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MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells Lijun Zhang 1, Gaozhi Li 1, Ke Wang, Yixuan Wang, Jingjing Dong, Honghui Wang, Liqun Xu, Fei Shi, Xinsheng Cao, Zebing Hu*, Shu Zhang** The Key Laboratory of Aerospace Medicine, Ministry of Education, Air Force Medical University, 710032, Xi’an, Shaanxi, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 October 2019 Accepted 8 November 2019 Available online xxx
Disuse osteoporosis is common in prolonged therapeutic bed rest, space flight and immobilization due to limb fracture, which is related to reduction of mechanical stress on bone. Mechanical unloading can inhibit the differentiation of osteoblasts, but the detailed mechanism is still unclear. Runt-related transcription factor-2 (Runx2), is an important transcription factor, which plays a crucial role in disuse osteoporosis induced by unloading conditions. In this study, we found that Runx2-targeting mechanosensitive miR-30 family members, miR-30b, miR-30c, miR-30d and miR-30e increased significantly, and were negatively correlated with the expression of Runx2 under unloading condition. Further studies found that the four miRNAs inhibited the expression of Runx2 and osteoblast differentiation under normal loading, and the knockdown of these miRNAs attenuated partly the inhibition of osteoblast differentiation induced by unloading condition in MC3T3-E1 cells. This study is the first to report miR-30 family members can regulate partly the dysfunction of osteoblasts under unloading condition, which is expected to be targets for the treatment of disuse osteoporosis. © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: miR-30 Runx2 Osteoblast differentiation Mechanical unloading
1. Introduction Disuse osteoporosis mainly refers to bone loss caused by the reduction of mechanical stress on bone, which is common in prolonged therapeutic bed rest, space flights and immobilization due to limb fractures [1]. Bone, a dynamic organ, undergoes continuous bone remodeling, which depends on the balance between osteogenesis mediated by osteoblasts and bone resorption mediated by osteoclasts [2]. Mechanical unloading can disturb bone remodeling by restraining osteoblast-mediated bone formation and promoting osteoclast-mediated bone resorption, which leads to disuse osteoporosis. Although there are many studies on bone loss caused by mechanical unloading, the specific mechanism is still not clear, and there are currently no effective countermeasures. Multiple unloading devices, such as 2D clinostats, rotating wall vessel bioreactors and random positioning machines, can be used
* Corresponding author. The Key Laboratory of Aerospace Medicine, Ministry of Education, Air Force Medical University, 710032, Xi’an, Shaanxi, China.. ** Corresponding author. E-mail addresses: [email protected] (Z. Hu), [email protected] (S. Zhang). 1 These authors contributed equally.
to simulate unloading conditions, which can hinder the function of osteoblasts, especially the differentiation of osteoblasts [3]. Osteoblast differentiation is regulated by a variety of transcription factors, including Runt-related transcription factor-2 (Runx2), osterix, osteocalcin (Ocn) and alkaline phosphatase (Alp). Runx2 is one of the most important osteoblast-specific transcription factors recognized by scholars. The Runx2 protein specifically binds cisacting elements on many osteogenic gene promoters and promotes the transcription of osteogenic target genes [4]. Accumulating evidence has shown that the expression of Runx2 under unloading conditions is significantly decreased [5]. Although several factors have been reported in previous articles to regulate osteoblast differentiation by directly or indirectly acting on Runx2 under unloading conditions, the regulation of Runx2-targeting miRNAs during osteoblast differentiation under unloading conditions has not been previously reported. MiRNAs are a group of noncoding RNAs with a length of approximately 20e24 nucleotides. Several experiments have shown that miRNAs can regulate the function of osteoblasts. MiR214 inhibits the activity of osteoblasts by targeting ATF4 [6], and miR-141 and miR-200 suppress the differentiation of osteoblasts by inhibiting the expression of the osteogenic transcription factor Dlx5 in MC3T3-E1 cells. In this study, we studied the changes and
https://doi.org/10.1016/j.bbrc.2019.11.057 0006-291X/© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
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functions of the miR-30 family under unloading conditions, and found that this family has the potential to directly relieve disuse osteoporosis. To further study the possible regulatory mechanism of mechanical unloading on osteoblast differentiation, our study predicted several miRNAs targeting Runx2 by bioinformatics analysis and identified the members of the miR-30 family, which have previously been reported to be associated with osteoblast differentiation, as promising candidates. This study identified that miR30b, miR-30c, miR-30d, miR-30e were upregulated under unloading conditions, whereas the level of miR-30a did not significantly change. Moreover, the expression of miR-30b, miR-30c, miR-30d, and miR-30e was negatively correlated with the changes of Runx2 after 72 h of unloading. We found that these miRNAs could inhibit the expression of Runx2 and its downstream target genes. Downregulation of miRNA-30 partially alleviated the inhibition of osteoblast differentiation under unloading conditions. Our findings provide potential therapeutic targets that may alleviate bone loss induced by unloading conditions. 2. Material and methods 2.1. Cell culture The preosteoblastic MC3T3-E1 cell line was cultured in a-MEM medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Hyclone, USA), and cells were placed in a cell incubator at 37 C with 5% CO2. The medium was changed after 2 days. When the cells reached 90% confluence, they were subcultured after digestion with trypsin (Millipore, USA) for the experiments. For the osteoblast differentiation experiments, cells at the proper confluency were cultured in an osteogenic medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 100 nM dexamethasone, 10 mM b-glycerophosphate, and 50 mg/ml ascorbic acid. 2.2. qRT-PCR Total RNA was extracted from MC3T3-E1 cells by RNAiso Plus (TaKaRa, Japan). Total RNA was transcribed into cDNA with a Prime Script™ RT Master Mix reagent kit (TaKaRa, Japan), and a Mir-X miRNA First-Strand Synthesis Kit (Clontech, USA) was used to reverse transcribe the miRNA. The expression of target genes was analyzed using a CFX96 real-time PCR detection system (BIO-RAD, USA) and SYBR Premix Ex Taq TM II (TaKaRa, Japan). U6 small nuclear RNA and GAPDH were used as internal controls. The primer sequences are listed in Supplementary Table 1 (Table S1). 