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miR-342-5p inhibits expression of Bmp7 to regulate proliferation, differentiation and migration of osteoblasts
Molecular Immunology 114 (2019) 251–259
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
miR-342-5p inhibits expression of Bmp7 to regulate proliferation, differentiation and migration of osteoblasts Xuesen Lia, Kang Lib, Guisheng Yub, Chuandong Yub, Chuanan Liub, a b
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Department of Orthopedics, The Affiliated Hospital of Qingdao University, Qingdao 266555, Shandong, China Department of Orthopedics, Heze Municipal Hospital, Heze 274031, Shandong, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fracture healing Osteoblasts microRNA-342-5p Bmp7 MEK/ERK
Background: Fracture healing is a complex process, and patients with fracture will undergo non-union or compromised regeneration. MicroRNA (miR)-342-5p is a Notch downstream molecule, and its roles in fracture healing remain unclear. We aimed to explore the functional roles of miR-342-5p in osteoblasts as well as the underlying mechanisms. Methods: The expression of miR-342-5p in differentiation of MC3T3-E1 cells or hMSCs was examined by quantitative reverse transcription PCR (qRT-PCR). The effects of aberrantly expressed miR-342-5p on cell proliferation, apoptosis, migration, and expressions of proteins associated with proliferation and osteogenic differentiation were determined by Cell Counting Kit-8, trypan blue staining, flow cytometry, Transwell assay, Western blot and qRT-PCR assays, respectively. The downstream factor and the target genes of miR-342-5p as well as the involvements of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were finally assessed. Results: miR-342-5p level was decreased during differentiation of MC3T3-E1 cells or hMSCs. After cell transfection, miR-342-5p overexpression significantly reduced cell viability, induced apoptosis, inhibited proliferation, migration and differentiation, and down-regulated Bmp7 expression. Subsequent experiments showed the effects of miR-342-5p inhibition on MC3T3-E1 cells were abrogated by Bmp7 knockdown. Additionally, COL4A6 and Bmp2 were predicated as target genes of miR-342-5p. Finally, phosphorylated levels of MEK and ERK were increased by miR-342-5p inhibition via up-regulating Bmp7 expression. Conclusion: miR-342-5p inhibition promoted proliferation, migration and differentiation of osteoblasts via regulating Bmp7, along with activation of the MEK/ERK pathway.
1. Introduction When the external force exceeds the bone tolerance, a complete or incomplete break is occurred in the continuity of the bone, resulting in fracture healing (Xiao et al., 2017). Bone fracture, frequently occurred in children, elderly populations and patients with osteoporosis, is one of the most traumatic injuries in human (Lloret et al., 2016). Despite of the non-union which accounts for 10%, many patients with fracture suffers compromised regeneration (Hankenson et al., 2015). Moreover, the possibility of delayed union and non-union is raised with age, metabolic conditions and trauma severity (Hankenson et al., 2015). With this status quo, improving healing process of fracture is of substantial benefit to patients and may significantly lower the social burden. MicroRNAs (miRNAs/miRs), single-stranded non-coding RNAs (approximately 22–24 nucleotides), are identified to participate in diverse
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biological and pathological processes via modulating gene expression at posttranscriptional level (Tsochandaridis et al., 2015). Recently, more and more miRNAs are proved to be involved in the process of fracture repair. For example, miR-92a silence could improve fracture healing through enhancing angiogenesis in young mice (Murata et al., 2014). Bone formation was promoted by miR-503 via suppression of Smurf1 expression (Sun et al., 2017). MiR-342-5p is reported to be included in the imprinted 14q32 miRNA cluster, and acts as an innovative Notch downstream molecule (Snowhite et al., 2017; Yan et al., 2016). The Notch pathway is an important pathway in fracture healing, and it regulates embryological bone development and many process recapitulated in fracture repair (Dishowitz et al., 2013; Matthews et al., 2014). All the observations described above make us hypothesize that miR-342-5p might be involved in the process of fracture healing. The fracture repair is a complex process, in which multiple
Corresponding author at: Department of Orthopedics, Heze Municipal Hospital, No. 2888 Caozhou Road, Heze 274031, Shandong, China. E-mail address: [email protected] (C. Liu).
https://doi.org/10.1016/j.molimm.2019.07.027 Received 10 May 2019; Received in revised form 25 July 2019; Accepted 27 July 2019 0161-5890/ © 2019 Published by Elsevier Ltd.
