miRNA-125b Regulates Osteogenic Differentiation of Periodontal Ligament Cells Through NKIRAS2/NF-κB Pathway

Periodontal ligament (PDL), the most important tissue influencing the orthodontic tooth movement, is composed of heterogeneous multipotent cell populations, which has the ability to undergo osteoblast differentiation [1]. Osteoblast differentiation of periodontal ligament cells (PDLCs) results in periodontal tissue remodeling and regenerating therefore it plays a vital role in maintaining homeostasis of periodontium. Varieties of factors have been found involved in the regulation of PDLCs osteoblast differentiation, however, the molecular mechanisms and signaling pathways are still unclear. A better understanding of the molecular mechanisms that govern osteogenesis might provide us with new perspectives on treatment of orthodontic teeth movement and periodontitis [2].

MicroRNAs consist of a group of endogenous small, non-coding RNAs of approximately 22 nucleotides that exert post-transcriptional effects on gene expression [3]. They are thought to take part in several biological processes [4]. Some microRNA can inhibit osteo-blast differentiation (e.g. miRNA-214 and miRNA-138) by targeting Osx [5] and Runx2 [6]; while the others (e.g. miRNA-378; miRNA-17; miRNA-21; miR-101; miRNA-146a; miR-132) can promote osteoblast differentiation via several pathways, by targeting BMP2 [7], Smurf1 [8], PLAP-1 [9], NF-κB signaling [10] or mTOR signaling pathway [11]. MiR-125b, one of the early discovered miRNAs, has been shown to inhibit osteoblast differentiation in the mouse MSC line ST2 [12], and it is a key regulatory factor of osteoblastic differentiation by directly targeting Cbfb [13] and indirectly acting on Runx2 at an early stage MSCs osteoblastic differentiation. Moreover, it has been proved that miR-125b is significantly down regulated during osteogenic differentiation of PDLCs [14]. However, the role of miR-125b in the osteogenic differentiation of PDLCs and the molecular targets of miR-125b are still not identified.

As a key transcription factor, nuclear factor kappa B (NF-κB) comprises two subunits, commonly p50 (NFκB1)/p65 (RelA), which is held in the cytoplasm by the inhibitor of NF-κB (IκB) in its inactive state [15]. It has been found involved in bone development and bone remodeling [16]. Suppressing activation of the NF-κB signal transduction pathway can potently augment osteoblast differentiation [17, 18]. NF-κB inhibitor interacting RAS-like 2 (NKI-RAS2) can interfere with proteosomal degradation of IκB [19, 20] and disturb NF-kB activity.

Several studies report that miR-125b plays a key role in inhibiting osteoblastic differentiation, but its role in regulating osteoblastic differentiation of PDLCs still remains unclear. At same time, the function of NKIRAS2 in osteogenic process of PDLCs is also unclear. And NKIRAS2 is one of the target gene of miR-125b [21] so whether osteogenic process of PDLCs is regulated through mR-125b/ NKIRAS2 pathway? In this study, we used primary cultured PDLCs as the host to explore the roles of miR-125b and the related mechanism.

The human permanent teeth were obtained from healthy individuals under 25 years of age for orthodontics reason at the School of Stomatology, China Medical University, under the approval of the Ethical Committee of School of Stomatology, China Medical University. After extraction, the teeth were immediately washed by phosphate-buffered saline (PBS; Sigma, USA) and Dulbecco’s modified Eagle’s medium (DMEM; Sigma, USA) supplemented with antibiotics (300 U/ml penicillin and 300 mg/ml streptomycin; Sigma, USA). The periodontal ligament was separated from the middle third of the root surface and cultured by the combination of tissue explant and enzymatic digestion method. The PDL tissues were digested with 3ml 0.2% collagenase type I enzyme (Sigma, USA) at 37°C in 50 min and vortexed every 10 min into the smallest pieces. The cells containing mixture were supplemented with culture medium and centrifuged at 1, 000 rpm. Precipitate was suspended with culture medium, then seeded onto a 25-cm² flask (Nunc, Denmark) and incubated in a humid environment. Cells growth and morphology were observed under an inverted microscope. PDLCs were used at passages 3 for following study.

