8]. SCAPs residing in the apical papilla play a critical role in tooth development and pulp regeneration in permanent teeth [1]. SCAPs were classi ed as pluripotent MSCs after a positive expression of CD146, CD90, CD73, STRO-1 and CD105 markers [8]. SCAPs t exhibit the properties of high proliferative potential, low immunogenicity, self-renewal capacity and multipotency of differentiation such as osteogenetic, odontogenetic, dentinogenetic, adipogenetic and chondrogenic [9,10]. In addition, considerable evidence indicates that after culture in mineral induction medium (MM) containing βglycerophosphate, dexamethasone, and L-ascorbate-2-phosphate, SCAPs are found to express a variety of osteo/odontogenic markers, such as ALP, osterix (OSX), runt-related transcription factor 2 (RUNX2) and dentin sialophosphoprotein (DSPP) [11]. Previous studies have found DSPP, RUNX2, ALP and OSX to be important transcriptional regulators of osteogenic and odontogenic differentiation [12]. SCAPs were used to reconstruct the spinal cord injury in animal model [13]. Therefore, SCAPs have been increasingly employed to investigate regenerative medicine.
MiRNAs are a class of highly conserved small noncoding RNAs molecules that operates as genome main regulators and ne tuners via post-transcriptional gene silencing [14]. MiRNAs containing [19][20][21][22] nucleotides RNA molecules, always act as a negative regulator in the process of target genes expression in a sequence-speci c manner by directly binding partially complementary sequences of target mRNAs in 3′UTRs leading to suppression of the expression of their target mRNAs and its corresponding protein in many biological processes [15,16]. MiRNAs extensively distributed in the body [17]. Evidences showed that miRNAs are ubiquitous and potent regulators of almost all biological processes, including proliferation, cell differentiation, metabolism, tumorigenesis, apoptosis, and tissue development [18][19][20].
Emerging evidence also suggests that miRNAs are active regulators in the self-renewal ability and differentiation potential of stem cells by post-transcriptionally targeting factors implicated in stem cell maintenance [12,21]. It is reported that increasing number of miRNAs exert a signi cant impact on apoptosis, proliferation, differentiation and bone resorption of osteoblasts [19,22]. There are studies have demonstrated that miRNAs almost function in the whole process of osteogenic differentiation [23,24].
Most of these miRNAs have been reported to promote or inhibit the formation and maturation of osteoblasts, and a few miRNAs are involved in the regulation of osteoblasts functions [25]. Recently, some studies have demonstrated the importance of miRNAs in regulating osteogenic differentiation [26]. For instance, miR-138 reduces ectopic bone formation in vivo through negatively regulating osteogenic differentiation of human MSCs [27]. MiR-124 also inhibits osteogenic differentiation of MSCs and in vivo bone formation [28]. Our team have demonstrated that miRNA let7b played the important role in the differentiation of SCAPs [29]. Previous studies reported that miR-124-3p.1 is involved in cancer development and has also been identi ed as a potential tumor suppressor in certain cancers [30][31][32].
Moreover, miR-124-3p.1 sensitizes carboplatin induced mitochondrial apoptosis by inhibiting CAV1 in ovarian cancer [33]. Recently, there are studies demonstrated that miR-124-3p.1 overexpression negatively regulated proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) [34]. However, the role of miR-124-3p.1 in the differentiation of SCAPs are still unclear. The purpose of this study was to explore the effect and mechanism of miR-124-3p.1 on proliferation and osteo/odontogenic differentiation of SCAPs.
Materials And Methods 2.1 Cell culture SCAPs were isolated from dental apical papilla tissue. Dental apical papillae were isolated and transferred into phosphate-buffered saline (PBS; Gibco) containing penicillin-streptomycin (Pen-Strep; Gibco) and trimmed to small pieces under sterile conditions. The samples containing stem cells were transferred into a ask containing α-modi ed Eagle's minimum essential medium (α-MEM; Gibco), 10% fetal bovine serum (FBS; BI) and 2% Pen-Strep.

Colony Forming Assay
After 10 days of culture, cells were stained with 0.1% toluidine blue (Sigma Aldrich, MO, USA).