2.3. Western blot analysis The total protein of the MC3T3-E1 cells was extracted with the M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, USA) containing 10% protease inhibitor cocktail (Roche, Switzerland). The protein concentration was measured by a Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein samples containing loading buffer were subjected to electrophoresis on NuPAGE™ Bis-Tris Protein Gels (Invitrogen, USA) for 2 h. Proteins were transferred to polyvinylidene difluoride membranes, and the membranes were blocked in 5% skim milk for 4 h. Then, the PVDF membranes were incubated with primary antibodies specific for Runx2 (1:1000, Cell Signaling Technology, USA), Ocn (1:500, Abcam, UK) and GAPDH (1:5000, Proteintech, China) overnight at 4 C. The next day, TBST was used to wash the membranes. Next, the PVDF membranes were incubated with a peroxidase-conjugated secondary antibody (1:5000,
ZSGB-BIO, China) for 1 h. After washing with TBST, the membranes were visualized with Super Signal West substrate (Thermo Fisher Scientific, USA). Densitometric analyses of the bands were quantified using Image J software. 2.4. Cell transfection The mimics and inhibitors of the miR-30 family were purchased from GenePharma (Shanghai, China). Mimic-30s (40 nM), inhibitor30s (80 nM) or their negative controls were transfected into MC3T3-E1 cells using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. Mimic-30s included mimic-30b, mimic-30c, mimic-30d and mimic-30e. Inhibitor-30s included inhibitor-30b, inhibitor-30c, inhibitor-30d and inhibitor30e. The sequences of the siRNAs and negative controls were listed Supplementary Table 2 (Table S2). 2.5. Alizarin red staining MC3T3-E1 cells were induced using osteogenic medium for 21 days. The cells were gently rinsed in DPBS three times and were then fixed in ice-cold 70% alcohol for 1 h. After washing with double-distilled H2O, the cells were stained with 40 mM Alizarin red S (Sigma-Aldrich, Missouri, USA) at room temperature for 15 min. Then, the cells were gently rinsed with double-distilled H2O three times and incubated in DPBS at room temperature for 15 min. Next, the cells were imaged with a digital camera. The relative areas of the mineralized nodules were measured by Image J software. 2.6. Alkaline phosphatase staining MC3T3-E1 cells were induced with osteogenic medium for 7 days. The cells were gently rinsed in PBS and then fixed in 4% polyformaldehyde at room temperature for 15 min. After washing with PBS three times, the cells were stained using a NBT/BCIP staining kit (Beyotime Biotechnology, China) based on the manufacturer’s instructions. The cells were imaged with a digital camera. 2.7. Alkaline phosphatase activity assay The total protein of the MC3T3-E1 cells was collected with MPER Mammalian Protein Extraction Reagent (Thermo Scientific, USA). A Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA) was used to measure the protein concentration. An alkaline phosphatase assay kit (Nanjing Jiancheng Technological, China) was used to quantify ALP activity. Alp activity refers to the amount of phenol produced by the reaction of 1 g protein with the substrate for 15 min. 2.8. 2D clinorotation 2D clinorotation (designed by the China Astronaut Research and Training Center, Beijing, China) was used to simulate the unloading environment for in vitro cells. Cells at 90% confluence were subcultured into culture flasks and then placed in a cell incubator at 37 C with 5% CO2. After cell adherence, a-MEM medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Hyclone, USA) was used to fill the culture flasks, and the culture flasks were placed upright in a cell incubator at 37 C overnight. The bubbles in the culture flasks were completely exhausted. Next, the culture flasks were rotated in a 2D clinorotation (Clino) for an appropriate amount of time. The control group was treated the same and incubated in a cell incubator at 37 C.
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
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2.9. Statistical analysis All experiments were performed at least three times. Data are expressed as the mean ± SD and were analyzed with SPSS 22.0 software. The two-group samples were compared using Student’s t-test, and multiple independent groups were compared with oneway ANOVA. P < 0.05 was considered significant.
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and Alp. The mRNA expression of Runx2, Ocn and Alp was markedly decreased in MC3T3-E1 cells treated with unloading for 72 h (Fig. 2A). Western blot analysis showed that the protein expression levels of Runx2 and Ocn were significantly reduced (Fig. 2B). Moreover, Alp activity showed similar trends (Fig. 2C). These results suggest that there is a remarkable negative correlation between the expression of the miR-30 family except miR-30a and Runx2, including its downstream osteogenic markers.
3. Results 3.1. Expression of the miR-30 family except miR-30a is upregulated under unloading conditions in MC3T3-E1 cells
3.3. The miR-30 family members except miR-30a inhibit osteoblast differentiation
To further explore the effect of unloading on Runx2, we predicted that multiple miRNAs act on Runx2 by bioinformatics analysis using miRDB, TargetScan and starBase(Table S3). We found that the miR-30 family members are important candidates in Runx2targeting miRNAs. To study the changes of miRNA-30 family expression in response to unloading, MC3T3-E1 cells were treated with 2D clinorotation for 48 h. The qRT-PCR analysis results showed that miR-30b, miR-30c, miR-30d and miR-30e were significantly upregulated, whereas the expression level of miR-30a did not significantly change (Fig. 1A). We focused on the roles of miR-30b, miR-30c, miR-30d and miR-30e and measured the changes of their expression in cells treated with unloading over 72 h. The expression of miR-30b and miR-30d were significantly upregulated at 24 h and increased over time to 72 h, as shown by qRT-PCR. The expression of miR-30c and miR-30e was enhanced at 72 h, but their highest expression was found at 48 h (Fig. 1B).