The characterization of PDLCs was tested by flow cytometry (BD FACSVerse, USA). After the cells were transferred, they were fixed for 15 min in 4% paraformaldehyde (Sigma, USA). The cells were incubated with 3% bovine serum albumin and then with primary antibodies (Abcam, UK) raised against CD45, CD90, CD146 for 1 h. Then we washed cells with buffer, and added the secondary antibody for 45 min. Finally, the cells were washed three times and analyzed with a flow cytometer. The identity of PDLCs was further confirmed based on their differentiation potential by Cell Counting Kit-8 (BestBio, China).

Osteoblastic differentiation was initiated by seeding PDLCs at a density of 5, 000 cells per cm² in the medium containing 10% FBS (Invitrogen, USA), 100nM dexamethasone (Baomanbio, China), 100 mM β-glycerol phosphate (Sigma, USA) and 50 μg/ml α-ascorbic acid 2-phosphate (Sanat Cruz, USA), 100 U/ml penicillin (Sigma, USA), and 100 mg/ml streptomycin (Sigma, USA) for 21d.

After 21 days of culture in the osteogenic medium, the cells were washed with PBS twice and fixed with 4 % paraformaldehyde for 30 min at 4 °C. After washed by deionized water for three times, the fixed cells were stained with 2% Alizarin red S (Sanat Cruz, USA) and Alkaline phosphatase staining was monitored using ALP staining kit (Cosmo Bio, Japan) according to the manufacturer’s instructions. Finally, the cells were washed and photographed.

After 21 days of culture in the osteogenic medium, the cells were washed three times with PBS and lysed by 0.1%TritonX-100. Lysates were clarified by centrifugation at 13, 000×g, 4 °C for 20min and sonication, and the ALP substrate solution (Yeasen, China) was added to each well (96-well plate) at 37 °C for 10-60 min. ALP activity was determined by absorbance measurement at 405 nm and normalized to total protein. ALP activity was normalized to total protein.

RT-qPCR was performed to measure the relative mRNA levels. Cultured cells were lysed by Trizol (In-vitrogen, USA) for RNA isolation. Next, the RNA was reversed-transcribed into complementary DNA using thePrimeScript RT reagent kit (TaKaRa, JP). RT-qPCR amplification conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. The relative amount of mRNA or microRNA was calculated by 2^-ΔΔCt method and normalized to the expression of U6 respectively. The primer pairs of the related genes are indicated in Table 1.

Lentivirus vectors were designed and constructed byGenomeditech (Shanghai, China). These lentivirus vectors contain a target gene or empty lentiviral vectors. (1×10⁶/well) PDLCs were seeded into 24-well plates for 24 h and then treated with 5μg/mL Polybrene (Sanat Cruz, USA) and lentivirus suspension, and cultured for 24h. Then the medium was replaced by fresh medium. The shRNA of NKIRAS2 were designed and synthesized by Genomeditech (Shanghai, China), and they were transfected into PDLCs according to the protocol.

Cell fractionation was performed using the nuclear extract kit (Active Motif, USA). Cells lysates were extracted with lysis buffer (50 mMTris, pH 7.5, 250 mMNaCl, 0.1% sodium dodecyl sulfate, 2mM dithiothreitol (DTT), 0.5% NP-40, 1mM PMSF and protease inhibitor cocktail) on ice for 30 min. After centrifugation at 15, 000g at 4 °C for 10 min, proteinconcentrations were collected and then subjected to SDS-PAGE gel andelectro-blotted on to polyvinylidenedifluoride (PVDF) membranes. The membranes were then blocked with 5% BSA (CST, USA) and incubated overnight with primary antibodies. Blots were then washed and incubated for 1 h with secondary antibody (goat anti-rabbit Alexa Fluor 680, Invitrogen, USA). Protein bands were visualized with chemiluminescent ECLreagent (Millipore, USA).

