LncRNA-KCNQ1OT1 Promotes the Odontoblastic Differentiation of Dental Pulp Stem Cells via Regulating miR-153-3p/RUNX2 Axis

Background and Objective: Long non-coding RNAs (LncRNAs) play a key role in the odontoblastic differentiation. This study aimed to explore the role of LncRNA-KCNQ1OT1 in the odontoblastic differentiation of human dental pulp stem cells (DPSCs) and its possible mechanism. Methods: The expression of LncRNA-KCNQ1OT1, miR-153-3p, RUNX2 in the odontoblastic differentiation was detected by qRT-PCR. Interaction between LncRNA-KCNQ1OT1 and miR-153-3p and interaction between miR-153-3p and RUNX2 were detected by dual-luciferase assay. The cell viability of DPSCs was detected by cell counting kit-8 (CCK-8), and the effect of LncRNA-KCNQ1OT1 and miR-153-3p on the odontoblastic differentiation of DPSCs was observed by alizarin red staining, alkaline phosphatase (ALP) activity assay and Western blot for RUNX2, DSPP, DMP-1. Results: During odontoblastic differentiation of DPSCs, the expression of LncRNA-KCNQ1OT1 increased, miR-153-3p expression decreased, and RUNX2 expression increased. Dual-luciferase assay showed that LncRNA-KCNQ1OT1 sponges miR-153-3p and miR-153-3p targets on RUNX2. After LncRNA-KCNQ1OT1 and miR-153-3p expressions of DPSCs were changed, the cell viability was not notably changed, but the odontoblastic differentiation was notably changed which was conrmed with alizarin red staining, ALP activity and Western blot for RUNX2, DSPP, DMP-1. Conclusion: LncRNA-KCNQ1OT1 promotes the odontoblastic differentiation of DPSCs via regulating miR-153-3p/RUNX2 axis, which may provide a therapeutic clue for odontogenesis.


Introduction
Human dental pulp stem cells (DPSCs) are a kind of adult stem cells with high proliferation, self-renewal ability and multi-differentiation potential, which are mainly derived from dental pulp tissue. DPSCs can not only differentiate into dentin, but also into bone, cartilage, fat, myogenic and other mesodermalderived cells [1,15]. Human DPSCs are usually taken from extracted third molars or healthy premolars that need to be extracted due to orthodontics. They are easier to obtain and have less ethical pressure. Therefore, DPSCs are ideal seed cells for tissue engineering [5,8,18]. The ability of DPSCs to differentiate into odontoblasts plays an important role in maintaining the dynamic balance of dental pulp tissue and tooth regeneration, and is the basis for future use of tissue engineering to achieve dentin regeneration [13,14,24]. The regulation of odontoblastic differentiation of DPSCs is still a di cult and hot point of research. Odontoblastic differentiation of DPSCs involves many factors such as biological scaffolds, regulatory genes and signal pathways [4,14,22,26,27]. MicroRNAs (miRNAs, miRs) as posttranscriptional inhibitors, recognize and bind to the 3'untranslated region (3'UTR) of the target gene to inhibit the translation of the target gene protein, and have complex regulatory effects on the body's physiological/pathological activities, including the process of odontoblastic differentiation [10][11][12]. Long non-coding RNAs (LncRNAs) as competing endogenous RNAs (ceRNAs) play key role in cell cycle, migration, proliferation, differentiation and apoptosis through sponging miRNAs to regulate miRNA targets. More and more studies have shown that LncRNAs participate in the differentiation process of odontoblasts through sponging miRNAs [3,6,27].
Studies have shown that runt-related transcription factor 2 (RUNX2) plays a key role in the odontoblastic differentiation. Its expression promotes the odontoblastic differentiation [2,7,16]. In this study, the software targetscan (http://www.targetscan.org) was used to analyze which miRNAs maybe target the RUNX2, and the result showed RUNX2 may be targeted by miR-153-3p. We used LncBase Predicted v.2 database (http://carolina.imis.athena-innovation.gr) to analyze the potential LncRNAs which interact with miR-153-3p, and the results showed LncRNA-KCNQ1OT1 contains potential binding site of miR-153-3p.
Jiang et al reported that miR-153-3p inhibited osteogenic differentiation of periodontal ligament stem cells via targeting KDM6A and regulating the ALP, RUNX2 and OPN transcription [9]. Studies showed LncRNA-KCNQ1OT1 regulated osteogenic differentiation by sponging miR-214 [20], miR-320a [21]. Yu et al reported that knockdown of lncRNA-KCNQ1OT1 in tendon stem cell inhibited the osteogenic differentiation by regulating miR-138/PPARγ or RUNX2 axis [23]. However, the role of lncRNA-KCNQ1OT1 and miR-153-3p in the differentiation of DPSCs into odontoblasts is still unclear. The study aimed to explore the effect of lncRNA-KCNQ1OT1 and miR-153-3p on the odontoblastic differentiation of DPSCs and con rm its regulation axis.

