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Abstract

Osteoporosis (OP) is one of the most common chronic metabolic bone diseases in the seniors and postmenopausal women. Plenty of microRNAs (miRNAs) have been confirmed to be involved in OP progression. However, the role of miR-655-3p in osteogenic differentiation and bone formation was still unclear. In this study, we aimed to investigate the cellular function of miR-655-3p and its underlying mechanism in OP. We found that miR-655-3p expression was downregulated in both ovariectomized (OVX) mice bone tissues and MC3T3-E1 cells treated with simulated microgravity (MG). MiR-655-3p overexpression facilitated cell differentiation but suppressed cell apoptosis of MC3T3-E1 cells induced by simulated MG. Mechanistically, we confirmed that lysine-specific histone demethylase 1 (LSD1) is a downstream target gene of miR-655-3p. Furthermore, overexpression of miR-655-3p activated the bone morphogenetic protein 2 (BMP-2)/decapentaplegic homolog (Smad) signaling pathway by suppressing LSD1 expression. Moreover, LSD1 knockdown accelerated osteogenic differentiation and inhibited apoptosis in MC3T3-E1 cells under simulated MG. Additionally, the OVX mouse model was established to investigate the role of miR-655-3p/LSD1 axis in vivo. The results demonstrated that LSD1 could reverse the effects triggered by the injection of adeno-associated virus-miR-655-3p on OP development. Further investigations revealed that miR-655-3p boosted osteogenic differentiation through LSD1/BMP-2/Smad signaling pathway. In summary, these findings implied a potential value of miR-655-3p in OP therapy.

Keywords

miR-655-3p LSD1 BMP-2/Smad signaling pathway osteoporosis

Introduction

Osteoporosis (OP) is a complex skeletal disease, characterized by low-traumata fractures and declined bone mineral density (BMD).^1,2 As a common disease in the seniors and postmenopausal women, the main risk factors like nutrition, inflammatory and mechanical stress, and hormone fluctuation were closely related to the occurrence or the development of OP.^3,4 Nowadays, OP treatment focuses on the restoration and maintenance of the balance between bone formation and bone resorption.⁵ However, the current treatment is still undesirable due to the lack of osteogenic drug targets. Therefore, it is urgent to explore the potential pathogenesis of OP to strengthen the prevention and improve OP treatment.

It is well known that epigenetic mechanisms, including MicroRNA (miRNA)-mediated posttranscriptional regulation, posttranslational histone modifications, and DNA methylation, are able to affect gene expression in a stable and potentially genetic manner without altering the DNA sequence.^6,7 It has been reported that environmental and stochastic stressors contribute to multiple pathologies in bone diseases through epigenetic mechanisms.^6,8,9 Therefore, OP may have a strong epigenetic component. miRNAs are single-stranded, short noncoding RNAs with about 22 nucleotides, playing important roles in posttranscriptional gene regulation.¹⁰ Some miRNAs have been confirmed to participate in the regulation of cellular processes in different kinds of diseases or tumors. For example, miR-335-5p inhibits osteoarthritis development by activating autophagy.¹¹ Attenuation of miR-10a-5p alleviates joint inflammation in rheumatoid arthritis.¹² Besides, recent studies proposed that miR-655-3p participates in various cancer progressions. For instance, miR-655-3p suppresses ovarian cancer cell migration and proliferation.¹³ MiR-655-3p hampered cell proliferation by regulating human cleft lip-related genes in cultured human lip cells.¹⁴ In addition, increasing research studies have demonstrated that miRNAs were implicated in osteogenic differentiation. For instance, miR-92a-1-5p inhibits osteogenic differentiation by modulating β-catenin.¹⁵ MiRNA-132-3p inhibition facilitates osteogenic differentiation of mesenchymal stem cells and rescues disuse osteopenia.¹⁶ MiR-193a acts as a suppressor in bone marrow-derived stroma cell differentiation.¹⁷ A recent study indicated that miR-655-3p was downregulated in OP, however, the role of miR-655-3p in OP remains undefined.

Accumulating studies have demonstrated that miRNAs elicit their impacts on the development of diverse diseases by targeting specific genes.¹⁸ For example, miR-145 modulates the progression of congenital heart disease by targeting FXN.¹⁹ MiR-500a-3p targets mixed lineage kinase domain-like (MLKL) to attenuate inflammatory response and alleviate kidney injury.²⁰ MiR-1827 represses osteogenic differentiation through targeting insulin-like growth factor 1 (IGF1) in maxillary sinus membrane stem cells.²¹ Moreover, increasing evidence has indicated that miR-655-3p participates in various disease progression in a same way. For instance, miR-655-3p targets pituitary tumor-transforming gene 1 (PTTG1) to suppress cell migration and invasion in non-small cell lung cancer.²² MiR-655-3p inhibits the progression of hepatocellular carcinoma by modulating a disintegrin and metalloprotease 10 (ADAM10) and β-catenin pathway.²³ Nevertheless, the underlying regulatory mechanism of miR-655-3p in OP development is still unclear.

In this study, we aimed to explore the biological role and the mechanism of miR-655-3p in OP. Our results revealed that miR-655-3p alleviated the development of OP by targeting lysine-specific demethylase 1 (LSD1) and activating bone morphogenetic protein 2 (BMP-2)/decapentaplegic homolog (Smad) signaling pathway, which may provide novel target and strategy for OP treatment.

Materials and method

Clinical samples

The peripheral blood from patients with OP (n = 40) or healthy volunteers (n = 20) in Traditional Chinese Medicine Hospital Dianjiang Chongqing (Chongqing, China) was collected. Before the blood collection, none of the enrolled patients received bed rest within 6 months. All patients had signed the informed consent before sample collection. This study was allowed by the Ethical Review Committee of Traditional Chinese Medicine Hospital Dianjiang Chongqing (Chongqing, China).