The primary antibodies used in this study were directedagainst NKIRAS2 (diluted 1: 500, Abcam, UK), IκB (diluted 1: 10000, Abcam, UK), phosphor-IκB(diluted 1: 2000, Cell Signaling, USA), p65(diluted 1: 2000, Abcam, UK), Runx2(1/500; Cell Signaling, USA), ALP(diluted 1: 5000, Abcam, UK), OPN (diluted 1: 1000, Abcam, UK), OCN(diluted 1: 500, Abcam, UK), LAMIN A and Osterix (diluted 1: 5000, Abcam, UK).

The luciferase assay is to determine whether miR-125b could directly regulateNKIRAS2. The binding sequences of NKIRAS2 mRNA 3′UTRs and miR-125b were amplified by RT-qPCR. Then the RT-qPCR product was inserted into the multiple cloning site of the pGL3 empty vector. Binding-region mutations were achieved from a Quik Change Site-Directed Mutagenesis Kit (Stratagene, USA). Luciferase assays were performed 48 h after transfection using the Dual Lucif-erase Reporter Assay System (Promega, USA). Luciferase activity was normalized to ß-galactosidase activity.

Data are presented as the mean ± SD from at least three independent experiments. Statistical comparisons were made between two groups with the t-test by SPSS software. P values less than 0.05 were considered statistically significant.

After 10 d, the cells began to appear spread the tissue and the cells reached confluence about 21d later. Cultured PDLCs were spindle shaped, fibroblast-like appearance and they were grown to a near-confluent state from single cells (Fig. 1A). They are characterized by the absence of positive markers (CD146/CD90) and the expression of a specific pattern of surface antigens (CD45). FACS analysis demonstrates that PDLCs expressed positive markers CD146 (48.1%), CD90 (99.73%) and expressed negative makers CD45 (4.6%) (Fig. 1B). The identity of PDLCs was further confirmed based on their differentiation potential by CCK-8 (Fig. 1C). After the cells being cultured in osteogenic differentiation media for 21 days, the osteogenic differentiation potential of PDLCs was evidenced by Alizarin red S staining and ALP staining (Fig. 1D), respectively. There was mineralized nodule formation inside the cells and the ALP activity increased significantly (Fig. 1E). Our results showed the expression of specific markers for the osteogenic differentiation were increased (Fig. 1F).

We performed RT-qPCR to determine the expression of miR-125b and NKIRAS2 during osteoblastic differentiation in PDLCs. After being cultured in osteogenic differentiation media for 1, 3, 7, 10, 14, 21days, the expression of miR-125b (Fig. 2A) was down regulated from day 1 and reached a bottom 14 days later. The gene NKIRAS2 was increased during osteoblast differentiation of PDLCs (Fig. 2B), similar to osteoblast marker genes (Fig. 1F) and coinciding with down-regulation of miR-125b (Fig. 2A).

According to analysis, NKIRAS2 has a 7-nt seed match site for miR-125b within its 3’UTR, and this putative target site is highly conserved among the vertebrates (Fig. 3A). To test whether miR-125b is capable of interacting with the 3’UTR sequence of NKIRAS2, luciferase reporter assays were performed. The results demonstrate that co-transfection with pre-miR-125b resulted in concentration- dependent down-regulation of luciferase activityrelative compared to that of co-transfection with pre-miR-125b negative control or pNKIRAS2-MUT, while co-transfection anti-miR-125b with NKIRAS2 3’UTR luciferase reporter resulted in concentration-dependent up-regulation of luciferase activity compared to that of co-transfection with anti-miR-125b negative control or pNKIRAS2-MUT (Fig. 3B). These data indicate that NKI-RAS2 is a direct target of miR-125b.

To determine the biological effect of miR-125b on osteogenic differentiation, miR-125b was overexpressed by transfecting lentivirus vectors. After transfected for 2 days, PDLCs were cultured in osteogenic differentiation media for additional 7 days, then ALP activity and the osteoblast marker gene expression were examined. Alizarin red S and ALP staining was examined after 21 days. RT-qPCR was used to confirm transfection efficiency (Fig. 4A). Alizarin red S (Fig. 4B), ALP staining (Fig. 4C) analysis indicated that overexpression of miR-125b attenuated the osteogenic differentiation of PDLCs.ALP activity (Fig. 4D) and the expression of osteoblast marker gene (Fig. 4E, F), such as ALP, OCN, and OPN were decreased in cells transfected with LV-miR-125b compared to LV-vector group. These results indicate that the miR-125b negatively regulates osteogenic differentiation in PDLCs.

