microRNA‐126 inhibits vascular cell adhesion molecule‐1 and interleukin‐1beta in human dental pulp cells

Abstract Background Vascular cell adhesion molecule (VCAM‐1) mediates pulpitis via regulating interleukin (IL)‐1β. microRNA (miR)‐126 was reported to regulate the VCAM‐1 under many different pathophysiological circumstances. We investigated variations of miR‐126 and VCAM‐1 in inflamed patient pulp tissues and determined potential roles of miR‐126 in pulpitis using human dental pulp cells (hDPCs) in vitro. Methods We quantitatively measured the transcripts of miR‐126 and VCAM‐1 in inflamed human pulp tissues using qRT‐PCR and compared with those from healthy human pulp tissues. In addition, we transfected miR‐126 in hDPCs using plasmid DNA (pDNA)‐encoding miR‐126 delivered by polyethylenimine (PEI) nanoparticles. Results The irreversible pulpitis significantly reduced miR‐126 and increased the transcript of VCAM‐1 in pulp tissues (p < 0.05). pDNA‐encoding miR‐126 delivered PEI nanoparticles and effectively upregulated the expression of miR‐126 in hDPCs (p < 0.05). The overexpression of miR‐126 could effectively suppress the transcripts and protein levels of VCAM‐1 and IL‐1β induced by Pg‐LPS at 100ng/mL in DPCs (p < 0.05). Conclusions miR‐126 is involved in pulpitis and downregulated the VCAM‐1 and IL‐1β in DPCs. miR‐126 may be a potential target to attenuate the inflammation of pulpitis.

double-edged sword for healing pulpal tissues. It has been demonstrated that a relatively low amount of inflammation promotes dentin repair, whereas a high amount of chronic inflammation may inhibit repair mechanisms. 4,5 Thus, modulating the inflammatory reaction plays an important role in treating pulpitis and accelerating dentinogenesis. Inflammation involves inflammatory cell activation, cellular factor secretion, mediation of antigen-antibody reaction, accompanying increased blood flow, and dilation of venules and arterioles, enhancement of blood vessels permeability, and percolation of leukocytes into the tissues. 6,7 The vascular cell adhesion molecule-1 (VCAM-1/CD106) (a 90-kDa glycoprotein), a member of cell adhesion molecules (CAMS), plays a crucial role in accumulating inflammatory cells during inflammation by regulating the adhesion of lymphocytes monocytes, eosinophils, and basophils to vascular endothelium. [8][9][10][11][12] VCAM-1 was reported to actively involve the inflammation of pulpitis in tooth preparation. Lipopolysaccharide (LPS) increased VCAM-1 in human dental pulp cells (DPCs). 13,14 These evidence indicate that VCAM-1 may contribute to the excessive inflammation in pulpitis, 15 and thus, the modulation of VCAM-1 may effectively attenuate the inflammatory reaction of pulpitis.
MicroRNAs (miRs) are short non-coding RNAs that regulate physiologically and pathophysiologically through translational inhibition or degeneration of specific genes' mRNAs. 16 Growing evidence indicates that miRs play essential roles in pulpal inflammation and pulpal tissue repair. They may be used for pulpitis treatment. 17,18 miR-126 (also referred to as miR-126-3p) is derived from the egfl7 gene, harboring within intron 7 in all vertebrates. 19 miR-126 serves as a crucial regulator in endothelial cell functions, including vascular repair, angiogenesis, inflammatory activation, and apoptosis. Specifically, miR-126 represses SPRED1 and PIK2R2, which negatively regulate VEGF signaling via the MAPK/ERK and PI3K/AKT pathways, respectively, to promote the cells proliferation, migration, and angiogenesis. 19 miR-126 could also enhance the maturation and stabilization of growing blood vessels by suppressing the p21-activated kinase 1 gene and regulating angiopoietin-1 signaling. 20 Furthermore, miR-126 was reported to modulate the inflammation through directly targeting VCAM-1 to block the adhesion and infiltration of leukocytes into the vasculature wall. 19,21 However, the characteristics of miR-126 in the dental pulp and its regulatory roles in pulpal inflammation remain unknown.
In this study, we investigated the miR-126 and VCAM-1 variation in inflamed pulp tissues and determined the inhibitory function of miR-126 in VCAM-1 and pulpitis using human dental pulp cells (DPCs).