To further verify the effect of the miR-30 family except miR-30a on osteoblast differentiation, mimic-30s and inhibitor-30s were transfected into MC3T3-E1 cells to change the expression level of miR-30s. After knocking down the expression of miR-30b, miR-30c, miR-30d and miR-30e, we found by qRT-PCR that the mRNA expression of Runx2, Ocn and Alp was significantly increased. By contrast, the gene expression levels of Runx2, Ocn and Alp were all decreased after transfection of mimic-30b, 30c, 30d and 30e (Fig. 3A). Western blotting analysis showed the protein expression of Runx2 and Ocn was increased in the inhibitor-30s group, while the protein expression of Runx2 and Ocn was reduced in the mimic30s group (Fig. 3B). ALP staining and Alp activity were significantly promoted in the inhibitor-30s group and, conversely, were inhibited in the group treated with mimic-30s (Fig. 3C and D). Matrix mineralization, as shown by Alizarin red staining, was markedly increased in the group treated with inhibitor-30s, whereas the mimic-30s group had the opposite effect (Fig. 3E).
3.2. Expression of the miR-30 family except miR-30a is negatively correlated with osteoblast differentiation under unloading conditions in MC3T3-E1 cells
3.4. Inhibition of the miR-30 family except miR-30a partially attenuates the alterations of osteoblast differentiation induced by unloading in MC3T3-E1 cells
To investigate the relationship between osteoblast differentiation and the miR-30 family except miR-30a, we examined the expression of Runx2 and its downstream osteogenic factors Ocn
To further confirm the role of the miR-30 family except miR-30a on osteoblast differentiation under unloading, MC3T3-E1 cells were transfected with inhibitor-30s and were treated with 2D
Fig. 1. Expression of the miR-30 family except miR-30a is upregulated under unloading conditions in MC3T3-E1 cells. (A) mRNA expression of the miR-30 family members in MC3T3-E1 cells under unloading conditions for 48 h by qRT-PCR (n ¼ 3). (B) The changes of miR-30b, miR-30c, miR-30d and miR-30e expression in MC3T3-E1 cells treated by unloading within 72 h by qRT-PCR (n ¼ 3). *P < 0.05, **P < 0.01 vs. control.
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
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Fig. 2. Expression of the miR-30 family except miR-30a is negatively correlated with osteoblast differentiation under unloading conditions in MC3T3-E1 Cells. (A) qRT-PCR analyses of miR-30b, miR-30c, miR-30d and miR-30e in MC3T3-E1 cells under unloading conditions for 72 h (n ¼ 3). (B) Western blot analysis of Runx2 and Ocn in MC3T3-E1 cells treated by unloading within 72 h (n ¼ 3). (C) ALP activity analysis in MC3T3-E1 cells under unloading conditions (n ¼ 3). *P < 0.05, **P < 0.01 vs. control.
clinorotation for 48 h. Knockdown of the miR-30 family except miR30a significantly reversed the reduction of Runx2, Ocn and Alp expression at the mRNA level induced by unloading, but gene expression was not fully restored to the control levels (Fig. 4A). In addition, the unloading-induced decrease of Runx2 and Ocn protein expression was partially blocked in cells transfected with inhibitor-30s (Fig. 4B). Inhibition of miR-30b, miR-30c and miR-30e partially attenuated the decreases of Alp activity induced by unloading, whereas the inhibitor-30e only had a slight impact (Fig. 4C). 4. Discussion The biological and pathological processes of bone are regulated by mechanical stimulation. Previous studies have shown that prolonged mechanical unloading can inhibit osteoblast differentiation and lead to disuse osteoporosis [7,8]. Mechanical unloading is more common in long-term bed rest and space flights. In particular, in long-duration spaceflight, the human body is affected by microgravity unloading, which leads to bone loss [9].Due to the limitations of funds and experimental conditions, microgravity unloading is mostly simulated on the ground. Hindlimb unloading is a classical animal model used to study unloading, which can lead to bone loss in the femur and tibia of the hind limb [10]. Common experimental devices to simulate cellular unloading in vitro include rotating wall vessel bioreactors, random positioning machines, 2D clinostats and so on [3], which can disrupt the function of osteogenesis and inhibit the differentiation of osteoblasts. In this study, we selected 2D clinostats to simulate cellular unloading in vitro. The expression of the miR-30 family was detected in MC3T3-E1 cells treated with 2D-clinorotation. The expression of miR-30b, miR-30c, miR-30d and miR-30e was significantly increased under unloading conditions. Runx2, also known as core binding factor a1, is an important transcription factor related to osteoblast differentiation. Runx2 can regulate the expression of the downstream target gene, Ocn and Alp to induce osteoblast differentiation (Bruderer et al., 2014, [11]. Moreover, Runx2 plays a crucial role in decreasing osteoblast differentiation under unloading conditions. In mesenchymal stem cells from the femurs of hindlimb unloading rats, Runx2 expression