Cell culture and transfection

The pre-osteoblast MC3T3-E1 cells were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco) under 37°C with 5% CO₂. To carry out the functional experiments on osteogenic differentiation, MC3T3-E1 cells were cultured in osteogenic medium containing 100 nM dexamethasone, 10 mM β-glycerophosphate (Sigma, St. Louis, MO, USA), and 50 μM ascorbic acid.

MiR-665-3p mimic, inhibitor or mock were purchased from Genepharma (Shanghai, China). To knockdown the expression of LSD1, MC3T3-E1 cells were transfected with si-LSD1 or si-NC using Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s guidance.

RNA extraction and quantitative real-time polymerase chain reaction

Total RNA was isolated from peripheral blood, cells, or bone tissues using TRIzol reagent (Takara, Japan). To prepare the cDNA for quantizing miRNA, Mir-X miRNA first-strand synthesis kit (Takara) was used. To quantify mRNA, cDNA was synthesized using a PrimeScript^® RT Master Mix reagent kit (Takara). The following quantitative analysis was carried out using SYBR^® Premix Ex TaqTM II (Takara) and CFX96 real-time polymerase chain reaction detection system (Bio-Rad, Hercules, CA, USA). Internal references were U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative expression was examined using 2⁻ ^ΔΔCt method.

The primer sequences are available under requirement.

Luciferase reporter assay

The wild-type (WT) or mutant (Mut) sequences of LSD1 3′ untranslated region (UTR) were cloned into luciferase reporter vectors (Promega, Madison, WI, USA). Subsequently, the WT or Mut LSD1 3′ UTR vectors were cotransfected with miR-655-3p mimics, anti-miR-655-3p, or mock into MC3T3-E1 cells. After 48 h transfection, the dual-luciferase reporter assay system (Promega) was used to detect luciferase activity. Plasmids were purchased from Genepharma and transfected into MC3T3-E1 cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).

Flow cytometry assay

MC3T3-E1 cells were treated by trypsin digestion with a 0.125% trypsin solution. Then, cells were washed using phosphate-buffered saline (PBS) and centrifuged for 5 min at 1000 r/min. Next, cells were gathered and stained using Annexin V-FITC apoptosis detection kit (BioVision, San Francisco, CA, USA) after resuspension in sterile PBS. Finally, the rate of cell apoptosis was evaluated by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

Microcomputed tomography analysis

Each mouse femur was fixed in 4% paraformaldehyde for 24 h and then scanned by a microcomputed tomography (μCT) scanner (Siemens, Germany), as described previously.²⁴ The microstructure of the femur was represented by the region of interest (ROI). The ROI was 15 µm above the proximal epiphyseal growth plate and selected as a 2.5 × 2.5 × 3 mm³ cube. The indices of BMD, relative bone volume (BV/TV), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), trabecular bone separation (Tb.Sp), and trabecular bone pattern factor (TbPF) were evaluated by COBRA software version 2.0 for μCT.

Histological preparation

The isolated femurs were decalcified in a decalcifying solution containing EDTA (Beyotime Biotechnology, Shanghai, China). Subsequently, these femurs were dehydrated, inlayed in paraffin, sliced, and dyed following the manufacturer’s instruction (Sigma-Aldrich, St. Louis, MO, USA) for Masson staining analysis.

Western blot analysis

Radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology), containing proteinase inhibitor cocktail (Roche Diagnostics, Germany), was used to extract cellular proteins. After 15 min, the protein concentration was measured by BCA protein assay kit (Beyotime Biotechnology). To extract the protein in bone, the bone marrow was flushed out, and then bone tissues were triturated with liquid nitrogen before adding RIPA. Next, equal amounts of proteins were subjected to NuPage bis-tris polyacrylamide gels (Invitrogen). Then, the proteins were transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). Membranes were then blocked with 5% nonfat milk and subsequently incubated with the primary antibodies overnight at 4°C. The primary antibodies were shown as follows: Runx2 (ab76252, Abcam, Shanghai, China), Bglap (ab93876, Abcam), Col1a1 (ab34710, Abcam), LSD1 (ab17721, Abcam), BMP-2 (ab14933, Abcam), p-Smad1 (ab73211, Abcam), p-Smad5 (ab66737, Abcam), Osterix (ab209484, Abcam), Bax (ab32503, Abcam), Cleaved Caspase-3 (ab49822, Abcam), Bcl-2 (ab196495, Abcam), and GAPDH (ab181602, Abcam). Next, the membranes were incubated with secondary antibodies for 1 h at room temperature. ECL detection kit (Thermo Scientific, 34077, Rochester, NY) was used to detect and image protein bands. GAPDH was regarded as the internal control.

Hematoxylin and eosin staining assay

Tissues were mixed with 4% formaldehyde and slides were staining with Mayer’s hematoxylin (Sigma-Aldrich). Then, slides were respectively blued in 0.1% sodium bicarbonate and counterstained with eosin Y solution (Sigma-Aldrich). Under the light microscope, the morphological changes of the slides were observed.

Enzyme-linked immunosorbent assay

To determine the alkaline phosphatase (ALP) activity, culture supernatant was collected from MC3T3-E1 transfected cells in 24-well plates. Then, the enzyme-linked immunosorbent assay (ELISA) kits obtained from Abcam were used in our study.

Simulated microgravity

Simulated microgravity (MG) environment for cells was built by 2-D clinorotation (exploited by China Astronaut Research and Training Center, Beijing, China). The procedure was performed, as described previously.²⁵ In brief, MC3T3-E1 cells, with a density of 1 × 10⁵ cells, were placed onto coverslips. After the cells cohered to the wall, the coverslips were plugged into a chamber filled with culture medium and maintained the distance of 12.5 mm away from the rotational axis of chamber. After the bubbles were completely removed, the lids of the containers were screwed tightly. Then, these chambers were placed into a clinostat and rotated around a horizontal axis at the speed of 24 r/min. The vertical rotation groups severed as controls.