Chondrogenic Differentiation
SCAPs were cultured in chondrogenic medium for 1 month. After that, cells were immersed in paraformaldehyde for 48 hours, then imbedded in para n, and sliced into 5 μm sections. Then, cells were stained with Alcian blue staining for 30 min.

Osteogenic differentiation
Cells were seeded in 12-well plates. Afterward, mineral induction medium consisting ofβ-glycerophosphate10 mM, ascorbate-2-phosphate50 mM, and dexamethasone 100 nM (Sigma), 10% FBS and 1% Pen-Strep was added to the wells. The culture plates were incubated in humidi ed condition for 21 days.

Adipogenic Differentiation
SCAPs were cultured in adipogenic medium (Cyagen, China) for 28 days. After stained with Oil Red O reagent (Cyagen) for 5 min, lipid droplets were observed under microscope.

Transfection
The miR-24-3p oligos (inhibitor, mimic, and inhibitor NC and mimic NC) and control siRNA (NC), siRNA for MACF1(siMACF1) were designed by Ribio (Ribio CO., China). Cells were transfected with oligos and siRNAs via riboFECTTM CP following the manufacturer's protocols. Western blot and qRT-PCR were used to verify the interference e ciency. Lentiviral vectors overexpressing MACF1 were constructed and produced by Shanghai Genechem Company (Shanghai, China). SCAPs were inoculated overnight and infected with lentiviruses with polybrene.

5-Ethynyl-2 -Deoxyuridine (EdU) assay
The EdU DNA Proliferation Detection kit (Ribo Biotechnology, China) was used to detect cell proliferation ratio. After culture, cells were xed and administrated with EdU labeling solution. Cells were photographed under inverted uorescence microscope (Leica, Germany).

Alizarin red staining
After washed and xed, SCAPs were stained with ARS solution (Sigma, USA). The red sediments of calcium deposition were observed under microscope. For quantitative analysis, mineralized nodules were dissolved by 10% cetylpyridinium chloride (CPC; Sigma, USA). Then OD value was measured at 562 nm.

Alkaline phosphatase activity
Cells were harvested and permeated in Triton X-100. After centrifugation, the supernatant transferred into fresh 1.5 ml tubes with relative working solution and then were incubated. The OD value was measured at 520 nm.

ALP staining
After washed and xed, cells were stained using the BCIP/NBT ALP staining kit (Beyotime, China). Cells were observed with microscope.
2.9 RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) RNA isolation was performed using the TRIzol™ (Invitrogen) method. The cDNAs were synthesized and used for PCR. qRT-PCR reactions were performed by SYBR Green method. GAPDH and U6 were used as internal controls. Bulge-Loop miRNA qPCR Primer kit (RiboBio) was used for measuring miRNA-124.3p.1 expression. Primers in this study are listed in Table 1. 2.10 Western blotting RIPA buffer was used to extract proteins with protease inhibitor cocktail. After PAGE, the proteins were blotted onto PVDF membranes. Membranes were blocked, incubated with speci c primary antibody and next day with the secondary HRP-conjugated antibody 2.11 Immuno uorescence staining Cells were xed, permeabilized. Then, cells were blocked and incubated with primary antibodies, uorescent dye-labeled designated secondary antibody and DAPI above

Luciferase Assay
The potential binding site of MACF1-wt and mutant sequence MACF1-mut was synthesized into pmiR-GLO (Promega, Madison, WI, USA). SCAPs were co-transfected with the MACF1-wt or MACF11-mut reporter gene plasmid and miR-124-3p.1 mimic. The activities were measured by a Promega luciferase assay (Promega, USA) were normalized against the activity of the Renilla luciferase gene.

Co-immunoprecipitation (Co-IP) assay
After cells lysed and centrifuged, supernatants were incubated with the anti-MACF1 antibody in rotation.
Next day, the supernatants were mixed with protein G plus A agarose (Beyotime, China) at in rotation. Then, the immunocomplexes were washed. Beads complexes were resuspended in 2 × loading buffer.

Animal procedures
SCAPs with miR-124-3p.1 mimic and miR-124-3p.1 mimic NC induced in MM for 7 days were harvested for the in vivo study. About 6.0 × 10 6 SCAPs were mixed with Bio-Oss Collagen scaffolds (Geistlich, Germany). And then the mixtures were implanted into the dorsal surface of BALB/c homozygous 5-weekold nude mice. Two months later, the implants were harvested and were detected under micro-CT analysis. After that, implants were decalci ed and embedded with para n. Para n sections were stained with hematoxylin and eosin (H&E) staining and Masson's trichrome staining.