| Collection of dental pulp tissue
This study was approved by the Institutional Review Boards of the University. Each patient signed written informed consent. Healthy dental pulp was obtained from 10 patients with an average age of 19.25 ± 3.3 years old from the extracted wisdom teeth or premolars of patients whose teeth were removed for orthodontic reasons.
The inflamed pulp tissues were extirpated from carious teeth of 10 patients with an average of 25.5 ± 11.6 years old diagnosed with irreversible pulpitis according to the American Association of Endodontists guidelines. 22 The dental pulp was exposed with a handpiece and extirpated with a nerve broach.

| Cell culture
Human DPCs were isolated and cultured as described previously. 23 Briefly, the pulp tissue was carefully stripped from the crown and root and cut into pieces smaller than 1mm. 3 The tissue fragments were then covered by the glass coverslips on the bottom of culture dishes and incubated with DMEM (Gibco) supplemented with 100 IU/ml penicillin (Gibco) and 20% fetal bovine serum (FBS, Gibco).
The culture medium was changed at five-day intervals. After reaching 70% confluence, the cells were collected by trypsinization (0.2% trypsin and 0.02% EDTA, Gibco) and split at a ratio of 1:4 and subcultured with DMEM supplemented with 10% FBS.

| Transfection of DPCs with plasmid DNA (pDNA) encoding miR-126
The pDNA-encoding miR-126 was constructed with pSilencer-4.1 and the miR-126-specific sequence commercially (OriGene). pSilencer-4.1 was used as a vector stably expressing miR-126, and the mature sequence of miR-126 (CAUUAUUACUUUUGGUACGCG) was synthesized by oligonucleotides. Empty pSilencer-4.1 vector (EV) was used as a control. Polyethyleneimine (PEI) was used to facilitate the transfection of plasmid DNA (pDNA)-encoding miR-126 as described in our previous studies. 24 Briefly, pDNA-encoding miR-126 and PEI at a ratio of 1:3 were mixed in an opti-MEM medium (Gibco) for 30 s and incubated for 20 min at room temperature to form nanoplexes. A total of 500 μl of opti-MEM containing 3 µg PEI and 9 µg pDNA of miR-126 was added to DPCs at 5 × 10 5 cells/well in a 6-well plate. After 4 h, DPCs were washed twice using PBS and then cultured with DMEM for 48 h. The overexpression of miR-126 was measured using qRT-PCR.

| Influence of miR-126 on VCAM-1 and IL-1β under LPS challenge
After transfection with the pDNA-encoding miR-126 at 9µg pDNA delivered by PEI in a 6-well plate for 48 h, the DPCs were treated

| qRT-PCR
Total RNA from human pulp tissue and cultured cells were col-

| Statistical analysis
The data were analyzed with commercially available statistics software (Statistical Analysis System 8.2). All quantitative results were expressed as mean ± standard deviation. The differences in relatively normalized expression for miR-126 and VCAM-1 between health and inflamed pulp were determined using Student's t test.
Statistical significance among groups with miR-126 treatment was determined using a one-way analysis of variance (ANOVA), and the Bonferroni post hoc test was used for multiple comparisons. A pvalue of < 0.05 was considered to be significant. Each experiment was performed in triplicate.

| Pulpal inflammation reduced miR-126 and increased VCAM-1
Pulpal tissues were collected from patients diagnosed with irreversible pulpitis and healthy pulpal tissue as controls. miR-126 was significantly decreased in inflamed pulp tissues ( Figure 1A), while the transcript of VCAM-1 was significantly increased (p < 0.05) ( Figure 1B).

