Hsa-miRNA-143-3p regulates differentiation of human stem cells 1 from the apical papilla through targeting Nuclear factor I-C 2

17 Background : Dental root development is independent and time-space-specific. Nuclear factor I-C 18 (NFIC) plays a key role in human root development through regulating the differentiation of stem 19 cells from the apical papillary (SCAPs). The function of microRNAs during the differentiation of 20 SCAP and post-transcriptional regulation of NFIC remain unclear. 21 Methods : We examined the microRNA expression profiles in human immature permanent teeth 22 and SCAPs differentiation. hSCAPs were treated with miR-143-3p over/low-expression viruses, 23 then the odonto/osteogenic differentiation of these stem cells and the involvement of NFIC 24 pathway were investigated. Next, luciferase reporter and its mutant plasmids were used to confirm 25 direct target gene of miR-143-3p. Mineralization induction assays ex vivo and in vitro were used to 26 investigate the functional significance of miR-143-3p. 27 Results : MiR-143-3p was screened by microarray expression profiling and bioinformatics 28 technology, which decreased during hSCAPs differentiation. Overexpression of miR-143-3p 29 inhibited the odontogenic differentiation of hSCAPs and downregulated the related genes, whereas 30 the functional inhibition of miR-143-3p yielded the opposite effect. The luciferase reporter gene 31 detection and bioinformatics approach identified NFIC as a potential target of miR-143-3p. Furthermore, NFIC overexpression reversed the inhibitory effect of miR-143-3p on the 33 odontogenic differentiation of hSCAP. Conclusions : MiR-143-3p maintains the stemness of hSCAPs and negatively modulates their 35 differentiation and mineralization by directly targeting transcription factor NFIC, which serves as an contribution towards a better understanding of the developmental mechanisms of root 37 formation.


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Tooth root development is a complex process and requires exact temporal and spatial 41 regulation of cell proliferation and differentiation. Many signaling molecules are involved in the 42 regulation of growth and eventually apical closure [1] . Stem cells from apical papilla (SCAPs) are 43 multipotent progenitor cells residing in the root apex of immature teeth. SCAPs can differentiate 44 into radicular pulp as well as the odontoblasts that are responsible for root dentinogenesis and 45 undergo morphological and functional changes [2] . These postnatal stem cells play a crucial role in 46 pulp-dentin complex healing and regeneration when the roots of young permanent teeth are 47 8 manufacturer's instructions. cDNA was synthesized using an AMV Reverse Transcriptase kit 135 (Fermentas, St. Leon-Rot, Germany). The qRT-PCR was performed on a LightCycler 480 (Roche,136 Indianapolis, USA) with the Fast Start Universal SYBR GreenMasterMix (Roche) according to 137 themanufacturer's instructions. The relative mRNA expression was calculated using the 138 comparative cycle threshold ( △△ Ct) method. Protein extracts were resolved by 10-12% 139 SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against NFIC, KLF4, 140 DMP1 (Abcam, Cambridge, UK) and DSP (Santa cruz, USA). 141

Statistical analyses 142
Statistical analysis was performed with SPSS software, version 16.0 (SPSS, Chicago, IL, 143 USA). p <0.05 was considered statistically significant. Student's t-test and chi-square test was 144 used to analyze differences between groups. All experiments were independently performed at 145 least three times. 146