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Gaithersburg, MD, USA) was used for estimation of MC3T3-E1 or hMSCs cell viability. Briefly, after cell transfection, these transfected cells were collected and transferred to 96-well plates with a density of 5 × 103 cells per well. After incubation for 12, 24, 48 and 96 h at 37 °C, the CCK-8 solution (10 μL) was added into the culture medium in 96well plates. The mixture was incubated at 37 °C for 1 h, and the absorbance at 450 nm was detected using a Microplate Reader (Bio-Rad).
processes are temporally coordinated (Hankenson et al., 2014). Those biological processes include inflammation, intramembranous ossification, chondrogenesis, endochondral ossification and remodeling (Dimitriou et al., 2005). Generally speaking, fracture healing is initiated by inflammation phase, followed by repair phase, and completed by remodeling phase (Sato et al., 2016). As the important cell line in bone tissues, osteoblasts are derived from mesenchymal stem cells and are essential for bone matrix synthesis and bone mineralization (Matic et al., 2016). Osteoblasts are the chief bone-making cells, and reduced osteoblast activity may induce impaired healing of fracture (He et al., 2017). Hence, the alteration of osteoblasts after managements is of great importance for the process of fracture healing. Murine MC3T3-E1 cells are pre-osteoblasts from C57BL/6 mouse calvaria. In our study, we aimed to explore the functional roles of miR342-5p in proliferation, migration and differentiation of MC3T3-E1 cells. As miRNAs are frequently involved in regulation of biological processes through regulating mRNA expression, the downstream molecules of miR-342-5p were studied. Moreover, the involved signaling cascades were also investigated.
2.5. Trypan blue staining Trypan blue staining was performed to count MC3T3-E1 cells or hMSCs in the supernatant. In brief, 1 × 105 cells were seeded in 60-mm dishes. After transfection for 48 h at 37 °C, these cells were collected, and stained with 0.4% trypan blue (Invitrogen, Carlsbad, CA, USA) for 3 min. The number of the cells was measured by a microscope using a hemocytometer (Hausser Scientific, Horsham, PA). 2.6. Cell apoptosis assay The apoptosis radio of MC3T3-E1 cells or hMSCs was determined using Annexin V-FITC/PI apoptosis detection kit (Beijing Biosea Biotechnology, Beijing, China). In brief, MC3T3-E1 cells or hMSCs were cultured in 6 well-plates, and were transfected with miR-342-5p mimic, miR-342-5p inhibitor and corresponding controls. After transfection for 48 h, these cells were washed twice with phosphate buffered saline (PBS, Sigma-Aldrich) and re-suspended in buffer. Afterward, 10 μL Annexin V-FITC and 5 μL PI were added to stain cells for 15 min at room temperature in the dark. Cell apoptosis was directly assessed by a FACS can (Beckman Coulter, Fullerton, CA, USA) and analyzed by the FlowJo software (Treestar, Ashland, OR, USA).
2. Materials and methods 2.1. Cell culture and differentiation induction Murine MC3T3-E1 cells (China Center for Type Culture Collection, Wuhan, China) were routinely grown in α-Minimum Essential Medium (α-MEM; HyClone, South Logan, UT, USA) containing 10% (v/v) fetal bovine serum (FBS; Gibco) and 1% penicillin and streptomycin (HyClone). Human stromal (skeletal, mesenchymal) stem cells (hMSCs) were procured from human bone marrow aspirate and hMSC-TERT cell line, as well as the cells culture referred to the introduction of Chen et al. (Chen et al., 2014). Cell culture was carried out in a humidified incubator at 37 °C with 5% CO2. Culture media was refreshed thrice a week. At approximately 80% confluence, in vitro differentiation was induced by cell maintaining in osteogenic differentiation media that consisted of α-MEM, 10% FBS, 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid (both Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin and streptomycin. Osteogenic differentiation was lasted for 28 days.