Ovariectomized mouse model

A total of 20 female mice (5-week-old, 18–22 g) were provided by Chongqing TengXin Biotech Company (Chongqing, China). Mice were housed in individual cages at 26°C with a 12-h light/dark cycle (relative humidity 50–65%). During the experiments, the Reduction, Replacement, and Refinement animal welfare principle was obeyed.²⁶ Mice were divided into sham operation group (sham, n = 10) and ovariectomized model group (OVX, n = 10) randomly. Mice in sham group were anaesthetized by injecting 5% chloral hydrate at a dose of 400 mg/kg animal body weight into abdominal cavity. Mice in OVX group were treated the same surgical protocols but extirpated both sides of the ovaries. Three months later, mice were anesthetized by intraperitoneal injection of 5% chloral hydrate again and underwent distal femur scanning using µCT (SkyScan; Bruker Corporation, Billerica, MA, USA) along the axis of femur to examine postmenopausal OP symptoms. Adeno-associated virus (AAV) was purchased from Hanheng Company (Hanheng Biotechnology Co., Ltd, Shanghai, China) and injected into mice 4 days before establishing the OVX mouse model. The expression of miR-655-3p or LSD1 was overexpressed in OVX mice by injection of AAV. Control mice were only treated with AAV. All procedures were conducted following the recommendations in the Guide for the Care and Use of Laboratory Animals (8th edition, 2011, National Research Council).

Statistical analysis

All statistical analyses were performed using SPSS 22.0 software. The quantitative data were expressed as mean ± standard deviation from three duplicate experiments. Differences between groups were analyzed using two-tailed t-test or one-way analysis of variance. The value of p <0.05 was considered statistically significant.

Results

MiR-655-3p is downregulated in OP tissues and MC3T3-E1 cells

To investigate the significance of miRNAs in mouse osteoblasts under simulated MG environment, the OVX mice model was established. First, Masson staining assay displayed that the distal femurs of OVX mice showed less osteoid staining than sham mice (Figure 1(a)). Then, the µCT analysis revealed significant reductions in BMD, BV/TV, Tb.Th, and Tb.N while showed notable enhancements in Tb.Sp and TbPF in OVX mice group compared to those in sham group (Figure 1(b) to (g)). Real-time quantitative polymerase chain reaction (RT-qPCR) showed that miR-655-3p displayed the most downregulation among these miRNAs in the peripheral blood of OP patients compared with healthy controls (Figure 1(h)). Hence, we chose miR-655-3p for further exploration. As shown in Figure 1(i), the expression of miR-655-3p was much lower in OVX mice tissues than that in the sham mice. To investigate the correlation between osteoblast differentiation and miR-655-3p, we cultivated mouse pre-osteoblast MC3T3-E1 cells in a simulated MG environment. RT-qPCR assay elucidated that miR-655-3p level was conspicuously declined in MG group at 48 h under clinorotation conditions (Figure 1(j)). Moreover, time-dependent experiment revealed that the expression of miR-655-3p was continuously downregulated under clinorotation conditions (Figure 1(k)). Taken together, miR-655-3p is lowly expressed in both OP tissues and MC3T3-E1 cells under simulated MG conditions.

Figure 1.

MiR-655-3p is downregulated in OP tissues and MC3T3-E1 cells. (a) Masson staining showed the representative images of the distal femurs of sham mice and OVX mice. (b to g) μCT analysis of the ROI region of the distal femurs of mice from each group. The three-dimensional indices were BMD, BV/TV, Tb.Th, Tb.N, Tb.Sp, and TbPF. *p < 0.01 versus sham. (h) RT-qPCR analysis was used to evaluate the expression of five candidate miRNAs and found that miR-655-3p expression was the most downregulated. *p < 0.01 versus healthy control. (i) The level of miR-655-3p in OVX mice was detected by RT-qPCR. *p < 0.01 versus sham. (j) RT-qPCR analysis was carried out to assess the expression of miR-655-3p under clinorotation conditions for 48 h (N = 3). *p < 0.01 versus CON. (k) RT-qPCR analysis was applied to measure MiR-655-3p level under clinorotation conditions for 72 h (N = 3). *p < 0.01 versus CON. BMD: bone mineral density; BV/TV: relative bone volume; Tb.Th: trabecular bone thickness; Tb.N: trabecular bone number; Tb.Sp: trabecular bone separation; TbPF: trabecular bone pattern factor; RT-qPCR: real-time quantitative polymerase chain reaction; miRNA: microRNA; OVX: ovariectomized model group; μCT: microcomputed tomography; ROI: region of interest; OP: osteoporosis.

MiR-655-3p enhances osteogenic differentiation and inhibits MC3T3-E1 cell apoptosis

To analyze the biological effects of miR-655-3p on osteogenic differentiation, we carried out follow-up experiments. RT-qPCR assay was used to determine the overexpression or knockdown efficiency of miR-655-3p mimics (miR-655-3p) or anti-miR-582-3p in MC3T3-E1 cells. The results demonstrated that miR-655-3p level was markedly increased by transfection of miR-655-3p mimics while significantly declined in anti-miR-655-3p treated cells (Figure 2(a)). As depicted in Figure 2(b) and (c), the protein expression of osteogenic genes (Runx2, Bglap, and Col1a1) was dramatically increased by miR-655-3p overexpression, whereas miR-655-3p suppression exerted opposite effects. Moreover, ALP activity was obviously increased in the group treated with miR-655-3p overexpression, whereas it was decreased in anti-miR-655-3p group (Figure 2(d)). In addition, the percentage of apoptotic cells assessed by flow cytometry was dramatically increased by transfection of anti-miR-655-3p in MC3T3-E1 cells (Figure 2(e) and (f)). The protein level of Bax and Cleaved Caspase-3 was remarkably increased by anti-miR-655-3p, whereas Bcl-2 protein level was conspicuously decreased in anti-miR-655-3p group compared with the mock group (Figure 2(g) and (h)). In conclusion, these results revealed that the overexpression of miR-655-3p promotes osteogenic differentiation and suppresses apoptosis in MC3T3-E1 cells.