significantly decreased [12]. It has also been found that Runx2 expression is reduced in vitro osteoblasts treated with a random positioning machine [13]. In this study, the expression of Runx2 significantly decreased and was negatively correlated with the expression of miR-30b, miR-30c, miR-30d and miR-30e in MC3T3E1 cells under unloading conditions up to 72 h. MiRNA is a kind of single-stranded endogenous noncoding RNA with a length of approximately 22 nt. MiRNA can bind to the sequence of 30 untranslated regions or coding regions of target gene mRNAs to inhibit their translation or to promote the degradation of RNA. Our previous studies have found that several miRNAs can regulate the differentiation of osteoblasts under unloading. The expression levels of miRNA-33e5p [14] and miRNA-132e3p [15] are decreased under unloading conditions and affect the differentiation of the preosteoblasts MC3T3-E1 and primary osteoblasts. Moreover, the interaction of miRNA-139e3p and lncRNA ODSM participates in unloading-induced bone loss by regulating the expression of the downstream target gene ELK1 [5]. It has been recognized that mechanical unloading inhibits the expression of Runx2 in osteoblasts. Although it has been reported that miRNA132e3p, miRNA-33e5p and microRNA-139e3p regulate osteoblast differentiation through different target genes under unloading, there are still few reports on miRNAs directly targeting Runx2 under unloading. In this study, we found that the miRNA-30 family can act on Runx2 through bioinformatics analysis. Further study confirmed that the miR-30 family except miR-30a can regulate the expression of Runx2 and osteoblast differentiation under unloading conditions. Recent studies have demonstrated that the miRNA-30 family is involved in osteoblast differentiation [16,17]. The miR-30 family includes miR-30a, miR-30b, miR-30c, miR-30d and miR-30e. In human coronary arteries, BMP2 reduces miRNA-30b and miRNA30c, targeting Runx2 to promote calcification of vascular smooth muscle cells [18]. MiR-30c can regulate the fate of osteoblasts and inhibit osteogenesis [19]. For postmenopausal osteoporosis, Dgcr5 negatively regulates miR-30d to upregulate Runx2, which induces osteoblast differentiation and delays the development of postmenopausal osteoporosis [20]. miR-30e inhibits the osteogenesis of mesenchymal stem cells by targeting Igf2 and differentiates them into an adipogenic lineage [21]. The findings presented in this study
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
Fig. 3. The miR-30 family members except miR-30a inhibit osteoblast differentiation. Inhibitor-30s, mimic-30s and their negative controls were transfected into MC3T3-E1 cells. (A) mRNA expression of Runx2, Ocn, and Alp in MC3T3-E1 cells by qRT-PCR (n ¼ 3). (B) Protein levels of Runx2, and Ocn in MC3T3-E1 cells by western blotting (n ¼ 3). (C) Representative images of Alp staining in MC3T3-E1 cells (n ¼ 3). (D) Alp activity analysis in MC3T3-E1 cells (n ¼ 3). (E) Representative images of Alizarin red staining and analysis of the relative mineralized area in MC3T3-E1 cells (n ¼ 3). *P < 0.05, **P < 0.01 vs. control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
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Fig. 4. Inhibition of the miR-30 family except miR-30a partially attenuates the alterations of osteoblast differentiation induced by unloading in MC3T3-E1 cells. Inhibitor-96 and their negative controls were transfected into MC3T3-E1 cells treated with unloading. (A) qRT-PCR analysis of Runx2, Alp, and Ocn in MC3T3-E1 cells (n ¼ 3). (B) Western blot analysis of Runx2 and Ocn in MC3T3-E1 cells (n ¼ 3). (C) Alp activity analysis of MC3T3-E1 cells (n ¼ 3). *P < 0.05, **P < 0.01 vs. control.
are consistent with previous reports. The results of this study demonstrated that miR-30b, miR-30c, miR-30d and miR-30e inhibited the expression of Runx2 and osteoblast differentiation in MC3T3-E1 cells under normal loading conditions. This study screened gravity-sensitive miRNA-30 family members, miR-30b, miR-30c, miR-30d and miR-30e, which were upregulated in MC3T3-E1 cells under unloading conditions. Although some studies have reported that overexpression of miR-30a could reduce the proliferation, migration and invasion of human osteosarcoma
and chondrosarcoma by targeting Runx2 [22,23], we found that the levels of miR-30a did not significantly change under unloading conditions. In previous articles, luciferase reporter assays have shown that miR-30 could directly bind to the promoter region of Runx2 in different kinds of cells [18,24,25], which suggests that the miR-30 family can directly act on Runx2. Consistently, rescue experiments showed that inhibitor-30s partly alleviated the inhibition of osteoblast differentiation under unloading conditions. In conclusion, this study reported, for the first time, that Runx2-
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
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targeting mechano-sensitive miRNAs, the miR-30 family except miR-30a, could respond to an unloading condition and modulate osteoblast differentiation in vitro. The expression of the miR-30 family members except miR-30a was significantly increased and was negatively correlated with that of Runx2 under unloading conditions. Moreover, the miR-30 family except miR-30a could block osteoblast differentiation by targeting Runx2. Specifically, the inhibition of miR-30b, miR-30c, miR-30d and miR-30e could partly reverse the unloading-induced decreases of osteoblast differentiation. Thus, we expect that the miR-30 family except miR-30a may be a potential therapeutic target of bone loss induced by unloading.
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Declaration of competing interest All the authors declare no conflict interest of this study.