MiR-143-3p expression is inversely associated with hSCAPs differentiation 148
Apical papilla tissues located in the root apex of immature teeth are reliable cell source for 149 pulp-dentine complex formation. MiRNA microarray assay was performed to analyze the miRNA 150 profiles of mesenchymal tissues isolated from root pulp (RP) and apical papilla (AP) of young 151 permanent teeth via Ion Torrent/MiSeq sequencing. Among the differentially expressed miRNAs, 152 miR-143-3p was significantly downregulated in the root pulp (Fig. 1A, B). Additionally, real-time 153 PCR was conducted to detect miR-143-3p expression in five RPs and matched APs, showing the 154 same trends that the levels of miR-143-3p decreased significantly in RPs compared with that in 155 APs (Fig. 1C). MiRNAs related to NFIC were predicted using bioinformatics database, showing 156 that miR-143-3p is one of the miRNAs retrieved from all three databases (Table 1). Therefore, 157 miR-143-3p was selected for further study. 158 To investigate the miR-143-3p expression patterns in vitro, hSCAPs were cultured in 159 mineralization medium. Alizarin red-S staining revealed the presence of red mineralized nodules 160 from day 7 after the differentiation induction (Fig. 1D). RT-PCR analysis indicated that the 161 expression level of miR-143-3p were gradually decreased during the mineralization of hSCAPs, 162 while the expression of odontogenic marker genes in dental root (NFIC and DSPP) had rising and 163 fluctuating trends before day 12 and then decreased (Fig. 1E). This indicates that miR-143-3p 164 expressions is down-regulated during hSCAPs differentiation in vitro and in vivo. 165

MiR-143-3p influences odontogenic differentiation of hSCAP in vitro and in vivo 175
To determine the effect of miR-143-3p on cellular behavior of hSCAPs, we stably 176

