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LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140–5p to upregulate BMP2
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2 Naidong Zhang, Xinyu Hu, Shuanghua He, Wenge Ding, Feng Wang, Yiwen Zhao, Zhihui Huang* Department of Traumatic Orthopedics, The First People's Hospital of Changzhou, Changzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 September 2019 Accepted 14 September 2019 Available online xxx
This study aims to explore the role of lncRNA MSC-AS1/microRNA-140e5p/BMP2 regulatory loop in promoting osteogenic differentiation of BMSCs. BMSCs were isolated from bone marrow of mice. Expression levels of MSC-AS1, microRNA-140e5p and BMP2 during osteogenic differentiation were detected by qRT-PCR. Meanwhile, regulatory effect of MSC-AS1 on osteogenic differentiation was detected through ALP staining and alizarin red staining. The binding sites between microRNA-140e5p and MSC-AS1 as well as between microRNA-140e5p and BMP2 were predicted by TargetScan, which were further confirmed by dual-luciferase reporter gene assay. In addition, protein levels of MSC-AS1/ microRNA-140e5p/BMP2 were detected by Western blot. Finally, rescue experiments were conducted to clarify the regulatory effects of MSC-AS1/microRNA-140e5p/BMP2 axis on osteogenic differentiation. MSC-AS1 and BMP2 were found to be remarkably up-regulated during osteogenic differentiation, while microRNA-140e5p was conversely down-regulated. Meanwhile, knockdown of MSC-AS down-regulated expression levels of osteogenesis-associated genes and weakened the mineralization capacity of BMSCs. MicroRNA-140e5p was verified to bind to the 30 UTR of MSC-AS1 and BMP2 genes. Knockdown of MSCAS1 in BMSCs could reduce the expression of microRNA-140e5p, while knockdown of microRNA-140 e5p also down-regulated BMP2 level. In addition, co-silence of MSC-AS1 and microRNA-140e5p reversed the inhibitory effect of MSC-AS1 knockdown on osteogenic differentiation and protein levels of p-Smad1/5/8, RUNX2 and Osterix. MSC-AS1 might promote the osteogenic differentiation of BMSCs through sponging microRNA-140e5p to up-regulate BMP2, thus alleviating the progression of osteoporosis. © 2019 Elsevier Inc. All rights reserved.
Keywords: osteoporosis MSC-AS1 MicroRNA-140e5p BMP2
1. Introduction Osteoporosis (OP) is a type of systemic bone disease characterized by systemic reduction of bone mass and degeneration of bone tissue microstructure, resulting in decreased bone strength, increased brittleness and increased risk of fracture [1]. With the aging population, the number of osteoporosis patients will continue to increase in the next few decades [2]. Osteoporosis mainly affects middle-aged people and the elderly, among which 45% of people over the age of 50 years suffer from osteoporosis.
* Corresponding author. Department of Traumatic Orthopedics, The First People's Hospital of Changzhou, 183 Jvqian Street, Changzhou, Jiangsu, 213003, China. E-mail address: [email protected] (Z. Huang).
Besides, female patients are more commonly affected than male patients, with an incidence rate of 9%e38%, higher than that of male patients [3]. Bone marrow-derived MSCs (BMSCs) are isolated from bone marrow and can be differentiated into osteoblasts, chondrocytes and adipocytes under specific circumstances [4]. BMSCs can be applied for bone repair and remodeling due to their osteogenic differentiation ability [5]. Therefore, it is one of the important therapeutic directions to promote the differentiation of BMSCs, thereafter correcting bone imbalance and alleviating osteoporosis. LncRNA is a class of ncRNA transcripts produced by RNA polymerase II, with the transcript length between 200 and 100 000 nt [6]. Current studies have found that lncRNAs can regulate gene expressions and participate in many important regulatory processes [7,8]. So far, some lncRNAs have been identified to be closely linked to OP [9]. Zhu et al. [10] found that lncRNA ANCR can
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Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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promote osteoblast differentiation by targeting EZH2 and Runx2. Xu et al. [11] found that lncRNA HIF1 alpha-as1 plays a key regulatory role in osteoblast differentiation by regulating HOXD10. LncRNA MSC-AS1 is newly discovered in recent years. Studies have found that MSC-AS1 serves as a ceRNA to regulate cell proliferation and GEM-induced apoptosis in pancreatic ductal adenocarcinoma cell lines by sponging miRNA-29b-3p [12]. However, the specific role of MSC-AS1 in osteogenic differentiation of BMSCs has not been fully explained. MicroRNAs (miRNAs) are a class of small non-coding RNAs of about 22 nucleotides, which are capable of regulating gene expressions and participating in multiple biological processes by complementary base pairing to target mRNAs [13]. Studies have found that miRNAs can regulate osteogenic differentiation and bone formation [14,15]. Besides, studies have found that microRNA140e5p can inhibit the differentiation of osteoblasts [16] and promote the adipocyte differentiation of hMSCs [17]. Bioinformatics predicted binding sites between microRNA-140e5p and BMP2 30 UTR. However, potential function ofmicroRNA-140e5p in the osteogenic differentiation by the BMP2/Smad signaling pathway is unclear. Therefore, this study mainly investigated whether lncRNA MSCAS1 can regulate the osteogenic differentiation of BMSCs through regulating BMP2/Smad signaling pathway by absorbing microRNA140e5p, thus providing a new potential target for OP treatment.