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Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (grant nos. 31570939, 81701856), the Key Research and Development Program of Shaanxi (Program No. 2018SF-039) and Young Talent fund of University Association for Science and Technology in Shaanxi, China (Grant No.20170402). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.057. Transparency document
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Transparency document related to this article can be found online at https://doi:10.1016/j.bbrc.2019.11.057 [19]
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MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells Lijun Zhang 1, Gaozhi Li 1, Ke Wang, Yixuan Wang, Jingjing Dong, Honghui Wang, Liqun Xu, Fei Shi, Xinsheng Cao, Zebing Hu*, Shu Zhang** The Key Laboratory of Aerospace Medicine, Ministry of Education, Air Force Medical University, 710032, Xi’an, Shaanxi, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 October 2019 Accepted 8 November 2019 Available online xxx
Disuse osteoporosis is common in prolonged therapeutic bed rest, space flight and immobilization due to limb fracture, which is related to reduction of mechanical stress on bone. Mechanical unloading can inhibit the differentiation of osteoblasts, but the detailed mechanism is still unclear. Runt-related transcription factor-2 (Runx2), is an important transcription factor, which plays a crucial role in disuse osteoporosis induced by unloading conditions. In this study, we found that Runx2-targeting mechanosensitive miR-30 family members, miR-30b, miR-30c, miR-30d and miR-30e increased significantly, and were negatively correlated with the expression of Runx2 under unloading condition. Further studies found that the four miRNAs inhibited the expression of Runx2 and osteoblast differentiation under normal loading, and the knockdown of these miRNAs attenuated partly the inhibition of osteoblast differentiation induced by unloading condition in MC3T3-E1 cells. This study is the first to report miR-30 family members can regulate partly the dysfunction of osteoblasts under unloading condition, which is expected to be targets for the treatment of disuse osteoporosis. © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: miR-30 Runx2 Osteoblast differentiation Mechanical unloading
1. Introduction Disuse osteoporosis mainly refers to bone loss caused by the reduction of mechanical stress on bone, which is common in prolonged therapeutic bed rest, space flights and immobilization due to limb fractures [1]. Bone, a dynamic organ, undergoes continuous bone remodeling, which depends on the balance between osteogenesis mediated by osteoblasts and bone resorption mediated by osteoclasts [2]. Mechanical unloading can disturb bone remodeling by restraining osteoblast-mediated bone formation and promoting osteoclast-mediated bone resorption, which leads to disuse osteoporosis. Although there are many studies on bone loss caused by mechanical unloading, the specific mechanism is still not clear, and there are currently no effective countermeasures. Multiple unloading devices, such as 2D clinostats, rotating wall vessel bioreactors and random positioning machines, can be used
* Corresponding author. The Key Laboratory of Aerospace Medicine, Ministry of Education, Air Force Medical University, 710032, Xi’an, Shaanxi, China.. ** Corresponding author. E-mail addresses: [email protected] (Z. Hu), [email protected] (S. Zhang). 1 These authors contributed equally.
to simulate unloading conditions, which can hinder the function of osteoblasts, especially the differentiation of osteoblasts [3]. Osteoblast differentiation is regulated by a variety of transcription factors, including Runt-related transcription factor-2 (Runx2), osterix, osteocalcin (Ocn) and alkaline phosphatase (Alp). Runx2 is one of the most important osteoblast-specific transcription factors recognized by scholars. The Runx2 protein specifically binds cisacting elements on many osteogenic gene promoters and promotes the transcription of osteogenic target genes [4]. Accumulating evidence has shown that the expression of Runx2 under unloading conditions is significantly decreased [5]. Although several factors have been reported in previous articles to regulate osteoblast differentiation by directly or indirectly acting on Runx2 under unloading conditions, the regulation of Runx2-targeting miRNAs during osteoblast differentiation under unloading conditions has not been previously reported. MiRNAs are a group of noncoding RNAs with a length of approximately 20e24 nucleotides. Several experiments have shown that miRNAs can regulate the function of osteoblasts. MiR214 inhibits the activity of osteoblasts by targeting ATF4 [6], and miR-141 and miR-200 suppress the differentiation of osteoblasts by inhibiting the expression of the osteogenic transcription factor Dlx5 in MC3T3-E1 cells. In this study, we studied the changes and
https://doi.org/10.1016/j.bbrc.2019.11.057 0006-291X/© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
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functions of the miR-30 family under unloading conditions, and found that this family has the potential to directly relieve disuse osteoporosis. To further study the possible regulatory mechanism of mechanical unloading on osteoblast differentiation, our study predicted several miRNAs targeting Runx2 by bioinformatics analysis and identified the members of the miR-30 family, which have previously been reported to be associated with osteoblast differentiation, as promising candidates. This study identified that miR30b, miR-30c, miR-30d, miR-30e were upregulated under unloading conditions, whereas the level of miR-30a did not significantly change. Moreover, the expression of miR-30b, miR-30c, miR-30d, and miR-30e was negatively correlated with the changes of Runx2 after 72 h of unloading. We found that these miRNAs could inhibit the expression of Runx2 and its downstream target genes. Downregulation of miRNA-30 partially alleviated the inhibition of osteoblast differentiation under unloading conditions. Our findings provide potential therapeutic targets that may alleviate bone loss induced by unloading conditions. 