DPSCs culture
This study was approved by the Ethics Committee of the A liated Hospital of Nantong University. We declared that all methods were carried out in accordance with the relevant guidelines and regulations.
The cell culture and identi cation was performed as described in our previous studies [12]. Brie y, the pulp from normal human impacted third molars was collected and digested with 3 mg/ml collagenase type I for 1 h at 37°C. Donor of the impacted third molar had given informed consent. Single-cell suspensions of dental pulp were cultured and passaged in Dulbecco modi ed Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C under 5% CO 2 .
The fourth passage cells were used in the following experiments.
After 48 hours, the transfected cells were taken for the following experiment.

Cell viability assay
The cell counting Kit-8 (CCK-8; Beyotime Biotechnology, China) was used to detect the viability of the DPSCs according to the manufacturer instructions, and the OD value was read at 450 nm.

Quantitative real-time PCR (qRT-PCR)
The total RNA in cells was extracted using Trizol (Invitrogen, USA), and quanti ed with agarose gel (Sigma-Aldrich, USA). For LncRNA and mRNA, cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix (Transgen, China). For miRNA, cDNA was synthesized using TransScript miRNA First-Strand cDNA Synthesis SuperMix (Transgen, China). GAPDH and U6 were used as the internal reference. The gene expression level was calculated with 2 −△△Ct method. The primer sequences were shown in the Table-1.

Alkaline phosphatase (ALP) activity assay
After 14 days of odontoblastic differentiation, the cells were lysed with 1% Triton X-100 for 15 min, and centrifuged at 10,000 g for 5 min, then the supernatant was collected and detected with ALP Assay Kit (Beyotime Biotechnology, China) according to the manufacturer instructions, and the OD value was read at 405 nm.

Alizarin red staining
After 14 days of odontoblastic differentiation, the cells were incubated with 2% alizarin red staining solution (Beyotime, China) for 10 min at room temperature. Then the cells were observed under an inverted microscope and the cell mineralization was quanti ed with alizarin red extracted with 100 mM cetylpyridinium chloride solution (Sigma, USA), and the OD value was read at 570 nm.

Statistical analysis
The SPSS 22.0 software was used to analyze the data of this research. The data of this study were presented as mean ± standard deviation (M ± SD). Comparison among groups was tested with independent t test or one-way analysis of variance (ANOVA) followed by Tukey's test. The Pearson analysis was used to analyze the correlation. Statistical graph was made using GraphPad Prism 7 software. P < 0.05 was considered statistically signi cant.