MiR-143-3p directly targets NFIC in hSCAPs differentiation 220
Four publicly available bioinformatics tools (miRDB, miRWalk, TargetScan, miRTarBase) 221 were used to analyze genes targeted by miR-143-3p. NFIC ranks among the predicted genes and 222 was retrieved in all four databases (Fig. 3A). The expression of NFIC was significantly lower in 223 miR-143-3p overexpressed hSCAPs and increased in miR-143-3p knockdown cells (Fig. 2D, E). 224 Then we obtained the sequence of miR-143-3p (UGAGAUGAAGCACUGUAGCUC) and found 225 the predicted consequential pairing of target region (top) and miRNA (bottom) between NFIC and 226 miR-143-3p on the TargetScanHuman website (Fig. 3B). The luciferase reporter assay was applied 227 to verify whether miR-143-3p could target the 3´UTR of NFIC directly. We cloned the 3´UTR 228 fragment (WT-NFIC) of NFIC containing a miR-143-3p binding site and mutant fragments 229 (MUT-NFIC) into luciferase reporter vectors and found that miR-143-3p could significantly 230 reduce WT-NFIC luciferase activity in hSCAPs but had no effect on that of MUT-NFIC group 231 (Fig. 3C). Next, to further investigate whether miR-143-3p exerted its effects through targeting 232 13 NFIC in hSCAPs, a plasmid containing full-length NFIC was transfected into the miR-143-3p 233 overexpressing hSCAPs, then we found that the cells showed the upregulated level of NFIC, 234 DSPP and KLF4, the downregulated DMP1 (Fig. 3D, E) and generated more calcium nodules (Fig.  235 3F) and higher ALPase activity (Fig. 3G)   Stem cells from apical papilla (SCAPs) are a population of stem/progenitor cells residing in 251 the apical papilla. As a key event during tooth root development, the differentiation of SCAPs is 252 regulated by various genes and signaling molecules. SCAPs share many features with dental pulp 253 stem cells but also feature their own specific mechanisms at cellular and molecular levels in the 254 dental root formation [13] . Ample evidence suggests that microRNAs are necessary for maintaining 255 homeostasis and proper functionality in many organs and are also implicated in tooth germ 256 development, differentiation and regeneration of pulp-dentin complex and periodontal tissue [14,15] . 257 In this study, to screen specific miRNAs that function in human tooth root formation, we first 258 performed miRNA microarray assay in roots of human young permanent teeth and found that the 259 expression of miR-143-3p decreased significantly in root pulp compared with that in apical dental 260 mesenchyme. Mineralization induction experiment showed that, miR-143-3p expression was 261 downregulated gradually during hSCAPs differentiation in vitro, which showed the opposite trend 262 with NFIC and DSPP, that are regarded as dentin-specific markers of odontoblasts differentiation. 263 In addition, the bioinformatic analysis was performed to suggest NFIC as the possible target gene 264 of miR-143-3p. These results implied that miR-143-3p may act as an essential factor in human 265 tooth root development and hSCAPs differentiation, and is related to the regulation of NFIC. The 266 gene of miR-143-3p is located on human chromosome 5q32 and can be transcribed and processed 267 into two isoforms: miR-143-3p and miR-143-5p. MiR-143-3p is common isoform in normal 268 tissues and highly expressed in mesenchymal cells [16] . MiR-143-3p has been shown to suppress 269 the differentiation of mouse pre-odontoblast cell line through Klf4 transcription factor signaling 270 pathways [17] , but the functions in root dentin development have rarely been reported. As a 271 15 DNA-binding transcription factor, NFIC has been considered as a crucial regulator of tooth root 272 formation. Nfic knockout mice cannot develop molar roots but crowns are normal, which may 273 result from the interference to the differentiation of odontoblasts due to disruption of NFIC [7,18] . 274 Our previous studies also demonstrated that NFIC expression was restricted within odontoblasts 275 and preodontoblasts and weakly within the pulp and apical papilla, and was involved in the 276 development of human root dentin and the regulation of odontoblastic differentiation of hSCAPs [9] . 277 Our findings, together with previous reports, suggest that miR-143-3p is likely to be a participant 278 in the regulation of tooth root development through interactions with NFIC. These findings 279 promoted us to hypothesize that miR-143-3p could have a negative effect on hSCAPs 280 differentiation and be related with NFIC signaling pathway. On the other hand, former studies on 281 the regulatory mechanism of Nfic mainly focused on its downstream signals [19] , for example, Nfic 282 activates hedgehog (Hh) attenuator Hhip in the mesenchyme, so that mesenchymal cells respond 283 correctly to the sonic hedgehog (Shh) signal from the epithelium to maintain growth patterns of 284 the apical papilla [20] . Our study provided new insights into understanding the developmental 285 mechanisms of root formation. 286 miR-143-3p has been found to act as a key regulator in various biological processes and 287 diseases, and become one of the best known of the tumor suppressor miRNA [21] by targeting 288 several oncogenes in various human cancers, such as breast cancer [22] , gastric cancer [23] , renal cell 289 cancer [24] . miR-143 serum levels were recently suggested as a biomarker for critical illness and 290 sepsis [25] . However, we have not found definite reports on cellular behavior of hSCAPs influenced 291 by miR-143-3p. In the present study, we describe the direct effects of miR-143-3p overexpression 292 and knockdown on the proliferation and differentiation of hSCAPs. The cell proliferation assay 293 16 based on the CCK-8 marker showed that miR-143-3p is not required for the hSCAPs proliferation. 