2. Material and methods 2.1. Cell culture The preosteoblastic MC3T3-E1 cell line was cultured in a-MEM medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Hyclone, USA), and cells were placed in a cell incubator at 37 C with 5% CO2. The medium was changed after 2 days. When the cells reached 90% confluence, they were subcultured after digestion with trypsin (Millipore, USA) for the experiments. For the osteoblast differentiation experiments, cells at the proper confluency were cultured in an osteogenic medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 100 nM dexamethasone, 10 mM b-glycerophosphate, and 50 mg/ml ascorbic acid. 2.2. qRT-PCR Total RNA was extracted from MC3T3-E1 cells by RNAiso Plus (TaKaRa, Japan). Total RNA was transcribed into cDNA with a Prime Script™ RT Master Mix reagent kit (TaKaRa, Japan), and a Mir-X miRNA First-Strand Synthesis Kit (Clontech, USA) was used to reverse transcribe the miRNA. The expression of target genes was analyzed using a CFX96 real-time PCR detection system (BIO-RAD, USA) and SYBR Premix Ex Taq TM II (TaKaRa, Japan). U6 small nuclear RNA and GAPDH were used as internal controls. The primer sequences are listed in Supplementary Table 1 (Table S1). 2.3. Western blot analysis The total protein of the MC3T3-E1 cells was extracted with the M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, USA) containing 10% protease inhibitor cocktail (Roche, Switzerland). The protein concentration was measured by a Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein samples containing loading buffer were subjected to electrophoresis on NuPAGE™ Bis-Tris Protein Gels (Invitrogen, USA) for 2 h. Proteins were transferred to polyvinylidene difluoride membranes, and the membranes were blocked in 5% skim milk for 4 h. Then, the PVDF membranes were incubated with primary antibodies specific for Runx2 (1:1000, Cell Signaling Technology, USA), Ocn (1:500, Abcam, UK) and GAPDH (1:5000, Proteintech, China) overnight at 4 C. The next day, TBST was used to wash the membranes. Next, the PVDF membranes were incubated with a peroxidase-conjugated secondary antibody (1:5000,
ZSGB-BIO, China) for 1 h. After washing with TBST, the membranes were visualized with Super Signal West substrate (Thermo Fisher Scientific, USA). Densitometric analyses of the bands were quantified using Image J software. 2.4. Cell transfection The mimics and inhibitors of the miR-30 family were purchased from GenePharma (Shanghai, China). Mimic-30s (40 nM), inhibitor30s (80 nM) or their negative controls were transfected into MC3T3-E1 cells using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. Mimic-30s included mimic-30b, mimic-30c, mimic-30d and mimic-30e. Inhibitor-30s included inhibitor-30b, inhibitor-30c, inhibitor-30d and inhibitor30e. The sequences of the siRNAs and negative controls were listed Supplementary Table 2 (Table S2). 2.5. Alizarin red staining MC3T3-E1 cells were induced using osteogenic medium for 21 days. The cells were gently rinsed in DPBS three times and were then fixed in ice-cold 70% alcohol for 1 h. After washing with double-distilled H2O, the cells were stained with 40 mM Alizarin red S (Sigma-Aldrich, Missouri, USA) at room temperature for 15 min. Then, the cells were gently rinsed with double-distilled H2O three times and incubated in DPBS at room temperature for 15 min. Next, the cells were imaged with a digital camera. The relative areas of the mineralized nodules were measured by Image J software. 2.6. Alkaline phosphatase staining MC3T3-E1 cells were induced with osteogenic medium for 7 days. The cells were gently rinsed in PBS and then fixed in 4% polyformaldehyde at room temperature for 15 min. After washing with PBS three times, the cells were stained using a NBT/BCIP staining kit (Beyotime Biotechnology, China) based on the manufacturer’s instructions. The cells were imaged with a digital camera. 2.7. Alkaline phosphatase activity assay The total protein of the MC3T3-E1 cells was collected with MPER Mammalian Protein Extraction Reagent (Thermo Scientific, USA). A Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA) was used to measure the protein concentration. An alkaline phosphatase assay kit (Nanjing Jiancheng Technological, China) was used to quantify ALP activity. Alp activity refers to the amount of phenol produced by the reaction of 1 g protein with the substrate for 15 min. 2.8. 2D clinorotation 2D clinorotation (designed by the China Astronaut Research and Training Center, Beijing, China) was used to simulate the unloading environment for in vitro cells. Cells at 90% confluence were subcultured into culture flasks and then placed in a cell incubator at 37 C with 5% CO2. After cell adherence, a-MEM medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Hyclone, USA) was used to fill the culture flasks, and the culture flasks were placed upright in a cell incubator at 37 C overnight. The bubbles in the culture flasks were completely exhausted. Next, the culture flasks were rotated in a 2D clinorotation (Clino) for an appropriate amount of time. The control group was treated the same and incubated in a cell incubator at 37 C.
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
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2.9. Statistical analysis All experiments were performed at least three times. Data are expressed as the mean ± SD and were analyzed with SPSS 22.0 software. The two-group samples were compared using Student’s t-test, and multiple independent groups were compared with oneway ANOVA. P < 0.05 was considered significant.
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and Alp. The mRNA expression of Runx2, Ocn and Alp was markedly decreased in MC3T3-E1 cells treated with unloading for 72 h (Fig. 2A). Western blot analysis showed that the protein expression levels of Runx2 and Ocn were significantly reduced (Fig. 2B). Moreover, Alp activity showed similar trends (Fig. 2C). These results suggest that there is a remarkable negative correlation between the expression of the miR-30 family except miR-30a and Runx2, including its downstream osteogenic markers.
3. Results 3.1. Expression of the miR-30 family except miR-30a is upregulated under unloading conditions in MC3T3-E1 cells
3.3. The miR-30 family members except miR-30a inhibit osteoblast differentiation
To further explore the effect of unloading on Runx2, we predicted that multiple miRNAs act on Runx2 by bioinformatics analysis using miRDB, TargetScan and starBase(Table S3). We found that the miR-30 family members are important candidates in Runx2targeting miRNAs. To study the changes of miRNA-30 family expression in response to unloading, MC3T3-E1 cells were treated with 2D clinorotation for 48 h. The qRT-PCR analysis results showed that miR-30b, miR-30c, miR-30d and miR-30e were significantly upregulated, whereas the expression level of miR-30a did not significantly change (Fig. 1A). We focused on the roles of miR-30b, miR-30c, miR-30d and miR-30e and measured the changes of their expression in cells treated with unloading over 72 h. The expression of miR-30b and miR-30d were significantly upregulated at 24 h and increased over time to 72 h, as shown by qRT-PCR. The expression of miR-30c and miR-30e was enhanced at 72 h, but their highest expression was found at 48 h (Fig. 1B).