electrophoresis (SDS-PAGE). After blockage in 5% skim milk for 1 h, the membrane was incubated with the primary antibodies and the corresponding secondary antibody. The protein bands were detected by gel imaging system through electrochemiluminescence (ECL) (Pierce, Rockford, IL, USA) and grey values were analyzed by Image J. 2.5. ALP staining BMSCs were induced for 7-day osteogenesis. BMSCs seeded in the 6-well plate were stained with ALP following the recommended protocols, washed in PBS and conterstained with hematoxylin. After washing and air dried, BMSCs were observed under a light microscope. 2.6. Alizarin red staining After osteogenic differentiation, BMSCs were washed in phosphate buffered saline (PBS) without CaCl2 and MgCl2 twice and fixed with 3.7% formalin for 10 min. Afterwards, cells were incubated with alizarin red staining solution at 37 C for 60 min in dark. Then cells were washed with deionized water for 5 times, observed and photographed by an optical microscope. 2.7. Dual-luciferase reporter gene assay
2. Methods 2.1. Cell culture and osteogenic differentiation BMSCs were isolated by whole bone marrow adherence method and cultured in a-MEM supplemented with 10% fetal bovine serum (FBS),100 mg/ml penicillin and 100 U/ml streptomycin in a 37 C, 5% CO2 incubator. After BMSCs were grown to 70%e80% confluence,aMEM containing 10 mmol/L dexamethasone, 50 mmol/L Vitamine C, 10 mmol/L b-sodium glycerophosphate was added for inducing osteogensis. The induction medium was changed every 3 days. 2.2. Cell transfection BMSCs were seeded in 6-well plates. MSC-AS1 shRNA and microRNA-140e5p inhibitor were obtained from GenePharma, Shanghai. Cell transfection was performed using Lipofectamine 2000 (Sigma) according to the manufacturer's standard protocol. 2.3. Quantitative real-time polymerase chain reaction (qRT-PCR) BMSCs were collected and total RNA was isolated by TRIzol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed and then qRT-PCR was performed using SYBR Green method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were used as internal references. Primers were listed as follows: MSC-AS1: forward: 50 -GCCAGTCAGAAAATGAGGAAC-30 , reverse: 50 CCAGTTGGGTGAACAGGAC-3’; miR-140e5p: forward: 50 TGCGGCAGTGGTTTTACCCTA-30 , reverse: 50 -CCA GTGCAGGGTCCGAGGT-3’; BMP2: forward: 50 -GCAAAGAAAAGGAACGGACATT-30 , reverse: 50 -GGGAAGCAGCAACGCTAGAA-3’. 2.4. Western blot assay The total proteins were extracted by radioimmunoprecipitation assay (RIPA) (Beyotime, Shanghai, China) lysate. Concentration of total protein was determined by bicinchoninic acid (BCA) method (Beyotime, Shanghai, China). Subsequently, protein sample was separated through sodium dodecyl sulphate-polyacrylamide gel
The plasmids for dual-luciferase reporter gene assay, including MSC-AS1-WT, BMP2-WT and MSC-AS1-MUT, BMP2-MUT (microRNA-140e5p targets wild-type MSC-AS1 30 -UTR and BMP2 30 -UTR) were synthesized by Genechem. MicroRNA-140e5p mimics and negative controls were obtained from Ribobio. Briefly, BMSCs were transfected with 0.05 mg firefly luciferase reporter gene, 0.05 mg MSC-AS1/BMP2 plasmid and 0.01 mg Renilla luciferase control vector. After 48 h, relative luciferase activity of cells was detected using the Dual-Luciferase Reporter System (Promega) according to the standard protocol. 2.8. Statistical analysis GraphPad Prism 6 (Version X; La Jolla, CA, USA) was used for statistical analysis. Differences between two groups were analyzed by using the Student's t-test. Comparison between multiple groups was done using one-way ANOVA test followed by Post Hoc Test (Least Significant Difference). p < 0.05 was considered statistically significant. 3. Results 3.1. Dynamic expressions of MSC-AS1, microRNA-140e5p and BMP2 during osteogenic differentiation Dynamic expressions of MSC-AS1, microRNA-140e5p and BMP2 in BMSCs during osteogenic differentiation at 0, 7, and 14 days by qRT-PCR, respectively. The results showed that the expression of MSC-AS1 gradually up-regulated with the prolongation of osteogenic differentiation (Fig. 1A). Meanwhile, microRNA-140e5p expression presented a time-dependent decrease during osteogenic differentiation (Fig. 1B). Detection of BMP2 expression showed a gradual increase on day 0, 7, and 14 of osteogenic induction (Fig. 1C). Expressions of osteogenesis-related genes such as RUNX2, OPN and OCN were gradually up-regulated with the prolongation of osteogenic induction (Fig. 1D). These results indicated that MSC-AS1 might regulate the osteogenic differentiation.
Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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Fig. 1. Expression levels of MSC-AS1, miR-140e5p and BMP2 in osteogenic differentiation of bone marrow mesenchymal stem cells. (A) The expression level of MSC-AS1 in BMSCs treated with osteogenic differentiation medium for 0, 7 and 14 days was detected by qRT-PCR, and the expression of MSC-AS1 gradually increased with the prolongation of osteogenic induction. (B) QRT-PCR was used to detect the expression level of miR-140e5p during osteoblast differentiation of BMSCs, and miR-140e5p was down-regulated in a time-dependent manner. (C) QRT-PCR was used to detect the expression of BMP2 during osteoblast differentiation of BMSCs, and the expression of BMP2 was up-regulated in a time-dependent manner. (D) QRT-PCR was used to detect the expression levels of osteogenesis-associated genes RUNX2, OPN and OCN during osteoblast differentiation of BMSCs.
3.2. Silencing of MSC-AS1 inhibits osteoblast differentiation of BMSCs To investigate the influence of MSC-AS1 on osteoblast differentiation, MSC-AS1 was knocked down in BMSCs at 14 days of osteogenic induction. QRT-PCR results showed that the level of MSC-AS1 remarkably decreased in BMSCs after knockdown of MSCAS1 (Fig. 2A). However, microRNA-140e5p expression was upregulated after knockdown of MSC-AS1 compared to the normal control group (Fig. 2B). After transfection of shMSC-AS1, qRT-PCR revealed a significant decrease in mRNA levels of BMP2 and osteogenesis markers (Fig. 2C and D). In addition, ALP staining showed a decrease in ALP content after silencing MSC-AS1 (Fig. 2E). Besides, alizarin red staining showed that MSC-AS1 silencing inhibited the mineralization ability of BMSCs (Fig. 2F). It is demonstrated that MSC-AS1 silencing inhibited the osteoblast differentiation of BMSCs, which may be affected by microRNA140e5p and BMP2. 3.3. MSC-AS1 directly adsorbs microRNA-140-5pto up-regulate BMP2 gene transcript To further investigate the mechanism of MSC-AS1 in regulating osteogenic differentiation, we predicted the binding site of microRNA-140e5p to MSC-AS1 and microRNA-140e5p to BMP2 by TargetScan (Fig. 3A). Further, dual-luciferase reporter gene assay confirmed that microRNA-140e5p inhibited the luciferase activity of wild-type MSC-AS1 and wild-type BMP2, while mutant-type MSC-AS1 and BMP2 were not affected (Fig. 3B). In addition, we determined the expression level of microRNA-140e5p after knockdown of MSC-AS1. Protein level of microRNA-140e5p was found to be remarkably up-regulated after knockdown of MSC-AS1 (Fig. 3C). Similarly, protein level of BMP2 was remarkably up-
regulated after knockdown of microRNA-140e5p (Fig. 3D). The above results demonstrated that MSC-AS1 could directly bind to microRNA-140e5p and thus regulate the expression of BMP2 in BMSCs. 3.4. MSC-AS1 induces osteogenic differentiation via microRNA140e5p regulation of BMP2/Smad pathway Co-transfection of microRNA-140e5p inhibitor and shMSC-AS1 was performed to clarify the regulatory effects of MSC-AS1/ microRNA-140e5p/BMP2 in osteogenic differentiation. Knockdown of MSC-AS1 remarkably decreased the expressions of osteogenesis genes, which were remarkably up-regulated after cotransfection with microRNA-140e5p inhibitor (Fig. 4A). The results indicated that down-regulation of microRNA-140e5p partially reversed the inhibitory effect of down-regulated MSC-AS1 on osteogenic differentiation. Furthermore, we examined whether MSC-AS1 could regulate the BMP2 signaling pathway by regulating microRNA-140e5p.Western blot analysis showed that silencing MSC-AS1 remarkably decreased the expression levels of BMP2, pSmad1/5/8, RUNX2 and Osterix proteins, which were partially reversed by knockdown of microRNA-140e5p (Fig. 4BeE). These results suggested that MSC-AS1 could promote osteogenic differentiation via modulating the BMP2/Smad pathway by absorbing microRNA-140e5p. 4. Discussion Osteoporosis is a metabolic disease caused by the imbalance of bone formation and bone absorption in the body, resulting in decreased bone mass and changes in bone structure [18]. At present, bisphosphonates, calcitonin and other drugs are widely used in clinic to slow down the development of OP through anti-bone
Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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Fig. 2. Silencing of MSC-AS1 inhibits osteogenic differentiation of BMSCs. (AeC) Expression levels of MSC-AS1 (A), miR-140e5p (B) and BMP2 (C) were detected after transfection of shMSC-AS1 by qRT-PCR. (D) Expressions of osteogenic markers Runx2, OPN and OCN in BMSCs transfected with shMSC-AS1 by qRT-PCR. (E) ALP staining showed a decrease in ALP content after knockdown of MSC-AS1 compared to the control group. (F) Alizarin red staining showed that the cell mineralization ability was weakened after silencing MSCAS1compared with the control group.
Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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Fig. 3. MSC-AS1 directly adsorbs miR-140e5p to up-regulate BMP2 gene transcript. (A) Potential binding sites for miR-140e5p to MSC-AS1 and BMP2 were predicted by the TargetScan. (B) Dual-luciferase reporter gene assay showed that overexpression of miR-140e5p significantly inhibited the activities of wild-type MSC-AS1 and wild-type BMP2 luciferase vectors, but had no effect on mutant-type MSC-AS1 and BMP2. (C) Western blot analysis showed that the expression of miR-140e5p was up-regulated after knockdown of MSC-AS1. (D) Western blot analysis showed that the expression of BMP2 was up-regulated after knockdown of miR-140e5p.
resorption. However, improvement of trabecular microstructure of OP patients is difficult to achieve. Human recombinant calcitonin, a bone-forming agent, is greatly restricted due to its narrow therapeutic window [19]. Therefore, improving the ability of bone formation has become the focus of OP treatment. A great number of lncRNAs related to the occurrence of bone diseases have been found through high-throughput sequencing or microarray technology. Some lncRNAs have been proven to regulate osteogenesis, lipid formation and osteoclast differentiation through epigenetic modification, miRNA adsorption and other ways, affecting the process of bone metabolism [20,21]. Studies have shown that lncRNA HIF1A and MALAT1 can promote osteogenic differentiation of BMSCs [22,23]. Here, we found that MSC-AS1 was gradually up-regulated during osteogenic differentiation, and MSCAS1 knockdown could inhibit the osteogenesis. Therefore, MSCAS1 may be an osteogenesis-associated lncRNA in BMSCs. The competitive endogenous RNA (ceRNA) theory hypothesizes that lncRNA can act as the ceRNA to absorb miRNA, thus upregulating target genes [24,25]. Abundant miRNAs have been identified to play a vital role in osteogenic differentiation [26]. For example, H19, as the endogenous competitive RNA of miRNA-141 and miRNA-22, promotes osteogenesis through the Wnt/betacatenin protein pathway [27]. MALAT1 promotes osteogenesis by acting as a sponge for miRNA-204 [28]. In this study, we found a
potential binding site between MSC-AS1 and microRNA-140e5p, and verified their binding by the dual-luciferase reporter gene assay. At the same time, relative expressions of MSC-AS1 and microRNA-140e5p during osteogenic differentiation were also investigated. The results showed that microRNA-140e5p showed a time-dependent decrease during osteogenic differentiation, and the expression of microRNA-140e5p increased after MSC-AS1 was knocked down. In addition, down-regulation of microRNA-140e5p can rescue the impaired osteogenic differentiation induced by knockdown of MSC-AS1. BMPs, a member of the TGF p superfamily, can regulate cell proliferation, differentiation and apoptosis, etc. [29]. The BMP2/ Smad signaling pathway plays a key part in osteogenesis and bone formation [30,31]. In general, the BMP2 ligand binds and activates the transmembrane receptors I and II [32,33], which in turn transmits signals through recruitment and phosphorylation of Smad1/5/8 [34e36]. Afterwards, p-smad1/5/8 and Smad4 constitute the p-smad1/8/4, which is transferred to the nucleus to regulate the expressions of RUNX2 and ALP [37e40]. Some studies have shown that silence of p-type Smad1/5/8 inhibits osteoblast differentiation [41,42]. Here, we found that BMP2 was the direct target of microRNA-140e5p. In addition, knockdown of MSC-AS1 could significantly down-regulate BMP2, p-smad1/5/8 and RUNX2, which were partially reversed by knockdown of microRNA-140e5p,
Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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Fig. 4. MSC-AS1 induces osteogenic differentiation through sponging miR-140e5p to regulate the BMP2/Smad pathway. (A) QRT-PCR detected that MSC-AS1 knockdown can downregulate the expressions of bone-associated markers Runx2, OPN and OCN, while transfection of miR-140e5p inhibitor can reverse the decline of Runx2, OPN and OCN expressions. (BeE) Western blot analysis showed that MSC-AS1 knockdown reduced the expression levels of BMP2, p-Smad1/5/8, Smad1/5/8, Runx2 and Osterix proteins, while transfection of miR-140e5p inhibitor reversed the decreased expressions of BMP2, p-Smad1/5/8, Smad1/5/8, Runx2 and Osterix.
suggesting that MSC-AS1 could induce osteogenic differentiation by regulating the BMP2/Smad pathway through sponging microRNA-140e5p.
Conflicts of interest
5. Conclusions
Appendix A. Supplementary data
In summary, the expression of MSC-AS1 was up-regulated during osteogenic differentiation. MSC-AS1 sponged microRNA140e5p to regulate the BMP2/Smad signaling pathway and thus regulate the osteogenic differentiation of BMSCs, providing a potential therapeutic target for osteoporosis.
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.058.
The authors declared no conflict of interest.
Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2 Naidong Zhang, Xinyu Hu, Shuanghua He, Wenge Ding, Feng Wang, Yiwen Zhao, Zhihui Huang* Department of Traumatic Orthopedics, The First People's Hospital of Changzhou, Changzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 September 2019 Accepted 14 September 2019 Available online xxx
This study aims to explore the role of lncRNA MSC-AS1/microRNA-140e5p/BMP2 regulatory loop in promoting osteogenic differentiation of BMSCs. BMSCs were isolated from bone marrow of mice. Expression levels of MSC-AS1, microRNA-140e5p and BMP2 during osteogenic differentiation were detected by qRT-PCR. Meanwhile, regulatory effect of MSC-AS1 on osteogenic differentiation was detected through ALP staining and alizarin red staining. The binding sites between microRNA-140e5p and MSC-AS1 as well as between microRNA-140e5p and BMP2 were predicted by TargetScan, which were further confirmed by dual-luciferase reporter gene assay. In addition, protein levels of MSC-AS1/ microRNA-140e5p/BMP2 were detected by Western blot. Finally, rescue experiments were conducted to clarify the regulatory effects of MSC-AS1/microRNA-140e5p/BMP2 axis on osteogenic differentiation. MSC-AS1 and BMP2 were found to be remarkably up-regulated during osteogenic differentiation, while microRNA-140e5p was conversely down-regulated. Meanwhile, knockdown of MSC-AS down-regulated expression levels of osteogenesis-associated genes and weakened the mineralization capacity of BMSCs. MicroRNA-140e5p was verified to bind to the 30 UTR of MSC-AS1 and BMP2 genes. Knockdown of MSCAS1 in BMSCs could reduce the expression of microRNA-140e5p, while knockdown of microRNA-140 e5p also down-regulated BMP2 level. In addition, co-silence of MSC-AS1 and microRNA-140e5p reversed the inhibitory effect of MSC-AS1 knockdown on osteogenic differentiation and protein levels of p-Smad1/5/8, RUNX2 and Osterix. MSC-AS1 might promote the osteogenic differentiation of BMSCs through sponging microRNA-140e5p to up-regulate BMP2, thus alleviating the progression of osteoporosis. © 2019 Elsevier Inc. All rights reserved.
Keywords: osteoporosis MSC-AS1 MicroRNA-140e5p BMP2
1. Introduction Osteoporosis (OP) is a type of systemic bone disease characterized by systemic reduction of bone mass and degeneration of bone tissue microstructure, resulting in decreased bone strength, increased brittleness and increased risk of fracture [1]. With the aging population, the number of osteoporosis patients will continue to increase in the next few decades [2]. Osteoporosis mainly affects middle-aged people and the elderly, among which 45% of people over the age of 50 years suffer from osteoporosis.
* Corresponding author. Department of Traumatic Orthopedics, The First People's Hospital of Changzhou, 183 Jvqian Street, Changzhou, Jiangsu, 213003, China. E-mail address: [email protected] (Z. Huang).
Besides, female patients are more commonly affected than male patients, with an incidence rate of 9%e38%, higher than that of male patients [3]. Bone marrow-derived MSCs (BMSCs) are isolated from bone marrow and can be differentiated into osteoblasts, chondrocytes and adipocytes under specific circumstances [4]. BMSCs can be applied for bone repair and remodeling due to their osteogenic differentiation ability [5]. Therefore, it is one of the important therapeutic directions to promote the differentiation of BMSCs, thereafter correcting bone imbalance and alleviating osteoporosis. LncRNA is a class of ncRNA transcripts produced by RNA polymerase II, with the transcript length between 200 and 100 000 nt [6]. Current studies have found that lncRNAs can regulate gene expressions and participate in many important regulatory processes [7,8]. So far, some lncRNAs have been identified to be closely linked to OP [9]. Zhu et al. [10] found that lncRNA ANCR can
https://doi.org/10.1016/j.bbrc.2019.09.058 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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promote osteoblast differentiation by targeting EZH2 and Runx2. Xu et al. [11] found that lncRNA HIF1 alpha-as1 plays a key regulatory role in osteoblast differentiation by regulating HOXD10. LncRNA MSC-AS1 is newly discovered in recent years. Studies have found that MSC-AS1 serves as a ceRNA to regulate cell proliferation and GEM-induced apoptosis in pancreatic ductal adenocarcinoma cell lines by sponging miRNA-29b-3p [12]. However, the specific role of MSC-AS1 in osteogenic differentiation of BMSCs has not been fully explained. MicroRNAs (miRNAs) are a class of small non-coding RNAs of about 22 nucleotides, which are capable of regulating gene expressions and participating in multiple biological processes by complementary base pairing to target mRNAs [13]. Studies have found that miRNAs can regulate osteogenic differentiation and bone formation [14,15]. Besides, studies have found that microRNA140e5p can inhibit the differentiation of osteoblasts [16] and promote the adipocyte differentiation of hMSCs [17]. Bioinformatics predicted binding sites between microRNA-140e5p and BMP2 30 UTR. However, potential function ofmicroRNA-140e5p in the osteogenic differentiation by the BMP2/Smad signaling pathway is unclear. Therefore, this study mainly investigated whether lncRNA MSCAS1 can regulate the osteogenic differentiation of BMSCs through regulating BMP2/Smad signaling pathway by absorbing microRNA140e5p, thus providing a new potential target for OP treatment.