Figure 2.

MiR-655-3p enhances osteogenic differentiation and inhibits MC3T3-E1 cell apoptosis. (a) RT-qPCR was conducted to detect the overexpression and knockdown efficiency of miR-655-3p. *p < 0.01 versus mock. (b and c) The protein level of osteoblast marker genes (Runx2, Bglap, and Col1a1) in MC3T3-E1 cells was measured by Western blot and quantitated by RT-qPCR. *p < 0.01 versus mock. (d) ALP activity in MC3T3-E1 cells was detected by ELISA. *p < 0.01 versus mock. (e and f) Flow cytometry analysis was carried out to determine cell apoptosis caused by transfection of anti-miR-655-3p. *p < 0.01 versus mock. (g and h) Protein expression of Bax, Bcl-2, and Cleaved Caspase-3 in MC3T3-E1 cells. *p < 0.01 versus mock. RT-qPCR: real-time quantitative polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay.

MiR-655-3p overexpression partially alleviates the alterations of MC3T3-E1 cells under simulated MG

As is known to all, osteogenic differentiation could be inhibited under simulated MG.²⁷ We then aimed to explore whether osteoblast differentiation could be affected by miR-655-3p under simulated MG. It was evident from Figure 3(a) to (d) that the mRNA and protein expression of Runx2, Bglap, and Col1a1 which identified as osteogenic markers were significantly reduced under the environment of simulated MG, while these effects could be offset by miR-655-3p overexpression. Additionally, upregulation of miR-655-3p notably reversed the decline of ALP mRNA expression and activity under simulated MG. These data revealed that overexpressing endogenous miR-655-3p could enhance MC3T3-E1 cell differentiation under simulated MG. Next, we evaluated cell apoptosis under simulated MG. Flow cytometry assay showed that miR-655-3p overexpression significantly recovered the elevated cell apoptosis induced by simulated MG (Figure 3(e) and (f)). Meanwhile, the enhancement of Bax and Cleaved Caspase-3 protein level in MC3T3-E1 cells under simulated MG was markedly reversed by miR-655-3p overexpression (Figure 3(g) and (h)). In a word, miR-655-3p overexpression partially alleviates MC3T3-E1 cell apoptosis while promotes osteogenic differentiation under simulated MG.

Figure 3.

MiR-655-3p overexpression partially alleviates the alterations of MC3T3-E1 cells under simulated microgravity. (a) RT-qPCR was conducted to detect the mRNA level of Runx2, Bglap, Col1a1, and ALP in MC3T3-E1 cells treated with miR-655-3p overexpression under simulated microgravity. *p < 0.01 versus CON. ^# p < 0.01 versus MG + NC. (b and c) The protein expression of Runx2, Bglap, and Col1a1 was detected in MC3T3-E1 cells under simulated MG. *p < 0.01 versus CON. ^# p < 0.01 versus MG + NC. (d) ELISA assay was used to evaluate the relative ALP activity under simulated MG. *p < 0.01 versus CON. ^# p < 0.01 versus MG + NC. (e and f) Cell apoptosis was tested by flow cytometry analysis and stained with annexin V-FITC/PI under simulated MG. *p < 0.01 versus CON. ^# p < 0.01 versus MG + mock. (g and h) Western blot assay was applied to assess protein level of Bax, Bcl-2, and Cleaved Caspase-3 in MC3T3-E1 cells under simulated MG. *p < 0.01 versus CON. ^# p < 0.01 versus MG + mock. ELISA: enzyme-linked immunosorbent assay; MG: microgravity; RT-qPCR: real-time quantitative polymerase chain reaction.

MiR-655-3p targets LSD1 and regulates BMP-2/Smad signaling pathway in OP

By searching StarBase website (http://starbase.sysu.edu.cn/), we discovered that miR-655-3p shared binding site with LSD1 3′ UTR in mouse (Figure 4(a)). Subsequently, the interaction between miR-655-3p and LSD1 was further confirmed by luciferase reporter assay. As displayed in Figure 4(b), LSD1 3′ UTR-WT luciferase reporter activity was markedly lowered by miR-655-3p overexpression, but significantly enhanced by transfection of anti-miR-655-3p, implying that miR-655-3p could directly bind with LSD1 3′ UTR. We then estimated LSD1 level in mice and found that the mRNA and protein expression of LSD1 were upregulated in OVX mice bone tissues compared with sham group (Figure 4(c) to (e)). Furthermore, we detected the mRNA and protein expression of LSD1 in MC3T3-E1 cells under simulated MG. The results showed that LSD1 mRNA and protein level were highly expressed in MC3T3-E1 cells under simulated MG (Figure 4(f) to (h)). Recent studies had verified that BMP-2/Smad signaling pathway played an important role in OP and its activation helped protect bone tissues. However, this effect could be suppressed by LSD1.^28,29 To explore whether miR-655-3p has capacity to activate BMP-2/Smad signaling pathway, we detected the protein level of LSD1 and the makers which associated with BMP-2/Smad signaling pathway first. Western blot assay elucidated that the protein level of BMP-2, p-Smad1, p-Smad5, Runx2, and Osterix was increased by miR-655-3p overexpression, while it was decreased in anti-miR-655-3p group. However, LSD1 protein level was declined in miR-655-3p upregulation group but enhanced by transfection of anti-miR-655-3p (Figure 4(i) and (j)). As shown in Figure 4(k), the mRNA expression of LSD1 was significantly reduced by miR-655-3p mimics but raised by anti-miR-655-3p. All the above results revealed that miR-655-3p targets LSD1 and regulates BMP-2/Smad signaling pathway in OP progression.

