Upregulated microRNA‐126 induces apoptosis of dental pulp stem cell via mediating PTEN‐regulated Akt activation

Abstract Introduction Human dental pulp stem cells (DPSCs) have potential applications in regenerative medicine. The molecular mechanisms underlying DPSCs viability and apoptosis are not completely understood. Here, we investigated the role of miR‐126 in DPSCs viability and apoptosis. Material and methods Senescent DPSCs were compared with early passage DPSCs. real‐time PCR and microARRAY were performed to identify the differential expression of miR‐126, and western blot was performed to detect the expression of PTEN. MTT assay was utilized to reveal the proliferative rate of both senescent and early passage DPSCs. Flow cytometry was used to examine the apoptotic rate of DPSCs. Dual‐luciferase reporter assay was carried out to detect the interaction of miR‐126 and PTEN. Results Senescent DPSCs showed a high level of apoptosis. Further study showed that miR‐126 is upregulated in senescent DPSCs and its overexpression in early passaged DPSCs induced apoptosis. Phosphatase and tensin homolog gene (PTEN) was identified as a target of miR‐126. PTEN was downregulated in senescent DPSCs, whereas miR‐126 inhibition upregulated PTEN level, and subsequently activated Akt pathway and suppressed the apoptotic phenotype of senescent DPSCs. In addition, PTEN overexpression rescued apoptosis of DPSCs at later stage. Conclusion Our results demonstrate that the miR‐126‐PTEN‐Akt axis plays a key role in the regulation of DPSCs apoptosis and provide a candidate target to improve the functional and therapeutic potential of DPSCs.

multipotent stem cells and can differentiate into various cell types including neurons, cardiomyocytes, chondrocytes, osteoblasts, and liver cells, and they are therefore valuable stem cell sources for regenerative therapies for the treatment of dental and other diseases. [5][6][7][8][9] However, the application of DPSCs in cell therapy is limited by their replicative and differentiation potentials, which are reduced or lost during culturing. Therefore, elucidating the mechanisms underlying DPSCs cell death and apoptosis is important for their application.
MicroRNAs (miRNAs) are a group of non-coding RNA molecules, with an approximate length of 22-25 nucleotides, which can post-transcriptionally regulate the expression of their target genes, ultimately leading to the degradation of the target mRNA. 10 An increasing number of reports have shown that miRNAs play an essential role in numerous molecular and biological processes, such as apoptosis, cell proliferation, migration, and necrocytosis. 11,12 MiR-126, frequently being downregulated in human tumors, has been demonstrated to possess fundamental roles in various cellular processes, especially in the modulation of proliferation and differentiation. [13][14][15][16][17] MiR-126 was also involved in the basal function of adipose tissue stem cell and mesenchymal stem cell. [18][19][20] However, the mechanism of miR-126 in regulating the function of human DPSCs remain needs to be further investigated. The present study attempts to probe the role of miR-126 in the features of DPSCs. It appears that expression of miR-126 was markedly increased in the senescent DPSCs. In turn, senescent DPSCs treated with miR-126 inhibitor showed apparent upregulated viability and reduced apoptosis. Our findings indicated that miR-126 could exert a regulatory function in cell viability of senescent DPSCs.

| Cells and cell culture methods
Normal human third molars were collected from adults (18-26 years of age) at Qilu Hospital under approval of the Ethics Committee of Qilu Hospital, School of Medicine, Shandong University. All patients gave their written informed consent to participate. DPSCs were obtained as described previously. 21 Cells were cultured in alpha-minimum essential medium (a-MEM; Hyclone, Logan, UT, USA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO 2 . The medium was replaced every 2 days. Cells were subcultured at a ratio of 1:3 until they reached 75%-85% confluence. Population doubling (PD) analysis was performed as described previously. 22 Cells were declared senescent and apoptotic when they were senescence-associated β-galactosidase (SA-β-gal) positive and had decreased Bcl-2, and increased Bax protein expression.

| miRNA array
Total RNA was extracted using the phenol-chloroform method (TRIzol; Invitrogen; Thermo Fisher Scientific, Inc.). The quality of the RNA was assessed by capillary electrophoresis (Agilent Technologies Inc.). Libraries for small RNA sequencing were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England BioLabs, Inc.) according to the manufacturer's protocol. The libraries were quantified using the Agilent Bioanalyzer 2100 system with DNA high-sensitivity chips. The raw sequence files were subjected to quality control analysis with the Fast QC quality control tool. To avoid low-quality data, adaptors were removed by Cutadapt Differential expression sequence was used to calculate differential expression levels and to evaluate the statistical significance of detected alterations between the control and case samples.