To investigate the role of NKIRAS2 in the osteogenic differentiation of PDLCs, we use RNA interference technique for suppressing NKIRAS2 levels in PDLCs.PDLCs were transfected with sh-NKIRAS2 and control for 2 days then cultured in osteogenic differentiation media for additional 7 and 21 days. RT-qPCR was used to confirm transfection efficiency (Fig. 5A). Alizarin red S, ALP staining and ALP activity showed that suppression of NKIRAS2 decreased ALP activity and mineralized bone matrix formation of PDLCs (Fig. 5B, C). RT-qPCR analysis confirmed that osteoblast marker genes were decreased and western blot analysis showed that Runx2, Osterix, OCN, OPN, ALP protein levels were decreased (Fig. 5D, E). These results indicated that the suppression of NKIRAS2 negatively regulates osteogenic differentiation in PDLCs.

To demonstrate whether the increase of PDLCs osteoblastic differentiation induced by the down-regulation of miR-125b expression was due to NKIRAS2, we co-transfected anti-miR-125b and sh-NKIRAS2 into PDLCs cells. As shown in (Fig. 6A), the expression of miR-125b and NKIRAS2 was blocked after co-transfection. After 14d osteogenic differentiation, the extent of osteoblast marker genes increase was far less than that effected by anti-miR-125b transfection alone (Fig. 6B), and microscopic analysis of the Alizarin red S, ALP staining revealed lower levels of mineralization in NKIRAS2 knockdown PDLCs than in control cells (Fig. 6C). All these data suggested that NKIRAS2 is essential for the role of miR-125b inhibitor to promote osteogenesis.

PDLCs were transfected with sh-NKIRAS2 and cultured in osteogenic differentiation media for additional 7 days, the nuclear and cytoplasmic fractions of cells were analyzed by western blotting. The level of phosphorylated IκB was higher in cells transfected with sh-NKIRAS2 than that were not. It suggests that NKIRAS2 can postpone phosphorylation process of IκB and inhibit degradation of IκB. The cells transfected with sh-NKIRAS2 were found to express higher level of nuclear p65 and lower level of cytoplasmic p65 relative to cells not transfected (Fig. 7). The results suggest that NKIRAS2 may inhibit the translocation of p65 into the nucleus.

The activation of the NF-κB pathway is characterized by the stabilization of p50(NFKB1)/ p65(RelA)(i), the translocation of p50/p65 into the nucleus (ii), the interaction of p50/ p65and its target genes(iii). It had been proved that NKIRAS2 can block the p50/p65 trans-location into the nucleus and NKIRAS2 is the target gene of miR-125b. Therefore, we investigated the level of p65 in nucleus to demonstrate whether miR-125b inhibits PDLCs osteo-blastic differentiation by enhancing NF-κB signaling. Nuclear and cytoplasmic fractions of cells cultured in osteogenic differentiation media were analyzed by western blotting. When the miRNA-125b was up regulated, the level of IkB was decreased and the level of nuclear p65 was increased (Fig. 8A). When the miRNA-125b was down regulated, the level of IkB was increased and the level of nuclear p65 was decreased (Fig. 8B).

In this study, we demonstrated that miR-125b and the related target NF-κB pathway play an important role in theosteogenic differentiation of PDLCs.PDLCs have been regarded as an important candidate with capability of cyto-differentiation and mineralization to form mineralized tissue cells [22]. PDLCs consist of heterogeneous multipotent cell populations, which have the potential to undergo osteogenic differentiation. Osteoblast differentiation results in the expression of tissue-specific gene such as alkaline phosphatase (ALP), osteo-calcin (OCN), osteopontin (OPN), periostin (PON), and others. Cell differentiation involves complex pathways regulated at both transcriptional and post-transcriptional levels. Recently, it has been reported that miRNAs have an influence on the cells osteoblast differentiation [23], through negative regulation of gene expression at the post-transcriptional level.