Figure 4.

MiR-655-3p targets LSD1 and regulates BMP-2/Smad signaling pathway in OP. (a) The binding site between miR-655-3p and LSD1 was shown. (b) The luciferase activity of the LSD1 WT and LSD1 Mut reporters in MC3T3-E1 cells treated with miR-655-3p mimics and anti-miR-655-3p. *p < 0.01 versus mock. (c) The expression of LSD1 in OVX mice. *p < 0.01 versus sham. (d and e) The protein level of LSD1 in OVX mice tissues was detected by Western blot. *p < 0.01 versus sham. (f) The mRNA expression of LSD1 was assessed under simulated MG. *p < 0.01 versus CON. (g and h) LSD1 protein level was evaluated under simulated MG. *p < 0.01 versus CON. (i and j) Western blot assay was conducted to measure the protein level of LSD1, BMP-2, p-Smad1, p-Smad5, Runx2, and Osterix by miR-655-3p overexpression or knockdown. *p < 0.01 versus mock. ^# p < 0.01 versus mock. (k) LSD1 mRNA expression in MC3T3-E1 cells treated with miR-655-3p mimics and anti-miR-655-3p. *p < 0.01 versus mock. ^# p < 0.01 versus mock. OVX: ovariectomized model group; LSD1: lysine-specific demethylase 1; WT: wild-type; Mut: mutant; BMP-2: bone morphogenetic protein 2; MG: microgravity; OP: osteoporosis.

LSD1 knockdown accelerates osteogenic differentiation and inhibits apoptosis in MC3T3-E1 cells under simulated MG

To further explore the function of LSD1 under simulated MG, we used si-LSD1 vectors to knockdown LSD1 in MC3T3-E1 cells (Figure 5(a)). As shown in Figure 5(b) to (d), si-LSD1 transfection conspicuously upregulated the mRNA and protein level of osteogenic genes (Runx2, Bglap, Col1a1, and ALP) in MC3T3-E1 cells under simulated MG. Similar trends of ALP activity were showed by ELISA assay (Figure 5(e)). In MG + siLSD1 group, osteoblasts apoptosis was dramatically decreased (Figure 5(f)). Moreover, the protein level of Bax and Cleaved Caspase-3 was declined, whereas Bcl-2 protein level was enhanced in MG + si-LSD1 group (Figure 5(g) and (h)). Overall, LSD1 suppresses osteogenic differentiation but promotes MC3T3-E1 cell apoptosis under simulated MG environment.

Figure 5.

LSD1 knockdown accelerates osteogenic differentiation and inhibits apoptosis in MC3T3-E1 cells under simulated MG. (a) The knockdown efficacy of LSD1 was detected by RT-qPCR. *p < 0.01 versus si-NC. (b) RT-qPCR analysis of Runx2, Bglap, Col1a1, and ALP mRNA level in MC3T3-E1 cells under simulated MG. *p < 0.01 versus CON. ^# p < 0.01 versus MG + si-NC. (c and d) Western blot assay was conducted to evaluate the protein level of Runx2, Bglap, and Col1a1 in MC3T3-E1 cells under simulated MG. *p < 0.01 versus CON. ^# p < 0.01 versus MG + si-NC. (e) ALP activity analysis in MC3T3-E1 cells under simulated MG. *p < 0.01 versus CON. ^# p < 0.01 versus MG + si-NC. (f) Cell apoptosis of MC3T3-E1 cells under simulated MG. *p < 0.01 versus CON. ^# p < 0.01 versus MG + si-NC. (g and h) Protein level of Bax, Bcl-2, and Cleaved Caspase-3 in MC3T3-E1 cells under simulated MG. *p < 0.01 versus CON. ^# p < 0.01 versus MG + si-NC. LSD1: lysine-specific demethylase 1; RT-qPCR: real-time quantitative polymerase chain reaction; MG: microgravity.

LSD1 is essential for miR-655-3p during OP progression in vivo

To further confirm whether miR-655-3p/LSD1 axis affects OP progression in vivo, we established the OVX model in mice. We discovered that both miR-655-3p and LSD1 expression were upregulated in mice injected with AAV-miR-655-3p and AAV-LSD1, respectively. (Figure 6(a)). Hematoxylin and eosin staining assay was carried to show the structure of bone tissues (Figure 6(b)). Compared with control groups, significant reduction of BMD, BV/TV, Tb.Th, and Tb.N but notable enhancements of Tb.Sp and TbPF were found in OVX mice model. Additionally, LSD1 amplification could reverse the enhancement of BMD, BV/TV, Tb.Th, and Tb.N as well as the decline of Tb.Sp and TbPF caused by miR-655-3p overexpression in OVX mice model (Figure 6(c) to (h)). To sum up, miR-655-3p could suppress OP development by targeting LSD1 in vivo.

Figure 6.

LSD1 is essential for miR-655-3p during OP progression in vivo. (a) RT-qPCR assay was carried out to investigate the expression of miR-655-3p and LSD1 in mice injected with AAV. *p < 0.01 versus AAV-mock. ^# p < 0.01 versus AAV vector. (b) H&E staining was used to observe the pathological changes in knee bone or other joints with osteoporosis. (c to h) μCT analysis of the ROI region of the distal femurs of mice from each group. The three-dimensional indices were BMD, BV/TV, Tb.Th, Tb.N, Tb.Sp, and TbPF. *p < 0.01 versus sham + AAV-mock + AAV-vector. ^# p < 0.01 versus OVX + AAV-mock + AAV-vector. ^& p < 0.01 versus OVX + miR-655-3p + AAV-vector. BMD: bone mineral density; BV/TV: relative bone volume; Tb.Th: trabecular bone thickness; Tb.N: trabecular bone number; Tb.Sp: trabecular bone separation; TbPF: trabecular bone pattern factor; AAV: adeno-associated virus; OVX: ovariectomized model group; μCT: microcomputed tomography; LSD1: lysine-specific demethylase 1; RT-qPCR: real-time quantitative polymerase chain reaction; OP: osteoporosis.