2.7. Cell migration assay in vitro cell migration of MC3T3-E1 cells or hMSCs was examined by using the Tranwell chamber (Costar, Corning, NY, USA) with polycarbonate membrane inserts (8-μm pore size). In brief, 5 × 105 cells suspended in 200 μL FBS-free medium were plated onto the upper chamber. Meantime, the lower chamber was filled up with 600 μL complete medium. The Tranwell chamber containing cells was subject to a humidified incubator at 37 °C for 24 h, and the cells on the upper membrane surface were discarded using a cotton swab. The cells that migrated to the lower side of the insert were fixed by methanol and stained in crystal violet. Migrated cells were counted under a fluorescence microscope (Leica, Wetzlar, Germany) in five randomly chosen fields.
2.2. Clinical sample selection The fracture and healthy individuals were selected from Heze Municipal Hospital, and utilized in this research. The fracture patients include hand fracture (n = 20, aging from 25 to 56, 13 males and 7 females) and intra-articular calcaneal fracture (n = 16, aging from 24 to 59, 10 males and 6 females). The healthy controls (HCs) (n = 20, aging from 24 to 59, 12 males and 8 females) served as control group. The blood samples were achieved from these fracture patients and healthy individuals. Then, RT-qPCR assay was carried out for the determination of miR-342-5p expression after surgery for day 7, 14, 21 and 28. The Medical Ethics Committee of the Heze Municipal Hospital supported this research.
2.8. Detection of alkaline phosphatase (ALP) activity After transfection, MC3T3-E1 cells or hMSCs were cultured into 24well plates, and 200 μL of lysis buffer was added to the culture plate and kept on ice for 5 min. Subsequently, these cells were centrifuged at 1000 × g for 10 min at 4 °C. ALP activity was detected by using a LabAssayTM ALP kit (Wako PureChemical Industries Ltd., Osaka, Japan) according to the kit instruction. The absorbance of each well was measured with a Microplate reader (Bio-Rad, Hercules, CA, USA) at 405 nm.
2.3. Transient cell transfection MiR-342-5p mimic, its control (scramble miRNA), miR-342-5p inhibitor, its negative control (NC), and bone morphogenetic protein 7 (Bmp7)-specific small interfering RNA (si-Bmp7) were all purchased from GenePharma Company (Shanghai, China). Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) was utilized for cell transfection following the manufacturer’s suggestions.
2.9. Dual luciferase reporter assay Luciferase reporter plasmids containing 3′untranslated region (3′UTR) of COL4A6 and Bmp2 and empty luciferase vectors were obtained from Promega (Fitchburg, WI, USA). These vectors were cotransfected with miR-342-5p mimic or its control into cells using Lipofectamine 2000 (Invitrogen). Luciferase activity was analyzed via exploiting Dual-Luciferase Reporter Assay System (Berthold Centro, USA) after 48 h post-transfection following the manufacturer’s
2.4. Cell viability assay A Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, 252
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Fig. 1. MicroRNA (miR)-342-5p is down-regulated during differentiation of MC3T3-E1 cells. A. Osteogenic differentiation of MC3T3-E1 cells was induced by incubation in complete culture medium containing 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid. Expression of miR-342-5p was examined by quantitative reverse transcription PCR at 0, 4, 7, 14, 21 and 28 days post-induction. B. The 36 fracture patients and 20 healthy individuals were selected, and divided into three groups, such as hand fracture (n = 20), intra-articular calcaneal fracture (n = 16) and healthy controls (HCs) (n = 20). The quantitative reverse transcription PCR assay was then executed for the determination of miR-342-5p expression after surgery for day 7, 14, 21 and 28.