Statistical analysis
All data are expressed as the mean ± SD and were analyzed by one-way analysis of variance (ANOVA). p<0.05 was considered statistically signi cant.

Characterization of SCAPs
Primary SCAPs were cultured after 3 days ( Figure 1A above). SCAPs were displayed long spindle shape at passage 3 ( Figure 1A below). IF staining showed SCAPs were stained positively for MSCs surface molecule STRO-1 ( Figure 1B). SCAPs positively expressed the mesenchymal stem cell markers CD73, CD90, CD29 and CD105, but negatively expressed the hematopoietic cell markers CD45, CD34 ( Figure  1C). Colony-forming assays were conducted to detect the colony forming e ciency. The result showed single colony-forming unit of SCAPs ( Figure 1D). The results of staining showed that SCAPs had the potential of differentiation into chondrocytes osteoblasts and adipocytes ( Figure 1E, F, G).

Silencing of MACF1 inhibited the odontogenic and osteogenic differentiation of SCAPs, overexpression of MACF1 promoted the odontogenic and osteogenic differentiation of SCAPs
To explore the potential in uence of MACF1 on the osteogenic and odontogenic differentiation of SCAPs, MACF1 knockdown and MACF1 overexpression were established. qRT-PCR showed that a signi cant decrease of MACF1 expression in siMACF1 group and an obvious increase in the MACF1 overexpression group compared to relative control groups ( Figure 6A). Therefore, western blot analysis con rmed the protein expression level of MACF1 was signi cantly downregulated in siMACF1 group and was dramatically upregulated in MACF-1-over group ( Figure 6B, C). ALP staining and ALP activity assay showed that ALP activity in siMACF1 group was clearly decreased and ALP activity were obviously enhanced in the MACF1 overexpression group ( Figure 6D, E). ARS staining and relevant CPC quantitative analysis showed matrix mineralization was dramatically downregulated in siMACF1 group and a contrary trend was detected in MACF-1 overexpression group ( Figure 6F, G). Besides, qRT-PCR and western blot showed that deletion of MACF1 downregulated the expression of DSPP, RUNX2, ALP and OSX. MACF1 overexpression increased gene and protein expression associated with osteo/odontogenetic differentiation (DSPP, RUNX2, ALP and OSX) ( Figure 6H, I, J). These results indicated that MACF1 downregulation is negatively associated with the osteogenic and odontogenic differentiation in SCAPs. But MACF1 overexpression promoted the osteogenic and odontogenic differentiation of SCAPs in vitro.  Figure 7A). In consonance with the results of qRT-PCR, western blot and immuno uorescence staining showed that upregulation of MACF1 induced by miR-124-3p.1 inhibitor could be negatively regulated by knockdown of MACF1 ( Figure  7B, C, D). Analogously, the relative expression levels of DSPP, RUNX2, ALP and OSX were signi cantly upregulated after miR-124-3p.1 inhibitor. However, the levels of these above genes were markedly inhibited by MACF1 knockdown ( Figure 7E). In addition, western blot showed that downregulation of miR-124-3p.1 remarkably promoted the expression of DSPP, RUNX2, ALP and OSX. Meanwhile, the expression of DSPP, RUNX2, ALP and OSX was reversed by co-transfection of miR-124-3p.1 inhibitor and siMACF1 ( Figure 7F, G). Immuno uorescence staining further veri ed the above results ( Figure 7H, I, J). Our data suggested that MACF1 repression was able to partially inhibited the positive effect of miR-124-3p.1 downregulation on osteogenic and odontogenic differentiation.

Macf1 Interacts With Smad7 In Scaps
To further explore the mechanism that MACF1 regulates SCAPs' differentiation, western blot and CO-IP were conducted. The result of western blot showed that MACF1 knockdown not only signi cantly decreased expression of smad7 in the cytoplasm, but also inhibited the expression of smad7 in the nucleus ( Figure 9A, C). Next, we found that smad7 were increased in SCAPs as compared with NC-over cells, the enhancement was especially obvious in the nucleus ( Figure 9B, D). We found that smad7 were co-immunoprecipitated with MACF1 using the antibody recognizing MACF1 (anti-MACF1). Co-IP result showed that smad7 was detectable in the anti-MACF1 immunoprecipitated products, indicating that MACF1 can interact with smad7 in SCAPs. Moreover, MACF1 is related to smad7 nuclear translocation ( Figure 9E). The mechanism diagram in this study was showed in Figure 9F.