The effect of LncRNA-KCNQ1OT1 on the cell viability and odontoblastic differentiation of DPSCs
After transfection, the LncRNA-KCNQ1OT1 expression level was detected by qRT-PCR. LncRNA-KCNQ1OT1 expression level of DPSCs transfected with pcDNA-KCNQ1OT1 signi cantly increased, and that of DPSCs transfected with KCNQ1OT1 siRNA notably decreased ( Fig. 2.a). The viability of DPSCs was assessed with CCK-8 kit. Compared with control, the differences of OD value among groups were not statistically signi cant. The transfection did not affect the viability of DPSCs (Fig. 2.b).
After 14 days of culture, the odontoblastic differentiation of DPSCs was detected by ALP activity, Alizarin red staining and Western blot for RUNX2, DSPP, and DMP-1. The result of ALP activity assay showed the OD value of the pcDNA-KCNQ1OT1 group was higher than that of control and NC groups, while the OD value of the KCNQ1OT1 siRNA group was lower than that of control and NC groups (Fig. 2.c). The result of Alizarin red staining displayed that mineralized bone matrix increased in the pcDNA-KCNQ1OT1 group and decreased in the KCNQ1OT1 siRNA group comparison with the control and NC groups (Fig. 2.d). The result of Western blot showed the protein expression levels of RUNX2, DSPP, and DMP-1 in the pcDNA-KCNQ1OT1 group increased and in the KCNQ1OT1 siRNA group decreased compared with the control and NC groups (Fig. 2.e).
After 14 days of odontoblastic differentiation, the mineralized bone matrix, ALP activity and protein expression levels of RUNX2, DSPP and DMP-1 in the pcDNA-KCNQ1OT1 group increased comparison with the control group. While DPSCs were co-transfected with pcDNA-KCNQ1OT1 and miR-153-3p mimic, the mineralized bone matrix, ALP activity, RUNX2, DSPP, and DMP-1 protein expression levels were close to the control group. When DPSCs were co-transfected with pcDNA-KCNQ1OT1 and mimic NC, the mineralized bone matrix, ALP activity, RUNX2, DSPP, and DMP-1 protein expression levels were similar to the pcDNA-KCNQ1OT1 group (Fig. 3.d-f).
3.4 LncRNA-KCNQ1OT1 act as a sponge of miR-153-3p and miR-153-3p target on RUNX2 Luciferase reporter gene experiment was used to verify that LncRNA-KCNQ1OT1 act as a sponge of miR-153-3p and miR-153-3p target on RUNX2. The result showed that when the binding fragment of LncRNA-KCNQ1OT1 or core sequence of 3'UTR of RUNX2 was mutated, the luciferase relative activity of mimic group was similar to the NC group. However, in the WT-LncRNA-KCNQ1OT1 or WT-RUNX2 reporter gene system, the relative activity of luciferase in mimic group was signi cantly lower than that in NC group ( Fig. 4.a,b).