HRP-linked goat anti-rabbit IgG secondary antibodies (Amersham)
were incubated with the membranes for 1 hour at room temperature in PBST containing 5% BSA, followed by chemiluminescent

detection. A C-DiGit Blot Scanner and Super Signal West Femto
Maximum Sensitivity Substrate Kit (provided by Thermo) were used to detect bound antibodies.

| RNA extraction and Q-PCR
Total RNA was extracted from tissues using Trizol reagent according to the manufacturer's instructions (Invitrogen). Messenger RNA (mRNA) levels were determined using the Light-Cycler 480 Real-Time PCR system (Roche), with GAPDH as the internal control.

| MiR-126 mimic/inhibitor preparation
Mimic/Inhibitor of miR-126 and negative control (NC) was acquired from RiboBio. MiR-126 mimic/inhibitor and NC mimic/inhibitor were supplemented to 0.9% NaCl to a terminal level of 10 mg/mL for further apply.

| MTT Assay
The MTT assay was conducted to evaluate cell proliferation. Briefly, cells were treated with 20 μL of MTT (0.5 mg/mL), and the supernatant was discarded. DMSO (150 μL) was then added to each well, with rotation for 10 minutes, to dissolve the formazan dye. An Infinite M200 microplate reader (Tecan) was then used to measure the absorbance at 490 nm.

| Annexin V-FITC/PI flow cytometry
Cell death was evaluated using an annexin V-FITC/PI apoptosis detection kit. Briefly, after transfection, cells were resuspended in 20 µL of binding buffer and then incubated for 20 minutes with 5 µL of PI and 10 µL of annexin V-FITC in the dark room. Cell death was evaluated by flow cytometry (FC).

| Dual-luciferase reporter assay
The 3′-UTR of the PTEN gene underwent amplification prior to fusion with the GV126 luciferase gene. The binding site of the PTEN gene and miR-126 was mutated via site-directed mutagenesis, which served as a control. Thymidine kinase promoter (TaKaRa; pRL-TK vectors) and plasmids containing Renilla luciferase were applied to adjust for transfection efficiency. HEK293T cells were co-transfected with miR-126-mimic and NC-mimic with luciferase reporter vectors and the luciferase assay was conducted.

| Statistical analysis
Data are expressed as mean ± standard deviation (SD). Two-tailed Student's t test was used for mean comparisons. P < .05 demonstrated significant difference.

| MiR-126 is upregulated in DPSCs
We extracted DPSCs from normal human third molars which was obtained from adults. Cells were characterized in vitro to confirm their stem characters. Western blot (WB) analysis indicated that the isolated DPSCs were positive for cyclin-dependent kinase inhibitors p21 and p16, which are markers of senescent DPSCs, were markedly upregulated at PD54 compared with the levels at PD16. We also found that pro-apoptotic protein Bax was increased while anti-apoptotic protein Bcl-2 was decreased in DPSCs at PD54 (Figure 1A), suggesting there existed apoptosis at PD54. The number of SA-βgal-positive cells increased significantly at PD54 compared with that at PD16, indicating the successful induction of senescent cells ( Figure 1B).
The miRNA microarray analysis revealed that miR-126 was significantly upregulated in PD54 DPSCs, comparing with PD16 DPSCs ( Figure 1C). To confirm this data, Q-PCR analysis was performed for determining the miR-126 expression at PD16 and PD54. It was found that the level of miR-126 was upregulated in DPSCs at PD54 ( Figure 1D), suggesting that miR-126 may be played a role in apoptosis of senescent DPSCs.

| MiR-126 is essential for maintaining viability of DPSCs
To probe the role of miR-126 on cell proliferation and apoptosis of early and senescent DPSCs, the cells at PD16 were transfected with miR-126 mimic or control, while cells at PD54 were transfected with miR-126 inhibitor or control to regulate the miR-126 expression.
The Q-PCR data indicated that usage of miR-126 mimic increased miR-126 in DPSCs at PD16 (Figure 2A). Then, we performed MTT assay to detect the proliferative rate of DPSCs during 72 hours post-transfection. For control group, data displayed that there was no any influence, while miR-126 caused a reduced cell proliferative rate ( Figure 2B). For DPSCs at PD54, miR-126 inhibition significantly increased the cell replication of DPSCs during 72 hours post-transfection ( Figure 2C,D). These data suggested that miR-126 played an inhibitory role in DPSCs viability.

| MiR-126 targets PTEN
It has been reported that PTEN sensor played an important role in modulating apoptotic cell signal. 24 Bioinformatics analysis predicted that miR-126 may target 3′-UTR of PTEN gene ( Figure 4A).

Transfection of miR-126 mimic inhibited luciferase function,
which was fused with the PTEN 3′-UTR by 60%, compared to the other control groups ( Figure 4B). In light of the key role of PTEN in the apoptosis, we measured its expression in DPSCs by Q-PCR

| Overexpression of PTEN deprived the apoptotic property in DPSCs
To determine whether PTEN can play a role in the apoptosis in

| D ISCUSS I ON
The properties of human stem cells are precisely regulated by intracellular or extracellular mechanical and molecular signals, and are a promising target for stem cell-based therapies for various human diseases. 25 Accumulating miRNAs may affect biological reactions via modulation of their target agents. Previous research has demonstrated that miR-126 may target different genes, including ADAM9, 26 LRP6, PIK3R2, 27 CXCR4, 28  Previous studies have demonstrated that PTEN-Akt signaling pathway contributes to the carcinogenesis of variant cancers, such as pancreatic cancer, hepatocellular carcinoma, breast carcinoma, and gastric carcinoma. [30][31][32][33] Our results suggested that PTEN-

Akt pathway was negatively modulated by miR-126 in senescent
DPSCs, suggesting miR-126 is a critical negative regulator in transduction of PTEN-Akt pathway. These findings could be also supported by a previous report 34  Mean ± SD of the results of three independent experiments was used to describe the data. N = 3. *P < .05, compared to the indicated group