MiR-708 inhibits MC3T3-E1 cells against H2O2-induced apoptosis through targeting PTEN

Background The dysregulation of proliferation and apoptosis plays a significant role in the pathogenesis of postmenopausal osteoporosis (PO). MicroRNAs play an important role in regulating apoptosis of MC3T3-E1 cells. However, the role and potential mechanism of miR-708 for regulating H2O2-induced apoptosis is unknown. This study aimed to investigate the protective function of miR-708 in H2O2-induced apoptosis of MC3T3-E1 osteoblasts. Methods MC3T3-E1 was co-cultured with H2O2 for 8 h, then, flow cytometry, malondialdehyde (MDA), and glutathione peroxidase (Gpx) levels were measured to establish the oxidative model. MiRNA microarray was performed to assess differentially expressed miRNAs between control and H2O2-treated MC3T3-E1 cells. We then performed RT-PCR to identify the relative expression of miR-708 and PTEN. After transfected MC3T3-E1 with miR-708 mimics, flow cytometry, MDA, and Gpx level were performed to identify the apoptosis rate and oxidative stress in these groups. Furthermore, we small interfering RNA of PTEN to identify the role of PTEN in H2O2-induced apoptosis of MC3T3-E1 cells. Results H2O2 (100 nM) could significantly induce the apoptosis of MC3T3-E1 cells. Moreover, H2O2 could significantly increase the MDA level and downregulated Gpx level. RT-PCR found that H2O2 significantly decrease the level of miR-708. Compared with H2O2 group, H2O2 + miR-708 mimic significantly decreased the apoptosis rate. Conclusions miR-708 plays a protective role in H2O2-induced MC3T3-E1 osteoblasts apoptosis and its protective effect is proceeded by regulating ROS level and PTEN expression level.


Introduction
Postmenopausal osteoporosis (PO) is one of the most common bone diseases, characterized by low bone mineral density (BMD) and osteoporotic fracture with high morbidity and mortality [1][2][3]. Currently, there are no ideal methods to cure osteoporotic fracture [4,5]. Therefore, a comprehensive interpretation of the pathogenesis and molecular mechanism of osteoporosis is important to find the target marker of PO [6].
Bone remodeling is regulated by osteoblast and osteoclast. During bone metabolism, it is inevitably produced reactive oxygen species (ROS) [7]. Recent studies have found that ROS could induce osteoblast apoptosis and thus promote the progression of OP [8]. Moreover, ROS can significantly inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) [9]. microRNAs (miRNAs), one type of the non-coding RNAs, could regulate cellular ROS level and is closely related to OP. Lu et al. [10] revealed that miR-214 protects MC3T3-E1 osteoblasts against H 2 O 2 -induced apoptosis by suppressing oxidative stress and targeting ATF4. One of the microRNAs, miR-708, was found to have a negative role in regulating breast cancer [11] and osteosarcoma [12] metastasis. Another study revealed that miR-708 could directly targeting with Nrf2 in bovine granulosa cells, while Nrf2 is a redox-sensitive transcription factor regulating the expression of antioxidant genes [13]. Additionally, the abnormal expression of miR-708 has recently been associated with oxidative stress in neurodegenerative disorders [14].
In this report, we focused on the protective effect of miR-708 in H 2 O 2 -induced osteoblasts apoptosis and potential mechanism. We hypothesized that miR-708 inhibits MC3T3-E1 cells against H 2 O 2 -induced apoptosis through targeting PTEN.

Microarray hybridization
Total RNA was isolated from MC3T3-E1 cells and H 2 O 2 -treated MC3T3-E1 cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Microarray hybridization was performed by Agilent-070155 Mouse miRNA Microarray (miRBase Release 21.0, miRNA ID version) and performed by Cloudseq Company (Shanghai, China). After normalization, differentially expressed miRNAs were performed by the Limma package of R software. Then, heatmap and volcano plot of the differentially expressed miRNAs were drawn using the pheatmap package of the R software.

MC3T3-E1 cell culture
Murine osteoblastic MC3T3-E1 cells were maintained in a α-minimum essential medium (α-MEM) media supplement with 10% fetal bovine serum 100 U/ml penicillin at 37°C in a humidified atmosphere of 5% CO 2 . MC3T3-E1 cells were seeded in a 6-well plate and divided into two groups: control group and H 2 O 2 group. The concentration of H 2 O 2 was 100 mM according to a previous report [15]. And we treated with 100 mM H 2 O 2 for 8 h to induce the oxidative stress.