IgG, ab205718, HRP-conjugated goat anti-mouse IgG, ab6708, Abcam) for 2 h at room temperature. Proteins in the PVDF membranes were detected by an enhanced chemiluminescence (ECL) assay kit (Amersham Biosciences, Pittsburgh, PA, USA).
instructions. 2.10. RNA isolation and quantitative reverse transcription PCR (qRT-PCR) Total cellular RNAs from MC3T3-E1 cells or the blood samples of fracture patients and healthy individuals were extracted using an RNeasy kit (Qiagen, Hilden, Germany) according to the supplier’s instructions. Complementary DNA (cDNA) from miRNAs and total RNAs were respectively synthesized using a TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Scientific, Pittsburgh, PA, USA) and a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific), according to the respective instructions. For quantification of miR-342-5p level, real-time PCR was carried out using the TaqMan Universal Master Mix II (Thermo Scientific). A DyNAmo HS SYBR Green qPCR Kit (Thermo Scientific) was utilized for measurements of mRNA expressions (Runx2, Ocn, Bmp7, COL4A6 and Bmp2) following the supplier’s protocol. The housekeeping genes U6 and GAPDH were used as internal standard for miR-342-5p and mRNAs, respectively. The miR-342-5p primers were 5′-CGG AGG GGT GCT ATC TGT GAT TGA G-3′. The U6 snRNA primers were 5′-GCT TCG GCA GCA CAT ATA CTA AAA T-3′ (sense) and 5′-CGC TTC ACG AAT TTG CGT GTC AT-3′ (antisense). The GAPDH mRNA primers were 5′-GCA CCG TCA CGG CTG AGA AC-3′ (sense) and 5′-TGG TGA AGA CGC CAG TGG A-3′ (antisense). Relative expression was calculated according to the 2−ΔΔCt method (Livak and Schmittgen, 2001).
2.12. Statistical analysis Each experiment was carried out in triplicate. Results were presented as the mean ± standard deviation (SD). Statistical analysis was performed using Graphpad Prism 5 software (GraphPad, San Diego, CA, USA). Statistical significance was determined using analysis of variance (ANOVA) with Bonferroni post-hoc test. A P < 0.05 indicated statistically significant.
3. Results 3.1. Level of miR-342-5p was decreased during differentiation of MC3T3E1 cells Prior to studying the roles of miR-342-5p in MC3T3-E1 cells, alteration of miR-342-5p expression during differentiation of MC3T3-E1 cells was examined. As evidence form Fig. 1, miR-342-5p level was arrestingly decreased during osteogenic differentiation of MC3T3-E1 cells (Fig. 1A). To further elucidate the role of miR-342-5p in fracture healing, we included fracture and healthy individuals. We discovered that miR-342-5p expression level was clearly enhanced in hand fracture and intra-articular calcaneal fracture groups after surgery for day 7. However, miR-342-5p expression was gradually declined in fracture groups after surgery for day 14, 21 and 28 (Fig. 1B). These findings suggested the possible involvements of miR-342-5p in fracture healing.
2.11. Western blot analysis Proteins of MC3T3-E1 cells were extracted using RIPA buffer supplemented with 1% phenylmethylsulfonyl fluoride (both Thermo Scientific). After quantification, protein samples (≃30 μg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane and blocked with nonfat milk powder (5%) in Tris-buffered saline containing 0.05% Tween-20 (TBST). Then, PVDF membranes were incubated with appropriate primary antibody against proliferating cell nuclear antigen (PCNA; ab152112), cyclinA (ab137769), cyclinE1 (ab71535), cyclin-dependent kinase (CDK) 2 (ab32147), cyclinD1 (ab134175), CDK4 (ab137675), runt-related transcription factor-2 (Runx2; ab23981), Bmp7 (ab129156), Bmp2 (ab214821), mitogen-activated protein kinase kinase (MEK; ab178876), phospho (p)-MEK (ab194754), extracellular signal-regulated kinase (ERK; ab115799), pERK (ab214036), GAPDH (ab181603, all Abcam, Cambridge, UK) or osteocalcin (Ocn; orb235048, Biorbyt, Cambridge, UK) and COL4A6 (sc-398655, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at room temperature. After washing with TBST, PVDF membranes were incubated with secondary antibody (HRP-conjugated goat anti-rabbit
3.2. MiR-342-5p was aberrantly expressed in MC3T3-E1 cells or hMSCs MiRNAs were transiently transfected into MC3T3-E1 cells to generate cells aberrantly expressing miR-342-5p. In Fig. 2, miR-342 level in cells transfected with miR-342-5p mimic was significantly higher than that in the Scramble group (P < 0.01). Meanwhile, miR-342-5p level in cells transfected with miR-342-5p inhibitor was markedly lower than that in the NC group in MC3T3-E1 cells (P < 0.01). The same results were presented in hMSCs (P < 0.01 or P < 0.001, Supplementary Fig. 1). Those results observably illustrated that miR-342-5p level was changed after cell transfection.