| PEI facilitated the transfection of miR-126 into human DPCs
We used PEI nanoparticles to deliver pDNA-encoding miR-126 to primary human DPCs collected from 12 different patients (6 male and 6 female). After treating with 3 μg pSil-miR-126 or EV in 600 μl opti-MEM, DPCs maintain the fibroblast-like morphology (Figure 2A). For DPCs treated with miR-126, miR-126 expression was significantly increased than that with EV after 24 h. The expression level of miR-126 maintains a high level after 7 days (p < 0.05) ( Figure 2B).

| Overexpression of miR-126 attenuated the IL-1β and VCAM-1
The inhibitory function of miR-126 on VCAM-1 and IL-1β was investigated in DPCs with miR-126 overexpression. DPCs were transfected with pDNA-encoding miR-126 or EV for 48 h and subsequently exposed to Pg-LPS at 100 ng/ml. Transfection with miR-126 delivered by PEI nanoparticles significantly increased the expression of miR-126 in DPCs (p < 0.05). While PG-LPS reduced miR-126 in cells transfected with miR-126 and EV ( Figure 4A), miR-126 was significantly increased in cells with miR-126 transfection than that with EV (p < 0.05) ( Figure 4B). In addition, for cells induced by Pg-LPS, miR-126 significantly reduced VCAM-1 and IL-1β than EV (p < 0.05).

| DISCUSS ION
In this study, we found that the expression of miR-126 in inflamed patient pulp tissues with increased VCAM-1 was significantly downregulated compared with that of healthy pulp tissues. In addition, we confirmed the function of miR-126 overexpression in reducing VCAM-1 and interleukin 1 beta (IL-1β), a pro-inflammatory cytokine participating in pulpitis, in human DPCs under the stimulation of LPS.
miR-126 has been reported to reduce IL-6, IL-10, and tumor necrosis factorα (TNFα) in endothelial cells via the PI3K/Akt/eNOS signaling pathway. 25 It also directly targets VCAM-1 and high mobility group box 1 (HMGB1), which are genes associated with endothelial activation and inflammation. 26 In this study, we found that the inflamed pulpal tissues from irreversible pulpitis patients signifi- Interleukin-1β gene is a well-known pro-inflammatory cytokine in initiation and progression of inflammation, including macrophage recruitment, activation, and inducement of other pro-inflammation cytokines, such as IL-6, IL-8, ICAM-1, and modulating chemokine expression. [34][35][36] IL-1β was reported to be significantly increased in inflamed dental pulp tissue and DPCs under LPS stimulation. 37 Inhibition of IL-1β can relieve cell damage in inflammation, 38,39 and the imbalance between IL-1β agonist and antagonist levels can lead to exaggerated inflammatory responses. 40 It was reported that patients obtained beneficial effects from using IL-1β antagonists. 41 IL-1β is not the direct target gene of miR-126, and the regulation of miR-126 on IL-1β is varied by cell types and inflammation. However, in this study, we found the miR-126 overexpression could significantly decrease IL-1β in DPCs. These findings further supported the therapeutic potential of miR-126 in pulpitis treatment. One previous study has demonstrated that miR-126 participated in the regulation of cell viability of DPCs by directly repressing PTEN levels and indirectly by activating Akt signal that can function as pro-apoptotic factors. 42 Our study has proposed different mechanisms of the effect of miR-126 on DPCs.
In conclusion, in this study, we revealed miR-126 and VCAM-1 variations in patients with irreversible pulpitis. LPS effectively downregulated miR-126 that might contribute to the pulpitis's inflammatory progression by activating VCAM-1 and IL-1β.
Overexpression of miR-126 using non-viral nanoparticle PEI can effectively suppress VCAM-1 and reduce IL-1β. These results indicated that miR-126 might be a potential target to treat pulpitis, and future studies were needed to confirm the regulation of miR-126 in vivo.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this. Further enquiries can be directed to the corresponding author.