Discussion
Multiple studies have demonstrated that dental apical papillae directly contribute toward the formation of the tooth roots [35]. Furthermore, SCAPs are an effective cell resource for tissue regeneration [36]. In the present study, we found that miR-124-3p.1 inhibited the osteo-/odontogenic differentiation potential of SCAPs. Importantly, we found that miR-124-3p.1 played an inhibitory role in the differentiation of SCAPs by binding to the 3′-UTR of MACF1, further regulation of smad7 entry into the nucleus.
Recent evidence indicates that miRNAs play important role in osteoblast differentiation and bone formation [37]. Previous studies have demonstrated that miRNAs act as important regulators for the stemness of oral mesenchymal stem cells [37,38]. Recently, the critical role of miR-124-3p.1 in osteogenic differentiation of human BMSCs has been previously demonstrated [34]. We, therefore, assume that miR-124-3p.1 can inhibit osteogenic and odontogenic differentiation of SCAPs through the MACF1/smad7 axis. Our study showed that overexpression of miR-124-3p.1 decreased the odonto/osteogenic differentiation of SCAPs while miR-124-3p.1 inhibition elevated osteogenic and odontogenic differentiation. The function and molecular mechanisms of miR-124-3p.1 in osteogenic and odontogenic differentiation was remains unclear. According to bioinformatics prediction (TargetScan V7.2), we discovered that MACF1 was a predicted target gene of miR-124-3p.1. As a member of the spectraplakin family of cytoskeletal crosslinking proteins, MACF1 is widely expressed in different tissues [39]. Accumulating evidence indicates that MACF1 is a critical factor in modulating actin and microtubule cytoskeletal networks and regulating cytoskeletal distribution, cell migration, cell survival and cell differentiation [40]. It was reported that MACF1 correlates with various physiological and pathological processes [41]. MACF1 has a signi cant effect on osteogenesis the differentiation of primary osteoblasts [42]. MACF1 is an important regulator in various signal transduction and cellular processes [43]. There are reports showed that downregulation of MACF1 suppressed the differentiation of osteoblastic cell line [44,45]. Taken together, the evidence illustrated that MACF1 can regulate the differentiation of osteoblast via Wnt signaling [46]. Moreover, smad7 is identi ed as a new downstream target of MACF1 and MACF1 promotes bone formation by facilitating smad7 nuclear translocation [47]. Previously, smad7 acts as an inhibitor to inhibit bone formation by a negative feedback way [48]. However, recent studies have emphasized the positive role of smad7 in stem cells such as osteoblasts and myoblasts [49,50]. It is also reported that nucleus smad7 can enhance osteogenic differentiation potential [47,49].

Conclusion
In summary, we found that miR-124-3p.1 negatively regulated the osteogenic and odontogenic differentiation potential by negatively regulating MACF1/smad7 axis in SCAPs. Additional studies are required to further con rm whether miR-124-3p.1 and MACF1 may enhance odontogenesis of SCAPs in vivo. Further studies are also needed to con rm the relationship between smad7 and differentiation of SCAPs, which will provide a theoretical basis for treating dental diseases.
Declarations Acknowledgements Not applicable. We would like to give our sincere appreciation to the reviewers for their helpful comments on this article.

Con ict of interest
Authors declare no con icts of interest.

Authors Contributions
LN conceived and designed the experiments, collected and assembled data, and wrote the manuscript.
LZH, YM and GYC performed data analysis and interpretation. WYQ, WJT and YCT collected and analyzed data. XT and FL reviewed the manuscript. YJH conceived and designed the study, provided nancial support and study material, performed the data analysis and interpretation, and approved the nal version of the manuscript. All authors read and approved the manuscript.

Funding
This work was supported by the National Natural Science Foundation of China (grant numbers: 81900962 and 82170940).

Availability of data and materials
Datasets used and analyzed during the current study are available from the corresponding author on reasonable request.