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CyclinA, CyclinE1, CDK2, CyclinD1 and CDK4 were all observably lowered in cells overexpressing miR-342-5p, and levels of those proteins were all enhanced in cells knocking down miR-342-5p. Additionally, trypan blue staining results showed that the cell numbers of MC3T3-E1 was obviously declined by miR-342-5p overexpression, and increased by miR-342-5p inhibition (P < 0.01, Fig. 3C). Further, flow cytometry assay results displayed that the percentage of apoptotic cells of MC3T3-E1 was significantly induced by miR-342-5p overexpression relative to the Scramble group (P < 0.01, Fig. 3D). In hMSCs, we also discovered that cell viability and cell numbers were both prohibited by miR-342-5p overexpression and accelerated by miR-342-5p inhibition (P < 0.05 or P < 0.01, Supplementary Fig. 2A and B). Further, cell apoptosis was also induced by miR-342-5p overexpression (P < 0.01), and slightly suppressed by miR-342-5p repression (Supplementary Fig. 2C). Those results talked above suggested that miR-342-5p could inhibit MC3T3-E1 and hMSCs cell proliferation. 3.4. MiR-342-5p repressed MC3T3-E1 cells or hMSCs migration
Fig. 2. MicroRNA (miR)-342-5p is aberrantly expressed in MC3T3-E1 cells. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR342-5p inhibitor or its negative control (NC). Non-treated cells were served as control. Expression of miR-342-5p was examined by quantitative reverse transcription PCR. **, P < 0.01.
After cell transfection, migrated cells were counted. As shown in Fig. 4, relative migration of cells overexpressing miR-342-5p was significantly lower than that in the Scramble group, and relative migration of cells knocking down miR-342-5p was dramatically higher than that in the NC group (both P < 0.05). The similar results were showed in Supplementary Fig. 3, which disclosed that cell migration in hMSCs was also prohibited by miR-342-5p overexpression, but expedited by miR342-5p inhibition (P < 0.05 or P < 0.01). Data indicated that miR342-5p could repress MC3T3-E1 cell or hMSCs migration.
3.3. MiR-342-5p inhibited MC3T3-E1 cell or hMSCs proliferation and induced apoptosis After cell transfection, cell viability of MC3T3-E1 cells was measured at 12, 24 48 and 96 h. In Fig. 3A, miR-342-5p overexpression markedly decreased cell viability relative to the Scramble group (P < 0.05 or P < 0.01), and miR-342-5p silenced notably increased cell viability relative to the NC group (P < 0.05 or P < 0.01). Meanwhile, expression of proliferation-related proteins was also examined in cells after cell transfection. In Fig. 3B, levels of PCNA,
3.5. MiR-342-5p suppressed differentiation of MC3T3-E1 cells or hMSCs To investigate the effect of miR-342-5p during differentiation, we measured ALP activity after the transfection with miR-342-5p mimic and miR-342-5p inhibitor. As shown in Fig. 5, ALP activity was
Fig. 3. MicroRNA (miR)-342-5p inhibits MC3T3-E1 cell proliferation and induces apoptosis. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR-342-5p inhibitor or its negative control (NC). Non-treated cells were served as control. A. Cell viability was measured by a Cell Counting Kit-8. B. Expression of proteins associated with proliferation was determined by Western blot analysis. C. Cell numbers were counted by using trypan blue staining. D. Cell apoptosis was examined by flow cytometry. *, P < 0.05; **, P < 0.01; ns, no significance. PCNA, proliferating cell nuclear antigen; CDK, cyclin-dependent kinase. 254
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Fig. 4. MicroRNA (miR)-342-5p represses MC3T3-E1 cell migration. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR-342-5p inhibitor or its negative control (NC). Non-treated cells were served as control. Relative migration was measured by Transwell assay. *, P < 0.05.