294 On the other side, we observed that overexpression of miR-143-3p resulted in decreased 295 expression levels of odontogenic markers (NFIC and DSPP) and weakened mineralization ability 296 in vitro, manifested by less mineral nodules and weaker ALP staining in mineralized induction. 297 The findings above were consistent with the effects of knockdown NFIC in hSCAPs [9] . 298 Furthrtmore, downregulation of miR-143-3p showed the opposite effects on hSCAPs, that is, 299 promoted the odontogenic differentiation and mineralization abilities. When hSCAPs of 300 enforced-expression miR-143-3p were transplanted into the dorsum of immunocompromized mice, 301 dentin-like structure and odontoblast-like cells were not generated and no or weak expression of 302 NFIC were observed. These results demonstrated that miR-143-3p negatively regulated 303 odontogenic differentiation of hSCAPs. 304 Odontoblasts synthesize and secrete dentin extracellular matrix proteins, among which DSPP 305 and DMP1 are considered as typical marker for the odontogenic differentiation of mesenchyma 306 stem cells [26] , and DMP1 might regulate the expression of DSPP [27] . Our results showed that the 307 expression of NFIC, DSPP and KLF4 were inhibited and DMP1 was promoted by miR-143-3p. 308 The reason might be the synergistic effect of miR-143-3p on DSPP and DMP1 by directly acting 309 on NFIC. The function of KLF4 as a transcription factor is relative complex, and it is responsible 310 for promoting the differentiation of mouse pre-odontoblast and the expression of Dmp1 and Dspp, 311 but shows inhibitory effect on some embryonic stem cells to maintain their stemness [28] . ALP and 312 OCN expression was not affected by miR-143-3p, which may be because they are more related to 313 the dentin mineralization in the late-stage of root development [29] , or associated with regulation by 314 17 other post-transcriptional mechanisms. The interaction of these molecules in regulating root 315 development needs further investigation. 316 MiRNAs lead to translational inhibition of target genes through gene silencing mechanisms. 317 To explore the underlying mechanisms of regulation between miR-143-3p and NFIC, the 318 luciferase reporter assay was used and found that miR-143-3p directly represses the gene 319 expression through binding to 3'-UTRs of NFIC, furthermore, NFIC overexpression reversed the 320 effect of miR-143-3p on the differentiation capacity of hSCAP, indicating that miR-143-3p could 321 inhibit hSCAP differentiation by targeting NFIC. Although some signaling pathways for crown 322 and root development may be similar, NFIC pathway reflects the independence of root 323 development. NFIC knockout may disrupt the nexus of critical signaling pathway specific to tooth 324 root, leading to the absent root phenotype in Nfic null mouse [30] . So this study considers that the 325 regulation of miR-143-3p is involved in specific signaling events that are unique to root formation, 326 and reduced miR-143-3p may activate the NFIC signalling pathway during initiation of SCAP 327 differentiation. MiR-143-3p was identified miRNA markers of the human naive, that are 328 specifcally expressed in naive and primed pluripotent states and are downregulated upon 329 diferentiation [31] , which is in line with our observation of downregulation during root development, 330 that is, miR-143-3p can maintain the stemness of SCAPs and degrade its expression in the process 331 of differentiation into odontoblasts or pulp cells. 332 Although previous works have reported miR-143 up-regulation during cell differentiation as 333 promoters of differentiation, such as embryonic stem [32] and smooth muscle cells [33] , our data 334 showed that miR-143-3p served as the maintainer of the undifferentiated state of SCAP, which is 335 consistent with the reports where it suppressed the differentiation of mouse pre-odontoblast [17,34] . 336 18 Accordingly, miR-143-3p-dependent transcription may modulate in different ways depending on 337 the cell types. For example, miR-143-3p is upregulated with stimulation with TGF-β and may be a 338 mediator of glomerulonephropathy [35] , but TGF-β1 down-regulated miR-143 leading to the 339 progression of nasopharyngeal carcinoma [36] . These findings illustrate the dual effects of these 340 multifunctional miRNAs in different biological roles, which may be tissue-and cell-specific. It 341 awaits further investigation to understand the complete mechanisms of root complex formation. 342

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Altogether, our study demonstrated that miR-143-3p maintained the stemness of hSCAPs and 344 negatively modulated their differentiation and mineralization by directly targeting transcription 345 factor NFIC, which serves as an contribution towards a better understanding of the developmental 346 mechanisms of root formation. SCAPs are considered as a more potential cell source for tooth root 347 regeneration, showing to form more uniform dentin-like tissue and possess much higher 348 dentinogenic capacity, an example of which is that SCAPs recombined with biological scaffolds in 349 the empty root canal space could generate bioengineered roots that can provide anchorage for a 350 porcelain crown [37] . It is hoped that the results of this study could promote regenerative medicine, 351 concentrating on guiding differentiation of SCAPs into functional cells to repair damaged tissues. 352 Ethical approval and consent to participate All procedures were in accordance with the ethical standards of the 353 institutional research committee. Informed written consent was taken from each patient.

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Consent for publication Written informed consent for publication was obtained from all participants.

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Availability of data and material The datasets used and/or analyzed during the current study are available from 356 the corresponding author on reasonable request.