To further verify the effect of the miR-30 family except miR-30a on osteoblast differentiation, mimic-30s and inhibitor-30s were transfected into MC3T3-E1 cells to change the expression level of miR-30s. After knocking down the expression of miR-30b, miR-30c, miR-30d and miR-30e, we found by qRT-PCR that the mRNA expression of Runx2, Ocn and Alp was significantly increased. By contrast, the gene expression levels of Runx2, Ocn and Alp were all decreased after transfection of mimic-30b, 30c, 30d and 30e (Fig. 3A). Western blotting analysis showed the protein expression of Runx2 and Ocn was increased in the inhibitor-30s group, while the protein expression of Runx2 and Ocn was reduced in the mimic30s group (Fig. 3B). ALP staining and Alp activity were significantly promoted in the inhibitor-30s group and, conversely, were inhibited in the group treated with mimic-30s (Fig. 3C and D). Matrix mineralization, as shown by Alizarin red staining, was markedly increased in the group treated with inhibitor-30s, whereas the mimic-30s group had the opposite effect (Fig. 3E).
3.2. Expression of the miR-30 family except miR-30a is negatively correlated with osteoblast differentiation under unloading conditions in MC3T3-E1 cells
3.4. Inhibition of the miR-30 family except miR-30a partially attenuates the alterations of osteoblast differentiation induced by unloading in MC3T3-E1 cells
To investigate the relationship between osteoblast differentiation and the miR-30 family except miR-30a, we examined the expression of Runx2 and its downstream osteogenic factors Ocn
To further confirm the role of the miR-30 family except miR-30a on osteoblast differentiation under unloading, MC3T3-E1 cells were transfected with inhibitor-30s and were treated with 2D
Fig. 1. Expression of the miR-30 family except miR-30a is upregulated under unloading conditions in MC3T3-E1 cells. (A) mRNA expression of the miR-30 family members in MC3T3-E1 cells under unloading conditions for 48 h by qRT-PCR (n ¼ 3). (B) The changes of miR-30b, miR-30c, miR-30d and miR-30e expression in MC3T3-E1 cells treated by unloading within 72 h by qRT-PCR (n ¼ 3). *P < 0.05, **P < 0.01 vs. control.
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
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Fig. 2. Expression of the miR-30 family except miR-30a is negatively correlated with osteoblast differentiation under unloading conditions in MC3T3-E1 Cells. (A) qRT-PCR analyses of miR-30b, miR-30c, miR-30d and miR-30e in MC3T3-E1 cells under unloading conditions for 72 h (n ¼ 3). (B) Western blot analysis of Runx2 and Ocn in MC3T3-E1 cells treated by unloading within 72 h (n ¼ 3). (C) ALP activity analysis in MC3T3-E1 cells under unloading conditions (n ¼ 3). *P < 0.05, **P < 0.01 vs. control.
clinorotation for 48 h. Knockdown of the miR-30 family except miR30a significantly reversed the reduction of Runx2, Ocn and Alp expression at the mRNA level induced by unloading, but gene expression was not fully restored to the control levels (Fig. 4A). In addition, the unloading-induced decrease of Runx2 and Ocn protein expression was partially blocked in cells transfected with inhibitor-30s (Fig. 4B). Inhibition of miR-30b, miR-30c and miR-30e partially attenuated the decreases of Alp activity induced by unloading, whereas the inhibitor-30e only had a slight impact (Fig. 4C). 4. Discussion The biological and pathological processes of bone are regulated by mechanical stimulation. Previous studies have shown that prolonged mechanical unloading can inhibit osteoblast differentiation and lead to disuse osteoporosis [7,8]. Mechanical unloading is more common in long-term bed rest and space flights. In particular, in long-duration spaceflight, the human body is affected by microgravity unloading, which leads to bone loss [9].Due to the limitations of funds and experimental conditions, microgravity unloading is mostly simulated on the ground. Hindlimb unloading is a classical animal model used to study unloading, which can lead to bone loss in the femur and tibia of the hind limb [10]. Common experimental devices to simulate cellular unloading in vitro include rotating wall vessel bioreactors, random positioning machines, 2D clinostats and so on [3], which can disrupt the function of osteogenesis and inhibit the differentiation of osteoblasts. In this study, we selected 2D clinostats to simulate cellular unloading in vitro. The expression of the miR-30 family was detected in MC3T3-E1 cells treated with 2D-clinorotation. The expression of miR-30b, miR-30c, miR-30d and miR-30e was significantly increased under unloading conditions. Runx2, also known as core binding factor a1, is an important transcription factor related to osteoblast differentiation. Runx2 can regulate the expression of the downstream target gene, Ocn and Alp to induce osteoblast differentiation (Bruderer et al., 2014, [11]. Moreover, Runx2 plays a crucial role in decreasing osteoblast differentiation under unloading conditions. In mesenchymal stem cells from the femurs of hindlimb unloading rats, Runx2 expression
