LncRNA MIAT can regulate the proliferation, apoptosis, and osteogenic differentiation of bone marrow-derived mesenchymal stem cells by targeting miR-150-5p

ABSTRACT Osteoporosis (OP) is a systemic bone metabolic disease with complicated pathogenesis and is difficult to cure clinically. The regulatory mechanisms of OP are needed to be further investigated. In the present study, we focused on the role of myocardial infarction-associated transcript (MIAT) in OP development and examined the underlying mechanism. The serum expression levels of MIAT in samples from patients with OP and healthy controls were compared using quantitative reverse transcription-PCR (qRT-PCR). The dual-luciferase reporter assay was used to confirm the relationship between MIAT and its potential target microRNA, i.e., miR-150-5p. Moreover, bone marrow-derived mesenchymal stem cells (BMSCs) were cultured and transfected with MIAT shRNA, with or without miR-150-5p inhibitor. EdU staining and colony formation analysis were performed to determine the proliferation ability of these cells. Furthermore, the TUNEL assay and flow cytometry were used to assess BMSC apoptosis. Finally, RT-PCR and Western blot assays were employed to assess the expression of osteogenic differentiation biomarkers. Compared with controls, the expression of MIAT was significantly increased, whereas that of miR-150-5p was markedly decreased in patients with OP. MIAT and miR-150-5p expression levels exhibited a strong negative correlation. Furthermore, osteogenic differentiation indicators were suppressed in serum of OP patients. MIAT was downregulated, and miR-150-5p was upregulated in induced to osteogenic differentiation BMSCs. Furthermore, downregulation of MIAT dramatically promoted osteogenic differentiation, increased proliferation, and inhibited apoptosis in BMSCs; miR-150-5p inhibitor abrogated the effects of MIAT. In conclusion, lncRNA MIAT can regulate the proliferation, apoptosis, and osteogenic differentiation of BMSCs.


Introduction
As the most prevalent progressive bone disease, osteoporosis (OP) dramatically limits patient activity and greatly increases the medical burden [1]. OP is characterized by low bone mass, potentially resulting in bone rarefaction and fractures [2]. It is well known that bone is a continuously and dynamically modified tissue, predominantly consisting of osteoblasts and osteoclasts. Osteoblasts promote bone formation, whereas osteoclasts can induce bone resorption [3]. The imbalance between osteoblasts and osteoclasts has been indicated as the primary factor resulting in OP and other bone metabolic processes [4]. Bone marrow-derived mesenchymal stem cells (BMSCs), a known source of osteoblasts, maintain a balance between bone formation and bone resorption [5,6]. Therefore, an in-depth investigation into the roles of BMSCs in OP is critical for early diagnosis, as well as disease management [7].
Accordingly, in the present study, we focused on the roles of MIAT and miR-150-5p in OP. Interaction between MIAT and miR-150-5p, and their effects on osteoblast differentiation of BMSCs were investigated, which may provide a novel treatment idea for OP.

Patients
Herein, we enrolled 30 healthy volunteers and 30 patients with OP, and took blood samples and isolated serum samples. Patients with OP presenting with hip fractures requiring surgical intervention, a history of cardiovascular diseases, cancers, diabetes, or other metabolic problems were excluded. Each participant provided informed consent before study initiation. The study protocol was approved by the Ethics Committee of the Liaohe Hospital.

Isolation and culture of BMSCs
Gradient centrifugation was performed to isolate MSCs from the bone marrow of rats. In brief, four male Sprague-Dawley rats were housed under standard conditions and allowed to access food and water ad libitum. Bone marrow aspirates were placed in MEM medium (Gibco), supplemented with 10% FBS for 24 h at 95% humidity and 5% CO 2 at 37°C [22]. Suspended cells were removed, and then cells were cultured for approximately 10 days. On reaching ~75% confluency, cells were harvested for subsequent experiments.