MiR-655-3p inhibits OP progression by targeting LSD1 to activate BMP-2/Smad signaling pathway

Next, we aimed to validate the relationship between miR-665-3p, LSD1, and BMP-2/Smad signaling pathway in OP progression, AAV-miR-655-3p and AAV-LSD1 were injected into OVX mice. As depicted in Figure 7(a), the mRNA level of Runx2, Bglap, and Col1a1 was reduced in OVX mice model compared with sham + AAV-mock + AAV-vector group. Moreover, injection of AAV-LSD1 markedly blocked the AAV-miR-655-3p-induced increase of Runx2, Bglap, and Col1a1 mRNA level in vivo. ELISA assay showed similar changes in ALP activity (Figure 7(b)). In addition, the remarkable upregulation of Bax and Caspase-3 mRNA expression and downregulation of Bcl-2 mRNA level were observed in OVX mice, and the results induced by injection of AAV-miR-655-3p were neutralized in OVX + AAV-miR-655-3p + AAV-LSD1 group (Figure 7(c)). Next, Western blot assay was carried out to assess the level of proteins which related to BMP-2/Smad signaling pathway. As demonstrated in Figure 7(d) and (e), the protein level of BMP-2, p-Smad1, p-Smad5, Runx2, and Osterix was decreased while LSD1 protein expression was increased in vivo. Furthermore, LSD1 amplification could offset the enhancement of BMP-2, p-Smad1, p-Smad5, Runx2, and Osterix protein level or decline of LSD1 protein expression induced by the injection of AVV-miR-655-3p in OVX mice model. In conclusion, miR-655-3p inhibits osteoblast apoptosis, whereas it promotes differentiation via LSD1/BMP-2/Smad signaling pathway.

Figure 7.

MiR-655-3p inhibits OP progression by targeting LSD1 to activate BMP-2/Smad signaling pathway. (a) The mRNA level of Runx2, Bglap, and Col1a1 in OVX mice injected with AAV-miR-65-3p or AAV-LSD1. *p < 0.01 versus sham + AAV-mock + AAV-vector. ^# p < 0.01 versus OVX + AAV-mock + AAV-vector. ^& p < 0.01 versus OVX + AAV-miR-655-3p + AAV-vector. (b) ALP activity analysis in OVX mice injected with AAV. *p < 0.01 versus sham + AAV-mock + AAV-vector. ^# p < 0.01 versus OVX + AAV-mock + AAV-vector. ^& p < 0.01 versus OVX + miR-655-3p + AAV-vector. (c) The mRNA level of Bax, Bcl-2, and Caspase-3 in OVX mice injected with AAV. *p < 0.01 versus sham + AAV-mock + AAV-vector. ^# p < 0.01 versus OVX + AAV-mock + AAV-vector. ^& p < 0.01 versus OVX + miR-655-3p + AAV-vector. (d and e) The protein level of LSD1, BMP-2, p-Smad1, p-Smad5, Runx2, and Osterix in OVX mice injected with AAV-miR-65-3p or AAV-LSD1. *p < 0.01 versus sham + AAV-NC + AAV-vector. ^# p < 0.01 versus OVX + AAV-NC + AAV-vector. ^& p < 0.01 versus OVX + miR-655-3p + AAV-vector. AAV: adeno-associated virus; OP: osteoporosis; OVX: ovariectomized model group; BMP-2: bone morphogenetic protein 2; LSD1: lysine-specific demethylase 1.

Discussion

OP is a chronic metabolic skeletal disease characterized by diminished state of bone formation and heightened bone resorption.³⁰ With the aging of population, the living burden of osteoporotic patients increases exponentially. Despite several agents, including bisphosphonates, calcitonin, and so on, were used for osteoporotic treatment, the long-term safety risks of these drugs could not be guaranteed.³¹ Therefore, investigation of effective therapeutic targets and exploration of underlying mechanism for osteoporotic management are necessary.

Numerous studies have validated that miRNAs can regulate cellular processes in various diseases, including OP.^32,33 For example, miR-145 promotes chondrocyte viability and inhibits cartilage extracellular matrix (ECM) degradation in osteoarthritis.³⁴ MiR-365a-3p accelerates the development of OP by targeting RUNX2 to suppressing osteogenic differentiation.³⁵ MiR-133a facilitates osteoclast differentiation in the progression of postmenopausal OP.³⁶ A recent study demonstrated that miR-655-3p was one of the significant downregulated miRNAs involved in OP development.³² Herein, we constructed the OP mice model by OVX treatment and cell model by MG treatment to further explore the biological function of miR-655-3p in OP. Our study indicated that miR-655-3p was downregulated in OP tissues and MC3T3-E1 cells under clinorotation conditions. Moreover, miR-655-3p promoted osteoblast differentiation while inhibited apoptosis of MC3T3-E1 cells in a simulated MG environment. These findings implied that miR-655-3p might alleviate bone loss induced by MG.