electrophoresis (SDS-PAGE). After blockage in 5% skim milk for 1 h, the membrane was incubated with the primary antibodies and the corresponding secondary antibody. The protein bands were detected by gel imaging system through electrochemiluminescence (ECL) (Pierce, Rockford, IL, USA) and grey values were analyzed by Image J. 2.5. ALP staining BMSCs were induced for 7-day osteogenesis. BMSCs seeded in the 6-well plate were stained with ALP following the recommended protocols, washed in PBS and conterstained with hematoxylin. After washing and air dried, BMSCs were observed under a light microscope. 2.6. Alizarin red staining After osteogenic differentiation, BMSCs were washed in phosphate buffered saline (PBS) without CaCl2 and MgCl2 twice and fixed with 3.7% formalin for 10 min. Afterwards, cells were incubated with alizarin red staining solution at 37 C for 60 min in dark. Then cells were washed with deionized water for 5 times, observed and photographed by an optical microscope. 2.7. Dual-luciferase reporter gene assay
2. Methods 2.1. Cell culture and osteogenic differentiation BMSCs were isolated by whole bone marrow adherence method and cultured in a-MEM supplemented with 10% fetal bovine serum (FBS),100 mg/ml penicillin and 100 U/ml streptomycin in a 37 C, 5% CO2 incubator. After BMSCs were grown to 70%e80% confluence,aMEM containing 10 mmol/L dexamethasone, 50 mmol/L Vitamine C, 10 mmol/L b-sodium glycerophosphate was added for inducing osteogensis. The induction medium was changed every 3 days. 2.2. Cell transfection BMSCs were seeded in 6-well plates. MSC-AS1 shRNA and microRNA-140e5p inhibitor were obtained from GenePharma, Shanghai. Cell transfection was performed using Lipofectamine 2000 (Sigma) according to the manufacturer's standard protocol. 2.3. Quantitative real-time polymerase chain reaction (qRT-PCR) BMSCs were collected and total RNA was isolated by TRIzol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed and then qRT-PCR was performed using SYBR Green method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were used as internal references. Primers were listed as follows: MSC-AS1: forward: 50 -GCCAGTCAGAAAATGAGGAAC-30 , reverse: 50 CCAGTTGGGTGAACAGGAC-3’; miR-140e5p: forward: 50 TGCGGCAGTGGTTTTACCCTA-30 , reverse: 50 -CCA GTGCAGGGTCCGAGGT-3’; BMP2: forward: 50 -GCAAAGAAAAGGAACGGACATT-30 , reverse: 50 -GGGAAGCAGCAACGCTAGAA-3’. 2.4. Western blot assay The total proteins were extracted by radioimmunoprecipitation assay (RIPA) (Beyotime, Shanghai, China) lysate. Concentration of total protein was determined by bicinchoninic acid (BCA) method (Beyotime, Shanghai, China). Subsequently, protein sample was separated through sodium dodecyl sulphate-polyacrylamide gel
The plasmids for dual-luciferase reporter gene assay, including MSC-AS1-WT, BMP2-WT and MSC-AS1-MUT, BMP2-MUT (microRNA-140e5p targets wild-type MSC-AS1 30 -UTR and BMP2 30 -UTR) were synthesized by Genechem. MicroRNA-140e5p mimics and negative controls were obtained from Ribobio. Briefly, BMSCs were transfected with 0.05 mg firefly luciferase reporter gene, 0.05 mg MSC-AS1/BMP2 plasmid and 0.01 mg Renilla luciferase control vector. After 48 h, relative luciferase activity of cells was detected using the Dual-Luciferase Reporter System (Promega) according to the standard protocol. 2.8. Statistical analysis GraphPad Prism 6 (Version X; La Jolla, CA, USA) was used for statistical analysis. Differences between two groups were analyzed by using the Student's t-test. Comparison between multiple groups was done using one-way ANOVA test followed by Post Hoc Test (Least Significant Difference). p < 0.05 was considered statistically significant. 3. Results 3.1. Dynamic expressions of MSC-AS1, microRNA-140e5p and BMP2 during osteogenic differentiation Dynamic expressions of MSC-AS1, microRNA-140e5p and BMP2 in BMSCs during osteogenic differentiation at 0, 7, and 14 days by qRT-PCR, respectively. The results showed that the expression of MSC-AS1 gradually up-regulated with the prolongation of osteogenic differentiation (Fig. 1A). Meanwhile, microRNA-140e5p expression presented a time-dependent decrease during osteogenic differentiation (Fig. 1B). Detection of BMP2 expression showed a gradual increase on day 0, 7, and 14 of osteogenic induction (Fig. 1C). Expressions of osteogenesis-related genes such as RUNX2, OPN and OCN were gradually up-regulated with the prolongation of osteogenic induction (Fig. 1D). These results indicated that MSC-AS1 might regulate the osteogenic differentiation.
Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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Fig. 1. Expression levels of MSC-AS1, miR-140e5p and BMP2 in osteogenic differentiation of bone marrow mesenchymal stem cells. (A) The expression level of MSC-AS1 in BMSCs treated with osteogenic differentiation medium for 0, 7 and 14 days was detected by qRT-PCR, and the expression of MSC-AS1 gradually increased with the prolongation of osteogenic induction. (B) QRT-PCR was used to detect the expression level of miR-140e5p during osteoblast differentiation of BMSCs, and miR-140e5p was down-regulated in a time-dependent manner. (C) QRT-PCR was used to detect the expression of BMP2 during osteoblast differentiation of BMSCs, and the expression of BMP2 was up-regulated in a time-dependent manner. (D) QRT-PCR was used to detect the expression levels of osteogenesis-associated genes RUNX2, OPN and OCN during osteoblast differentiation of BMSCs.
3.2. Silencing of MSC-AS1 inhibits osteoblast differentiation of BMSCs To investigate the influence of MSC-AS1 on osteoblast differentiation, MSC-AS1 was knocked down in BMSCs at 14 days of osteogenic induction. QRT-PCR results showed that the level of MSC-AS1 remarkably decreased in BMSCs after knockdown of MSCAS1 (Fig. 2A). However, microRNA-140e5p expression was upregulated after knockdown of MSC-AS1 compared to the normal control group (Fig. 2B). After transfection of shMSC-AS1, qRT-PCR revealed a significant decrease in mRNA levels of BMP2 and osteogenesis markers (Fig. 2C and D). In addition, ALP staining showed a decrease in ALP content after silencing MSC-AS1 (Fig. 2E). Besides, alizarin red staining showed that MSC-AS1 silencing inhibited the mineralization ability of BMSCs (Fig. 2F). It is demonstrated that MSC-AS1 silencing inhibited the osteoblast differentiation of BMSCs, which may be affected by microRNA140e5p and BMP2. 3.3. MSC-AS1 directly adsorbs microRNA-140-5pto up-regulate BMP2 gene transcript To further investigate the mechanism of MSC-AS1 in regulating osteogenic differentiation, we predicted the binding site of microRNA-140e5p to MSC-AS1 and microRNA-140e5p to BMP2 by TargetScan (Fig. 3A). Further, dual-luciferase reporter gene assay confirmed that microRNA-140e5p inhibited the luciferase activity of wild-type MSC-AS1 and wild-type BMP2, while mutant-type MSC-AS1 and BMP2 were not affected (Fig. 3B). In addition, we determined the expression level of microRNA-140e5p after knockdown of MSC-AS1. Protein level of microRNA-140e5p was found to be remarkably up-regulated after knockdown of MSC-AS1 (Fig. 3C). Similarly, protein level of BMP2 was remarkably up-
regulated after knockdown of microRNA-140e5p (Fig. 3D). The above results demonstrated that MSC-AS1 could directly bind to microRNA-140e5p and thus regulate the expression of BMP2 in BMSCs. 3.4. MSC-AS1 induces osteogenic differentiation via microRNA140e5p regulation of BMP2/Smad pathway Co-transfection of microRNA-140e5p inhibitor and shMSC-AS1 was performed to clarify the regulatory effects of MSC-AS1/ microRNA-140e5p/BMP2 in osteogenic differentiation. Knockdown of MSC-AS1 remarkably decreased the expressions of osteogenesis genes, which were remarkably up-regulated after cotransfection with microRNA-140e5p inhibitor (Fig. 4A). The results indicated that down-regulation of microRNA-140e5p partially reversed the inhibitory effect of down-regulated MSC-AS1 on osteogenic differentiation. Furthermore, we examined whether MSC-AS1 could regulate the BMP2 signaling pathway by regulating microRNA-140e5p.Western blot analysis showed that silencing MSC-AS1 remarkably decreased the expression levels of BMP2, pSmad1/5/8, RUNX2 and Osterix proteins, which were partially reversed by knockdown of microRNA-140e5p (Fig. 4BeE). These results suggested that MSC-AS1 could promote osteogenic differentiation via modulating the BMP2/Smad pathway by absorbing microRNA-140e5p. 4. Discussion Osteoporosis is a metabolic disease caused by the imbalance of bone formation and bone absorption in the body, resulting in decreased bone mass and changes in bone structure [18]. At present, bisphosphonates, calcitonin and other drugs are widely used in clinic to slow down the development of OP through anti-bone
Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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Fig. 2. Silencing of MSC-AS1 inhibits osteogenic differentiation of BMSCs. (AeC) Expression levels of MSC-AS1 (A), miR-140e5p (B) and BMP2 (C) were detected after transfection of shMSC-AS1 by qRT-PCR. (D) Expressions of osteogenic markers Runx2, OPN and OCN in BMSCs transfected with shMSC-AS1 by qRT-PCR. (E) ALP staining showed a decrease in ALP content after knockdown of MSC-AS1 compared to the control group. (F) Alizarin red staining showed that the cell mineralization ability was weakened after silencing MSCAS1compared with the control group.
Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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Fig. 3. MSC-AS1 directly adsorbs miR-140e5p to up-regulate BMP2 gene transcript. (A) Potential binding sites for miR-140e5p to MSC-AS1 and BMP2 were predicted by the TargetScan. (B) Dual-luciferase reporter gene assay showed that overexpression of miR-140e5p significantly inhibited the activities of wild-type MSC-AS1 and wild-type BMP2 luciferase vectors, but had no effect on mutant-type MSC-AS1 and BMP2. (C) Western blot analysis showed that the expression of miR-140e5p was up-regulated after knockdown of MSC-AS1. (D) Western blot analysis showed that the expression of BMP2 was up-regulated after knockdown of miR-140e5p.
resorption. However, improvement of trabecular microstructure of OP patients is difficult to achieve. Human recombinant calcitonin, a bone-forming agent, is greatly restricted due to its narrow therapeutic window [19]. Therefore, improving the ability of bone formation has become the focus of OP treatment. A great number of lncRNAs related to the occurrence of bone diseases have been found through high-throughput sequencing or microarray technology. Some lncRNAs have been proven to regulate osteogenesis, lipid formation and osteoclast differentiation through epigenetic modification, miRNA adsorption and other ways, affecting the process of bone metabolism [20,21]. Studies have shown that lncRNA HIF1A and MALAT1 can promote osteogenic differentiation of BMSCs [22,23]. Here, we found that MSC-AS1 was gradually up-regulated during osteogenic differentiation, and MSCAS1 knockdown could inhibit the osteogenesis. Therefore, MSCAS1 may be an osteogenesis-associated lncRNA in BMSCs. The competitive endogenous RNA (ceRNA) theory hypothesizes that lncRNA can act as the ceRNA to absorb miRNA, thus upregulating target genes [24,25]. Abundant miRNAs have been identified to play a vital role in osteogenic differentiation [26]. For example, H19, as the endogenous competitive RNA of miRNA-141 and miRNA-22, promotes osteogenesis through the Wnt/betacatenin protein pathway [27]. MALAT1 promotes osteogenesis by acting as a sponge for miRNA-204 [28]. In this study, we found a
potential binding site between MSC-AS1 and microRNA-140e5p, and verified their binding by the dual-luciferase reporter gene assay. At the same time, relative expressions of MSC-AS1 and microRNA-140e5p during osteogenic differentiation were also investigated. The results showed that microRNA-140e5p showed a time-dependent decrease during osteogenic differentiation, and the expression of microRNA-140e5p increased after MSC-AS1 was knocked down. In addition, down-regulation of microRNA-140e5p can rescue the impaired osteogenic differentiation induced by knockdown of MSC-AS1. BMPs, a member of the TGF p superfamily, can regulate cell proliferation, differentiation and apoptosis, etc. [29]. The BMP2/ Smad signaling pathway plays a key part in osteogenesis and bone formation [30,31]. In general, the BMP2 ligand binds and activates the transmembrane receptors I and II [32,33], which in turn transmits signals through recruitment and phosphorylation of Smad1/5/8 [34e36]. Afterwards, p-smad1/5/8 and Smad4 constitute the p-smad1/8/4, which is transferred to the nucleus to regulate the expressions of RUNX2 and ALP [37e40]. Some studies have shown that silence of p-type Smad1/5/8 inhibits osteoblast differentiation [41,42]. Here, we found that BMP2 was the direct target of microRNA-140e5p. In addition, knockdown of MSC-AS1 could significantly down-regulate BMP2, p-smad1/5/8 and RUNX2, which were partially reversed by knockdown of microRNA-140e5p,
Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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Fig. 4. MSC-AS1 induces osteogenic differentiation through sponging miR-140e5p to regulate the BMP2/Smad pathway. (A) QRT-PCR detected that MSC-AS1 knockdown can downregulate the expressions of bone-associated markers Runx2, OPN and OCN, while transfection of miR-140e5p inhibitor can reverse the decline of Runx2, OPN and OCN expressions. (BeE) Western blot analysis showed that MSC-AS1 knockdown reduced the expression levels of BMP2, p-Smad1/5/8, Smad1/5/8, Runx2 and Osterix proteins, while transfection of miR-140e5p inhibitor reversed the decreased expressions of BMP2, p-Smad1/5/8, Smad1/5/8, Runx2 and Osterix.
suggesting that MSC-AS1 could induce osteogenic differentiation by regulating the BMP2/Smad pathway through sponging microRNA-140e5p.
Conflicts of interest
5. Conclusions
Appendix A. Supplementary data
In summary, the expression of MSC-AS1 was up-regulated during osteogenic differentiation. MSC-AS1 sponged microRNA140e5p to regulate the BMP2/Smad signaling pathway and thus regulate the osteogenic differentiation of BMSCs, providing a potential therapeutic target for osteoporosis.
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.058.
The authors declared no conflict of interest.
Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058
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Please cite this article as: N. Zhang et al., LncRNA MSC-AS1 promotes osteogenic differentiation and alleviates osteoporosis through sponging microRNA-140e5p to upregulate BMP2, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.058