Colony formation assay
In brief, BMSCs were seeded into 6-well plates and cultured for 7 days, with the culture medium replaced every 2 days. Thereafter, BMSCs were stained with crystal violet (0.1%) for 10 min [26]. Colonies were visualized and counted using a microscope (Nikon, Tokyo, Japan).

Alkaline phosphatase (ALP) assessment
The cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. Then, cells were seeded and stained with ALP for 20 min [27]. The images were captured using a scanner. An ALP Assay Kit was used to detect ALP activity in accordance with the manufacturer's protocol.

Cell apoptosis
Cells were stained with the propidium iodide (PI)/ Annexin V-FITC kit (Sigma) in the dark for 10 min at room temperature [28]. Then, the cells were counted by flow cytometry (BD Biosciences).

Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling (TUNEL) staining
TUNEL staining was used to assess BMSC death [28]. Briefly, BMSCs were fixed and treated with a TUNEL staining kit (Beyotime). Then, images from six randomly selected fields were captured using a microscope (Olympus).

Western blotting
Protein extracts (15 μg/lane) were subjected to 10% sodium dodecyl sulfate gel electrophoresis. The protein extracts were then transferred to a polyvinylidene fluoride membrane (Millipore) and incubated with primary antibodies at 4°C overnight. On day 2, the membrane was incubated with secondary antibodies for 2 h at room temperature. Protein bands were visualized using an ECL Western blot Kit (CWBIO). Each protein level was normalized to that of GAPDH.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). Data are expressed as mean ± standard deviation (SD). Comparison of two groups was performed using Student's t-tests. Comparison of more than two groups was performed using analysis of variance. Statistical significance was set at P < 0.05.

MIAT and miR-50-5p were aberrant expressed in patients with OP
Based on the qRT-PCR analysis, we observed that MIAT expression was increased (Figure 1a, p < 0.01), while miR-150-5p was decreased (Figure 1b, p < 0.01) in serum samples from patients with OP. Additionally, correlation analysis revealed that MIAT and miR-150-5p expression levels were negatively correlated (Figure 1c, r = -−0.4941, p = 0.0055). Furthermore, osteogenic differentiation-related proteins including Runx2, ALP, and OCN were dramatically lower in serum of patients with OP than those in healthy control group (Figure 1d-F, p < 0.001).

MIAT is downregulated during osteogenic differentiation of BMSCs
The expression level of MIAT in BMSCs was determined by qRT-PCR. As shown in Figure 3, osteogenic differentiation of BMSCs was confirmed by increased levels of osteogenic  (Figure 3a-c). On the contrary, the expression of MIAT was decreased (Figure 3d), while miR-150-5p was increased during osteo-genic differentiation in a time-dependent manner (Figure 3e).

MIAT promotes the proliferation of BMSCs by targeting miR-150-5p
Based on EdU staining (Figure 5a and b) and the colony formation assay (Figure 5c and d), shMIAT infection significantly enhanced BMSC proliferation in the shMIAT group when compared with the shNC group; the miR-150-5p inhibitor partially inhibited the proliferative behaviors of shMIAT.   OCN (a-c). MIAT was decreased and miR-150-5p was increased time-dependently during the osteogenic differentiation of BMSCs (c and d). *p < 0.05, **p < 0.01, ***p < 0.001.

MIAT inhibits the apoptosis of BMSCs by targeting miR-150-5p
TUNEL staining (Figure 6a and b) and flow cytometry (Figure 6c and d) showed that apoptosis of BMSCs was markedly decreased in the shMIAT group. Notably, the miR-150-5p inhibitor partially blocked the anti-apoptotic effects of shMIAT.