LSD1 is the first histone demethylase to be discovered. LSD1 knockdown could inhibit cancer or disease development, and thus it is an attractive target for providing novel therapeutics.^37
–39 It has been reported that LSD1 served as a negative regulator of osteogenesis.²⁸ Likewise, in our study, we discovered that the mRNA and protein expression of LSD1 were obviously upregulated both in OVX mice and under MG. LSD1 deficiency facilitated osteogenic differentiation while suppressed apoptosis in MC3T3-E1 cells under simulated MG. Plentiful studies revealed that miRNAs could directly target mRNAs in various diseases.^40,41 Moreover, it has been reported that LSD1 suppressed the function of miR-137 in osteogenic regulation.²⁶ Therefore, we conjectured that miR-655-3p might target LSD1 and then participated in OP development. By searching StarBase, we discovered that miR-655-3p could share binding site with LSD1 and then we further confirmed the binding capacity between them.

Previous research studies have demonstrated that BMP-2/Smad signaling network is closely related to osteogenic differentiation. For instance, conidium lactone effectively induces osteogenic differentiation of bone marrow mesenchymal stem cells through BMP-2/Smad signaling pathway.⁴² Gremlin2 suppresses the osteogenesis of human bone marrow-derived mesenchymal stem cells by BMP-2/Smad/Runx2 signaling network.⁴³ Additionally, it is evident from a recent study that activating BMP-2/Smad44 signaling pathway helps to protect bone tissues, while LSD1 could repress this effect.²⁸ We then speculated that miR-655-3p might activate BMP-2/Smad signaling pathway in a similar way. As expected, our finding suggested that miR-655-3p was capable of negatively regulating LSD1 mRNA and protein expression. Besides, miR-582-3p could activate BMP-2/Smad signaling pathway through increasing BMP-2, p-Smad1, and p-Smad5 protein levels. Furthermore, we established the OVX mice model to validate whether miR-655-3p activated BMP-2/Smad signaling pathway by suppressing LSD1 in vivo. The data demonstrated that LSD1 amplification could offset the increase of BMD, BV/TV, Tb.Th, and Tb.N as well as the decrease of Tb.Sp and TbPF resulted from miR-655-3p overexpression in vivo. Additionally, overexpression of LSD1 could reverse the effects on BMP-2/Smad signaling activation induced by injection of AVV-miR-655-3p in OVX mice model.

Taken together, our study showed the first evidence that miR-655-3p promoted osteogenic differentiation via LSD1/BMP-2/Smad signaling pathway during OP progression (Figure S1 in the Online Supplemental Material). These findings may supply a novel biological target and possible mechanism for OP therapy.

Supplemental material

Supplemental Material, Supplement_Figure_1 - MiR-655-3p inhibits the progression of osteoporosis by targeting LSD1 and activating BMP-2/Smad signaling pathway

Supplemental Material, Supplement_Figure_1 for MiR-655-3p inhibits the progression of osteoporosis by targeting LSD1 and activating BMP-2/Smad signaling pathway by X-J Wang, J-W Liu and J Liu in Human & Experimental Toxicology

Footnotes

Authors’ note

XW and JL are co-first authors.

Acknowledgment

The authors thank all participators for their help.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

J Liu

Supplemental material

Supplemental material for this article is available online.

References

Dalle Carbonare

Valenti

Zanatta

, et al. Circulating mesenchymal stem cells with abnormal osteogenic differentiation in patients with osteoporosis. Arthritis Rheum 2009; 60(11): 3356–3365.

Rachner

Khosla

Hofbauer

. Osteoporosis: now and the future. Lancet 2011; 377(9773): 1276–1287.

Nagaraja

Risin

. The current state of bone loss research: data from spaceflight and microgravity simulators. J Cell Biochem 2013; 114(5): 1001–1008.

Henriksen

Thudium

Christiansen

, et al. Novel targets for the prevention of osteoporosis—lessons learned from studies of metabolic bone disorders. Expert Opin Ther Targets 2015; 19(11): 1575–1584.

Marie

Kassem

. Osteoblasts in osteoporosis: past, emerging, and future anabolic targets. Eur J Endocrinol 2011; 165(1): 1–10.

van Meurs

Boer

Lopez-Delgado

, et al. Role of epigenomics in bone and cartilage disease. J Bone Miner Res 2019; 34(2): 215–230.

Letarouilly

Broux

Clabaut

. New insights into the epigenetics of osteoporosis. Genomics 2019; 111(4): 793–798.

Michou

. Epigenetics of bone diseases. Joint Bone Spine 2018; 85(6): 701–707.

Feng

Zheng

. The emerging role of microRNAs in bone remodeling and its therapeutic implications for osteoporosis. Biosci Rep 2018; 38(3): BSR20180453.

10.

Jackson

Grabowska

Ratan

. MicroRNA in prostate cancer: functional importance and potential as circulating biomarkers. BMC Cancer 2014; 14: 930.

11.

Zhong

Long

, et al. miRNA-335-5p relieves chondrocyte inflammation by activating autophagy in osteoarthritis. Life Sci 2019; 226: 164–172.

12.

Hussain

Zhu

Jiang

, et al. Down-regulation of miR-10a-5p in synoviocytes contributes to TBX5-controlled joint inflammation. J Cell Mol Med 2018; 22(1): 241–250.

13.

Zha

Chen

. MiR-655-3p inhibited proliferation and migration of ovarian cancer cells by targeting RAB1A. Eur Rev Med Pharmacol Sci 2019; 23(9): 3627–3634.

14.

Gajera

Desai

Suzuki

, et al. MicroRNA-655-3p and microRNA-497-5p inhibit cell proliferation in cultured human lip cells through the regulation of genes related to human cleft lip. BMC Med Genomics 2019; 12(1): 70.

15.

Lin

Tang

Tan

, et al. MicroRNA-92a-1-5p influences osteogenic differentiation of MC3T3-E1 cells by regulating β-catenin. J Bone Miner Metab 2019; 37(2): 264–272.

16.

Zhang

Wang

, et al. Targeted silencing of miRNA-132-3p expression rescues disuse osteopenia by promoting mesenchymal stem cell osteogenic differentiation and osteogenesis in mice. Stem Cell Res Ther 2020; 11(1): 58.