In this study, we analyzed the expression of miR-125b in osteoblastic differentiation using PDLCs and found that miR-125b was decreased during the first 14 days and then elevated at the end of detection. The study also showed that the osteoblastic differentiation in PDLCs was inhibited by overactivity of miR-125b. They all demonstrated that miR-125b was a negative regulator in osteogenesis process. Furthermore, it has been reported NKIRAS2, which is a target gene of miR-125b, suppresses NF-κB activation by inhibiting its transcriptional activation [24]. Since NF-κB signaling is critical for PDLCs osteogenesis [9], NKIRAS2 caught our attention, and the osteogenic differentiation mechanism mediated by miR-125b was investigated. When we transfected the shRNA of NKIRAS2 into PDLCs, the osteoblastic differentiation had been inhibited. Cells transfected with sh-NKIRAS2 had decreased amounts of IκB protein, but increased the phosphorylated form of IκB protein. Furthermore, the decreased the amount of IκB proteins in sh-NKIRAS2 transfected cells also have sequestered the increased p65 in the nucleus. These results suggest that NKIRAS2 associated with IκB, slowing down the degradation of IκB, and inhibited canonical NF-κB signaling pathway.

The goal of this study was not only to identify miR-125b regulated the osteoblastic differentiation of PDLCs but also to predict whether miR-125b could regulate a specific signaling pathway. We silenced the NKIRAS2 and transfected the LV-anti-miR-125b to PDLCs simultaneously. The mRNA expression of Runx2, Osterix, OCN, OPN, and ALP increased and the protein expression level of NKIRAS2 reduced compared with the blank control group. However, the extent of osteoblast marker genes increase was far less than that effected by LV-anti-miR-125b transfection alone. All these data suggested that miR-125b inhibits PDLCs osteoblastic differentiation by target NKIRAS2.

Degradation of IκB by a phosphorylation-dependent represents an important event for the unmasking of NF-κB translocation sequences and the initiation of transcription by the nuclear factor [25]. To further investigate whether miR-125b inhibits PDLCs osteoblastic differentiation through enhancing NF-κB signaling, we confirmed the effects of miR-125b induced IκB degradation and nuclear translocation of p65. The results showed that miR-125b could inhibit PDLCs osteogenesis and theNF-κB pathway was involved in this process. As miR-125b expression was upregulated, the activity of NF-κB pathway was increased. MiR-125b induces the degradation of IκB and nuclear translocation of NF-κB p65/p50 heterodimers.

The osteoblastic differentiation of PDLCs can be influenced by many factors, such as inflammatory microenvironment. Liu [7] found that the multi-differentiation potential of mesenchymal stem cells (MSCs) isolated from periodontitis-affected periodontal ligament tissue was significantly lower than that of MSCs isolated from healthy human periodontal ligament tissue. Furthermore, inflammatory cytokines can regulate osteogenesis of PDLCs through different signaling pathways, such as MAPKs and Smad signaling pathways [26]. The “One shot, multiple targets” signature of miRNA heralds that there are other signaling pathways regulating the osteogenesis of PDLCs, for example, Smad4 is a target of miR-125b. In the future work, we will focus on the investigation of the roles of the other signaling pathways via which miR-125b regulates osteoblastic differentiation and whether miR-125b works in an inflammatory microenvironment.

When we analyzed the expression of miR-125b in osteoblastic differentiation, the result showed that the expression of miR-125b was decreased during early stage (≤14 days) while increased after 14 days of osteogenic differentiation of PDLCs. It suggested the miRNA could modulate osteogenic function initiating differentiation rather than after the differentiation [27, 28].

Collectively, in the present study we have demonstrated a previously unknown role for miR-125b in inhibiting PDLCs osteogenic differentiation. We have proved that this is likely to be due to the fact that NKIRAS2, a target of miR-125b, inhibits NF-κB signaling and promotes osteogenic differentiation. Furthermore, we find that miR-125b activates the NF-κB pathway by negatively regulating NKIRAS2 expression, inhibiting the osteogenic differentiation of periodontal ligament cells. Our results suggest that therapeutic inhibition of miR-125b in osteoblasts may promote bone formation and even reverse periodontitis by exerting an anabolic effect.

The authors declare that they have no competing interests.