MIAT may regulate osteogenic differentiation of BMSCs by targeting miR-150-5p
ALP staining and ALP activity assay were conducted to evaluate osteogenic differentiation of BMSCs. Based on ALP staining and ALP activity assay, ALP activity was significantly increased in the shMIAT group when compared with the shNC   -150-5p. EdU staining (a and b) and colony formation (c and d) showed that compared with shNC group, the proliferation of BMSCs was significantly increased in shMIAT group, which was reversed by miR-150-5p inhibitor. *p < 0.05, **p < 0.01, ***p < 0.001. Note: The petri dish is 35 mm in diameter. The scale length is 50 μm.
group. Moreover, transfection of miR-150-5p inhibitor abrogated the MIAT-induced effects on ALP activity (Figure 7a and b). Finally, the role of MIAT in the expression of osteogenic markers was evaluated by RT-PCR and Western blotting. Compared with shNC, shMIAT significantly increased the BMSC expression of osteogenic markers, RUNX2 and ALP (mRNA expression (Figure 7c) and protein expression (Figure 7d) at day 14, and the osteogenic effects of MIAT were partially abrogated by transfection with miR-150-5p.

Discussion
In the present study, we reported that lncRNA MIAT was significantly increased in patients with OP and could affect the growth, cell death, and osteogenic differentiation of BMSCs by regulating miR-150-5p expression.
Herein, we observed that MIAT targeted miR-150-5p, which is in line with previous study [18]. In addition, the expression of MIAT was increased, while that of miR-150-5p was decreased in patients with OP. Osteogenic differentiation indicators were also increased in serum of patients with OP. Furthermore, the expression of MIAT was decreased, while miR-150-5p was increased in BMSCs during osteogenic differentiation in a time-dependent manner. Thence, we speculated that MIAT and miR-150-5p may participate in osteogenic differentiation of OP.
Runx2 is an osteogenic marker related to osteoblast activation during osteogenesis [35], and ALP is a marker for early osteogenic differentiation of BMSCs and plays a role in the in vitro calcification of BMSCs [36]. In present study, MIAT was decreased and miR-150-5p was increased timedependently during the osteogenic differentiation of BMSCs. Furthermore, MIAT knockdown significantly promoted osteogenic differentiation of BMSCs. Interestingly, co-transfection of miR-150-5p abrogated the osteogenic ability of MIAT shRNA. Therefore, our findings revealed that MIAT might affect the osteogenic differentiation of BMSCs by targeting miR-150-5p.
Abnormal bone reconstruction [37,38] is another important factor in the development of OP. In the present study, MIAT knockdown significantly increased BMSC growth and decreased apoptosis, while miR-150-5p partially blocked the proliferative and anti-apoptotic effects of MIAT shRNA. Collectively, our results indicate that MIAT can affect the growth and apoptosis of BMSCs by targeting miR-150-5p.
There are some limitations in this study. Firstly, the clinical samples were not ethnically diverse, and only Han was included. In future experiments, the sample size should be further expanded and the number of ethnic groups should be increased to verify the results of this study. Secondly, whether MIAT could regulate OP process in vivo by binding with miR-150 should be further investigated. Finally, the downstream gene of miR-150 that affect osteogenic differentiation of BMSCs should be studied. Compared with shNC group, the ALP activity was significantly increased in shMIAT group, which was reversed by miR-150-5p inhibitor (a and b). Compared to shNC, shMIAT significantly increased the mRNA expression of RUNX2 and ALP (c), which were reversed by miR-150-5p inhibitor. MIAT knockdown significantly promoted the protein expression of RUNX2 and ALP by targeting miR-150-5p (d). *p < 0.05, **p < 0.01, ***p < 0.001. Note: The petri dish is 35 mm in diameter. The scale length is 200 μm.

Conclusions
In summary, MIAT was downregulated while miR-150-5p was upregulated in a time-dependent manner during the osteogenic differentiation of BMSCs. Furthermore, MIAT knockdown significantly increased ALP activity, proliferation, osteogenic differentiation, and osteogenesis and inhibited apoptosis in BMSCs by targeting miR-150-5p. These results suggest the potential role of MIAT in the treatment of OP.

Research Highlights
(1) Compared with controls, the expression of lncRNA MIAT is significantly increased in patients with osteoporosis, whereas that of miR-150-5p is markedly decreased.

Data Availability Statement
All data generated or analyzed during this study are available from the corresponding author upon reasonable request.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.