significantly decreased [12]. It has also been found that Runx2 expression is reduced in vitro osteoblasts treated with a random positioning machine [13]. In this study, the expression of Runx2 significantly decreased and was negatively correlated with the expression of miR-30b, miR-30c, miR-30d and miR-30e in MC3T3E1 cells under unloading conditions up to 72 h. MiRNA is a kind of single-stranded endogenous noncoding RNA with a length of approximately 22 nt. MiRNA can bind to the sequence of 30 untranslated regions or coding regions of target gene mRNAs to inhibit their translation or to promote the degradation of RNA. Our previous studies have found that several miRNAs can regulate the differentiation of osteoblasts under unloading. The expression levels of miRNA-33e5p [14] and miRNA-132e3p [15] are decreased under unloading conditions and affect the differentiation of the preosteoblasts MC3T3-E1 and primary osteoblasts. Moreover, the interaction of miRNA-139e3p and lncRNA ODSM participates in unloading-induced bone loss by regulating the expression of the downstream target gene ELK1 [5]. It has been recognized that mechanical unloading inhibits the expression of Runx2 in osteoblasts. Although it has been reported that miRNA132e3p, miRNA-33e5p and microRNA-139e3p regulate osteoblast differentiation through different target genes under unloading, there are still few reports on miRNAs directly targeting Runx2 under unloading. In this study, we found that the miRNA-30 family can act on Runx2 through bioinformatics analysis. Further study confirmed that the miR-30 family except miR-30a can regulate the expression of Runx2 and osteoblast differentiation under unloading conditions. Recent studies have demonstrated that the miRNA-30 family is involved in osteoblast differentiation [16,17]. The miR-30 family includes miR-30a, miR-30b, miR-30c, miR-30d and miR-30e. In human coronary arteries, BMP2 reduces miRNA-30b and miRNA30c, targeting Runx2 to promote calcification of vascular smooth muscle cells [18]. MiR-30c can regulate the fate of osteoblasts and inhibit osteogenesis [19]. For postmenopausal osteoporosis, Dgcr5 negatively regulates miR-30d to upregulate Runx2, which induces osteoblast differentiation and delays the development of postmenopausal osteoporosis [20]. miR-30e inhibits the osteogenesis of mesenchymal stem cells by targeting Igf2 and differentiates them into an adipogenic lineage [21]. The findings presented in this study
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
Fig. 3. The miR-30 family members except miR-30a inhibit osteoblast differentiation. Inhibitor-30s, mimic-30s and their negative controls were transfected into MC3T3-E1 cells. (A) mRNA expression of Runx2, Ocn, and Alp in MC3T3-E1 cells by qRT-PCR (n ¼ 3). (B) Protein levels of Runx2, and Ocn in MC3T3-E1 cells by western blotting (n ¼ 3). (C) Representative images of Alp staining in MC3T3-E1 cells (n ¼ 3). (D) Alp activity analysis in MC3T3-E1 cells (n ¼ 3). (E) Representative images of Alizarin red staining and analysis of the relative mineralized area in MC3T3-E1 cells (n ¼ 3). *P < 0.05, **P < 0.01 vs. control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
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Fig. 4. Inhibition of the miR-30 family except miR-30a partially attenuates the alterations of osteoblast differentiation induced by unloading in MC3T3-E1 cells. Inhibitor-96 and their negative controls were transfected into MC3T3-E1 cells treated with unloading. (A) qRT-PCR analysis of Runx2, Alp, and Ocn in MC3T3-E1 cells (n ¼ 3). (B) Western blot analysis of Runx2 and Ocn in MC3T3-E1 cells (n ¼ 3). (C) Alp activity analysis of MC3T3-E1 cells (n ¼ 3). *P < 0.05, **P < 0.01 vs. control.
are consistent with previous reports. The results of this study demonstrated that miR-30b, miR-30c, miR-30d and miR-30e inhibited the expression of Runx2 and osteoblast differentiation in MC3T3-E1 cells under normal loading conditions. This study screened gravity-sensitive miRNA-30 family members, miR-30b, miR-30c, miR-30d and miR-30e, which were upregulated in MC3T3-E1 cells under unloading conditions. Although some studies have reported that overexpression of miR-30a could reduce the proliferation, migration and invasion of human osteosarcoma
and chondrosarcoma by targeting Runx2 [22,23], we found that the levels of miR-30a did not significantly change under unloading conditions. In previous articles, luciferase reporter assays have shown that miR-30 could directly bind to the promoter region of Runx2 in different kinds of cells [18,24,25], which suggests that the miR-30 family can directly act on Runx2. Consistently, rescue experiments showed that inhibitor-30s partly alleviated the inhibition of osteoblast differentiation under unloading conditions. In conclusion, this study reported, for the first time, that Runx2-
Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057
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targeting mechano-sensitive miRNAs, the miR-30 family except miR-30a, could respond to an unloading condition and modulate osteoblast differentiation in vitro. The expression of the miR-30 family members except miR-30a was significantly increased and was negatively correlated with that of Runx2 under unloading conditions. Moreover, the miR-30 family except miR-30a could block osteoblast differentiation by targeting Runx2. Specifically, the inhibition of miR-30b, miR-30c, miR-30d and miR-30e could partly reverse the unloading-induced decreases of osteoblast differentiation. Thus, we expect that the miR-30 family except miR-30a may be a potential therapeutic target of bone loss induced by unloading.
[8]
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Declaration of competing interest All the authors declare no conflict interest of this study.
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Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (grant nos. 31570939, 81701856), the Key Research and Development Program of Shaanxi (Program No. 2018SF-039) and Young Talent fund of University Association for Science and Technology in Shaanxi, China (Grant No.20170402). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.057. Transparency document
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Transparency document related to this article can be found online at https://doi:10.1016/j.bbrc.2019.11.057 [19]
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Please cite this article as: L. Zhang et al., MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.057