17.

Wang

Zhao

, et al. miR-193a inhibits osteogenic differentiation of bone marrow-derived stroma cell via targeting HMGB1. Biochem Biophys Res Commun 2018; 503(2): 536–543.

18.

Michlewski

Caceres

. Post-transcriptional control of miRNA biogenesis. RNA 2019; 25(1): 1–16.

19.

Wang

Tian

, et al. MiRNA-145 regulates the development of congenital heart disease through targeting FXN. Pediatr Cardiol 2016; 37(4): 629–636.

20.

Jiang

Liu

, et al. hsa-miR-500a-3P alleviates kidney injury by targeting MLKL-mediated necroptosis in renal epithelial cells. Faseb J 2019; 33(3): 3523–3535.

21.

Zhu

Peng

, et al. miR-1827 inhibits osteogenic differentiation by targeting IGF1 in MSMSCs. Sci Rep 2017; 7: 46136.

22.

Wang

Cao

, et al. miR-655-3p inhibits cell migration and invasion by targeting pituitary tumor-transforming 1 in non-small cell lung cancer. Biosci Biotechnol Biochem 2019; 83(9): 1703–1708.

23.

Zheng

Xia

, et al. MicroRNA-655-3p functions as a tumor suppressor by regulating ADAM10 and β-catenin pathway in hepatocellular carcinoma. J Exp Clin Cancer Res 2016; 35(1): 89.

24.

Wang

, et al. MicroRNA-139-3p regulates osteoblast differentiation and apoptosis by targeting ELK1 and interacting with long noncoding RNA ODSM. Cell Death Dis 2018; 9(11): 1107.

25.

Wang

Sun

, et al. miRNA-132-3p inhibits osteoblast differentiation by targeting Ep300 in simulated microgravity. Sci Rep 2015; 5: 18655.

26.

Hovell

. Reduction, refinement and replacement. Vet Rec 2013; 172(26): 691.

27.

Shi

Xie

, et al. Microgravity induces inhibition of osteoblastic differentiation and mineralization through abrogating primary cilia. Sci Rep 2017; 7(1): 1866.

28.

Fan

Wang

, et al. MiR-137 knockdown promotes the osteogenic differentiation of human adipose-derived stem cells via the LSD1/BMP2/SMAD4 signaling network. J Cell Physiol 2019; 235(2): 909–919.

29.

Chai

Wan

Wang

, et al. Gushukang inhibits osteocyte apoptosis and enhances BMP-2/Smads signaling pathway in ovariectomized rats. Phytomedicine 2019; 64: 153063.

30.

Mukherjee

Chattopadhyay

. Pharmacological inhibition of cathepsin K: a promising novel approach for postmenopausal osteoporosis therapy. Biochem Pharmacol 2016; 117: 10–19.

31.

Lin

Zhu

Wang

, et al. Chinese single herbs and active ingredients for postmenopausal osteoporosis: from preclinical evidence to action mechanism. Biosci Trends 2017; 11(5): 496–506.

32.

Jin

, et al. Systematic analysis of lncRNAs, mRNAs, circRNAs and miRNAs in patients with postmenopausal osteoporosis. Am J Transl Res 2018; 10(5): 1498–1510.

33.

Condrat

Thompson

Barbu

, et al. miRNAs as biomarkers in disease: latest findings regarding their role in diagnosis and prognosis. Cells 2020; 9(2): 276.

34.

Liu

Ren

Zhao

, et al. LncRNA MALAT1/MiR-145 adjusts IL-1β-induced chondrocytes viability and cartilage matrix degradation by regulating ADAMTS5 in human osteoarthritis. Yonsei Med J 2019; 60(11): 1081–1092.

35.

Cheng

Yang

. MiRNA-365a-3p promotes the progression of osteoporosis by inhibiting osteogenic differentiation via targeting RUNX2. Eur Rev Med Pharmacol Sci 2019; 23(18): 7766–7774.

36.

Zhang

Huang

. MiRNA-133a is involved in the regulation of postmenopausal osteoporosis through promoting osteoclast differentiation. Acta Biochim Biophys Sin (Shanghai) 2018; 50(3): 273–280.

37.

Wang

Zhang

Yan

, et al. Design, synthesis and biological evaluation of curcumin analogues as novel LSD1 inhibitors. Bioorg Med Chem Lett 2019; 29: 126683.

38.

Ferdous

Grossniklaus

Boatright

, et al. Characterization of LSD1 expression within the murine eye. Invest Ophthalmol Vis Sci 2019; 60(14): 4619–4631.

39.

Majello

Gorini

Saccà

, et al. Expanding the role of the histone lysine-specific demethylase LSD1 in cancer. Cancers (Basel) 2019; 11(3): E324.

40.

Duan

Cao

Tang

, et al. miRNA-mRNA crosstalk in myocardial ischemia induced by calcified aortic valve stenosis. Aging (Albany NY) 2019; 11(2): 448–466.

41.

Jaber

Zhao

Lukiw

. Alterations in micro RNA-messenger RNA (miRNA-mRNA) coupled signaling networks in sporadic Alzheimer’s disease (AD) hippocampal CA1. J Alzheimers Dis Parkinsonism 2017; 7(2): 312.

42.

Wang

Bao

. Cnidium lactone stimulates osteogenic differentiation of bone marrow mesenchymal stem cells via BMP-2/Smad-signaling cascades mediated by estrogen receptor. Am J Transl Res 2019; 11(8): 4984–4991.

43.

Wang

Xiao

Wang

, et al. Gremlin2 suppression increases the BMP-2-induced osteogenesis of human bone marrow-derived mesenchymal stem cells via the BMP-2/Smad/Runx2 signaling pathway. J Cell Biochem 2017; 118(2): 286–297.

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