RNA isolation and real-time PCR
Total RNA was isolated by Trizol reagent (Invitrogen, Thermo Fisher, USA) according to the manufacturers' instruction. RNA was analyzed by NanoDrop-2000 (Thermo Fisher, USA) for RNA quantification and purity. The cDNA was synthesized using the PrimeScript RT Master Mix (Takara, Japan) according to the manufacturer's protocol. Synthesized cDNA was subsequently analyzed via qPCR using a SYBR Premix Ex TaqTM II (TliRNaseH Plus) kit (cat. no. RR820a; Takara Bio, Inc.). β-actin was used as an internal reference and the relative mRNA expression of target genes was calculated using the 2 −△△ct method. Primer sequence can be seen in Table 1.

Western blot assay
Total proteins were isolated by using RIPA and PMSF (100:1). Same concentration of proteins was transferred to sodium dodecyl sulfate (SDS)-polyacrylamide gels. Subsequently, the protein was transferred in gel to polyvinylidene fluoride (PVDF) membranes. Then PVDF membrane was blocked by nonfat-dried milk and then incubated with primary antibodies at 4°C overnight. PVDF membrane was washed with TBST and then incubated with secondary antibodies at room temperature for 1 h. Eventually, the electrochemical luminescence (ECL) solution was prepared in the darkroom. The exposure time was determined according to the fluorescence intensity.

ROS measurement
MC3T3-E1 cells in the above groups were collected by centrifuge. MDA and GPx levels were determined by MDA and GPx determination kits according to the manufacturer's instruction. the concentration of MDA and the activity of GPx were detected by spectrophotometer at 580 nm.

Luciferase reporter gene assay
Potential targets of miR-708 were predicted by performing a search in the following online database: miRanda (http://www.microrna.org/microrna/home.do) and Tar-getScan (http://www.targetscan.org/). 3′-UTR of the PTEN gene containing putative miR-708 targeting site was amplified by chemical synthesis and was inserted into the psiCHECK2 vector (Promega, Madison, WI, USA). When the confluence was up to 70%, MC3T3-E1 cells were transfected with related mixtures including 50 ng PTEN wild-type or PTEN mutant-type 3'-UTR reporter plasmids, miR-708 mimics or miR-708 NC in a final concentration of 20 nM, and Lipofectamine 3000 Fig. 1 a Data normalization for differentially expressed miRNAs (data before normalization and after normalization. b Volcano plot the differentially expressed miRNAs. c Heatmap of the differentially expressed miRNAs for 48 h. Luciferase activity was detected using the dualluciferase reporter gene kit (Beyotime, Shanghai, China).

Statistical analysis
All of the data are presented as means ± SD. Statistical analysis was performed using GraphPad Prism 7.00 (GraphPad Software, San Diego, USA). Student's t test was used between two groups, while ANOVA followed by Dunnett's test for multiple comparisons was conducted. A value of p < 0.05 was considered significant.

Differentially expressed miRNAs
As shown in Fig. 1a and b, after data normalization, 74 miRNAs were identified, including 63 miRNAs and 11 miRNAs were downregulated and upregulated respectively ( Fig. 1a and b). Volcano plot of the differentially expressed miRNAs can be seen in Fig. 1c. Heatmap of the differentially expressed miRNAs can be seen in Fig.  1d, and the miR-708 was the downregulated miRNA.

H 2 O 2 -induced MC3T3-E1 apoptosis and elevated oxidative stress
After treatment with H 2 O 2 to MC3T3-E1 cells for 24 h, MC3T3-E1 cells were harvested and performed Annexin-V-FITC analysis. Compared with the control group, adding H 2 O 2 could significantly increase the apoptosis rate ( Fig. 2a and b). Moreover, we measured the MDA and Gpx between the control and H 2 O 2 groups. Results have shown that, compared with the control group, adding H 2 O 2 could significantly increase the MDA (Fig. 2c, P < 0.05), while significantly decreased the Gpx level (Fig. 2d, P < 0.05).

MiR-708 was decreased and PTEN was increased in H 2 O 2treated MC3T3-E1 cells
We further explored the miR-708 and PTEN expression between control and H 2 O 2 groups. Compared with control group, H 2 O 2 could significantly decrease the relative expression of miR-708 (Fig. 3a), while significantly increased the relative expression of PTEN (Fig. 3b).