Discussion
Odontoblasts are terminally differentiated cells from DPSCs and are one of the main cells that form dental tissues. Odontoblastic differentiation is the prerequisite for dentin formation. Studying the in uence of various signaling pathways on the differentiation of odontoblasts, regulating the expression of various signaling pathways, and promoting the differentiation of odontoblasts will be of great signi cance for the treatment of various dentin-related diseases. More and more researches prove that LncRNAs and miRNAs play key roles in odontoblastic differentiation [3,6,27]. In this study, we found that LncRNA-KCNQ1OT1 promoted the odontoblastic differentiation of DPSCs via regulating miR-153-3p/RUNX2 axis.
RUNX2 is one of the members of the Runt family, which is an speci c transcription factor of odontoblastic differentiation of DPSCs [2]. In this study, during the odontoblastic differentiation of DPSCs, the expression of RUNX2 increased, which is consistent with the research results of other scholars [19]. Studies showed many miRNAs regulated the odontoblastic differentiation via targeting on RUNX2 [10,17,25]. In this study, we used targetscan software to found miR-153-3p have the binding site of RUNX2 3'UTR. Jiang et al reported that miR-153-3p inhibited osteogenic differentiation of periodontal ligament stem cells via targeting KDM6A and regulating the ALP, RUNX2 and OPN transcription [9]. The odontoblastic differentiation process is similar to the osteogenic differentiation. Therefore, in this study, the expression level of miR-153-3p during odontoblastic differentiation of DPSCs was detected and the result showed it decreased in the process and miR-153-3p expression level was negatively correlated with RUNX2 expression level. The luciferase reporter gene experiment con rmed that RUNX2 is a target of miR-153-3p. The results indicated that miR-153-3p was indeed involved in the odontoblastic differentiation of DPSCs, and it was a negative regulatory factor.
The mechanism by which the expression of miR-153-3p decreases during the differentiation of odontoblasts is still unclear. LncRNAs as ceRNAs can sponge miRNAs. We used LncBase Predicted v.2 database to found LncRNA-KCNQ1OT1 contained the potential binding site of miR-153-3p. Study showed that knockdown of LncRNA-KCNQ1OT1 in tendon stem cell inhibited the osteogenic differentiation [23]. The result of qRT-PCR in this study showed LncRNA-KCNQ1OT1 increased during odontoblastic differentiation of DPSCs and its expression level was negatively correlated with miR-153-3p. The luciferase reporter gene experiment con rmed that LncRNA-KCNQ1OT1 sponges miR-153-3p. The results indicated that LncRNA-KCNQ1OT1 positively regulated the odontoblastic differentiation of DPSCs. Therefore, we constructed pcDNA-KCNQ1OT1 or KCNQ1OT1 siRNA to transfect DPSCs to enhance or downregulate LncRNA-KCNQ1OT1 expression level. The result of CCK-8 showed LncRNA-KCNQ1OT1 expression changes had no effect on the cell viability. The odontoblastic differentiation of DPSCs was detected by alizarin red staining, ALP activity and Western blot for RUNX2, DSPP and DMP-1, and the ndings showed down-regulated LncRNA-KCNQ1OT1 expression inhibited the odontoblastic differentiation of DPSCs, while LncRNA-KCNQ1OT1 overexpression promoted the odontoblastic differentiation of DPSCs. When DPSCs were co-transfected with pcDNA-KCNQ1OT1 and miR-153-3p mimic, DPSCs overexpressed LncRNA-KCNQ1OT1 and miR-153-3p at the same time, LncRNA-KCNQ1OT1's promotion of odontoblast differentiation was reversed.

Conclusion
In summary, our data demonstrated that LncRNA-KCNQ1OT1 promotes the odontoblastic differentiation of DPSCs via regulating miR-153-3p/RUNX2 axis, which may provides a therapeutic clue for odontogenesis.

Declarations
The study was approved by the Ethics Committee of the A liated Hospital of Nantong University.

Consent to participate
Donor of the impacted third molar had given informed consent.

Consent for publication
Donor of the impacted third molar had given informed consent.
Code availability N/A.

Availability of data and material
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.  protein expression levels in the pcDNA-KCNQ1OT1 group increased and in the KCNQ1OT1 siRNA group decreased comparison with the control and NC groups. Bar = 50 mm. * vs. control group, P < 0.05, # vs. pcDNA group, P < 0.05, @ vs. siRNA NC group, P < 0.05, & vs. pcDNA-KCNQ1OT1 group, P < 0.05.  LncRNA-KCNQ1OT1 act as a sponge of miR-153-3p and miR-153-3p target on RUNX2. (a) In the WT-LncRNA-KCNQ1OT1 reporter gene system, the relative activity of luciferase in mimic group was signi cantly lower than that in NC group. When the binding fragment of LncRNA-KCNQ1OT1 was mutated, the luciferase relative activity of mimic group was similar to the NC group. (b) In the WT-RUNX2 reporter gene system, the relative activity of luciferase in mimic group was signi cantly lower than that in NC group. When the core sequence of 3'UTR of RUNX2 was mutated, the luciferase relative activity of mimic group was similar to the NC group. * vs. NC group, P < 0.05.

Supplementary Files
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