MiR-708 decreased H 2 O 2 -induced apoptosis and ROS level in MC3T3-E1 cells
Compared with the control group, adding H 2 O 2 significantly increased the apoptosis rate. There was no statistical difference between the miR-708 mimic and the control group in terms of the apoptosis rate ( Fig. 4a

PTEN is regulated by MiR-708
To further explore the relationship between miR-708 and PTEN, we used agomir-miR-708 and antagomir-miR-708 to explore the PTEN relative expression. Compared with agomir-NC, agomir-miR-708 could decrease the relative expression of PTEN (P < 0.05, Fig. 5). And when adding the antagomir-miR-708, the relative expression of miR-708 was significantly downregulated.

Inhibition the expression of PTEN reversed the apoptosis rate caused by H 2 O 2
Compared with the control group, si-PTEN could significantly decrease the protein expression of PTEN, which indicated that the si-PTEN could significantly downregulated the PTEN (Fig. 6a). Compared with the H 2 O 2 group, extra adding si-PTEN could significantly  decrease the apoptosis rate (Fig. 6b). Moreover, miR-708 could bind to the 576-582 of PTEN 3'UTR (Fig. 6c). The luciferase reporter gene assay in Fig. 6d further showed that the combination of PTEN-WT and miR-708 mimic largely decreased fluorescence intensity compared with the combination of PTEN-1-WT and miR-708 NC. However, the combination of PTEN-MUT and miR-708 mimic or miR-708 NC both have no effect on fluorescence intensity, indicating that there exists targeting relationship between PTEN and miR-708.

Discussion
In our study, the inhibitory effect of miR-708 on MC3T3-E1 cells apoptosis was found to be mediated by the PTEN. Previous studies have found that oxidative stress could promote inhibit osteogenesis of bone marrow mesenchymal stem cells and promote the apoptosis of osteoblast [16]. Microarray hybridization was performed and found that miR-708 was downregulated in the H 2 O 2 treated group. We found that miR-708 was decreased in the H 2 O 2 group than the control group by PCR. Meanwhile, PTEN was the target gene of miR-708. Yang et al. [17] Fig. 5 a Relative expression of miR-708 in agomir-NC and agomir-miR-708 groups. b Relative expression of PTEN in agomir-NC and agomir-miR-708 groups. c Relative expression of miR-708 (c) and PTEN (d) in antagomir-NC and antagomir-miR-708 groups Fig. 6 Western blot analyses of PTEN and cytochrome C control and si-PTEN groups.b Relative apoptosis rate in control, H 2 O 2, si-PTEN, and H 2 O 2+ si-PTEN groups. d Luciferase reporter gene assay was conducted to further demonstrate the targeting relationship between PTEN and miR-708 revealed that miR-21 promotes osteogenesis via the PTEN/PI3K/Akt/HIF-1α pathway and enhances bone regeneration in critical-size defects. PTEN has been implicated as an important regulator of osteoblast differentiation [18] and osteoblast apoptosis [19,20]. And miR-17/PTEN axis could also promote osteoblasts viability [21]. We found that miR-708 could bind to the 576-582 of PTEN 3′ UTR and relative luciferase activity further identified that miR-708 could directly target with PTEN. Previously study found that PTEN is an inhibitor of the AKT signaling pathway and suppresses the expression of AKT [22]. Liu et al. [23] reported that PTEN modulates neuron apoptosis involving the PI3K/Akt/mTOR signaling pathway. Thus, PI3K/Akt is the downstream signaling pathway of PTEN.
MiR-708 possesses many physiological functions including regulating cell proliferation, apoptosis, and autophagy. Sun et al. [24] identified that miRNA-708 functions as a tumor suppressor in colorectal cancer by targeting ZEB1 through Akt/mTOR signaling pathway. Saini et al. [25] revealed that miR-708 induces apoptosis and suppresses tumorigenicity in renal cancer cells. In this study, we found that miR-708 could significantly reduce the H 2 O 2 -induced apoptosis of MC3T3-E1 cells.

Conclusions
MiR-708 inhibits MC3T3-E1 osteoblasts against H 2 O 2induced apoptosis through directly targeting PTEN. Future studies should be focused on the effects of miR-708 for osteogenesis in vivo.