might be negatively modulated by miR-342-5p expression. Thus, we hypothesized that miR-342-5p might function through negatively regulating Bmp7. For verification, cells were transfected with NC, miR342-5p inhibitor or miR-342-5p inhibitor plus si-Bmp7. Following results showed miR-342-5p silence-induced increases of cell viability (Fig. 7B), up-regulations of proteins associated with proliferation (Fig. 7C), decrease apoptosis (Fig. 7D), increases migration (Fig. 7E), and osteogenic differentiation (Fig. 7F) were all notably abrogated by Bmp7 silence. Therefore, we concluded that miR-342-5p inhibition might affect MC3T3-E1 cells via up-regulation of Bmp7. Besides of this, we used the bioinformatics software of TargetScan (http://www. targetscan.org/) to find the target genes of miR-342-5p. We found that COL4A6 and Bmp2 3’UTR were both existed targeting sequences (Supplementary Fig. 5A and B). To further confirm whether COL4A6 or Bmp2 is direct target of miR-342-5p, the dual luciferase reporter assay was carried out. Results showed that miR-342-5p overexpression clearly restrained the luciferase activity of reporter genes containing 3’UTR-Wt of COL4A6 or Bmp2 (P < 0.05), but did not affect that of the reporters fused to the mut versions (Supplementary Fig. 5C and D). Further, the mRNA and protein levels of COL4A6 or Bmp2 in miR-324-5p mimic or inhibitor transfected cells were also determined. Results revealed that COL4A6 and Bmp2 mRNA or protein levels were both declined by miR324-5p overexpression (P < 0.05 or P < 0.01), meanwhile enhanced by miR-324-5p suppression (P < 0.01). These relevant data hinted that COL4A6 and Bmp2 were target genes of miR-324-5p.
Fig. 5. MicroRNA (miR)-342-5p inhibits ALP activity in MC3T3-E1 cells. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR342-5p inhibitor or its negative control (NC). Non-treated cells were served as control. ALP activity was detected by using LabAssayTM ALP kit. *, P < 0.05.
significantly suppressed in miR-342-5p mimic-transfecting MC3T3-E1 cells, and promoted in miR-342-5p inhibitor-transfecting MC3T3-E1 cells. Likewise, ALP activity was also restrained in miR-342-5p mimictransfecting hMSCs (P < 0.01), and aggrandized in miR-342-5p inhibitor-transfecting hMSCs (P < 0.05, Supplementary Fig. 4). In addition, the expression of proteins associated with osteogenic differentiation was measured after transfection. When compared with respective controls, expressions of Runx2 and Ocn at mRNA and protein levels were remarkably down-regulated by miR-342-5p overexpression while were notably up-regulated by miR-342-5p silence (all P < 0.05, Fig. 6A–B). Data illustrated that miR-342-5p could suppress differentiation of MC3T3-E1 cells.
3.7. MiR-342-5p silence activated the MEK/ERK pathway through upregulating Bmp7 MC3T3-E1 cells were transfected with NC, miR-342-5p inhibitor or miR-342-5p inhibitor plus si-Bmp7, and the involvements of the MEK/ ERK pathway in the regulation of miR-342-5p were finally explored. In Fig. 8, expressions of p-MEK and p-ERK were both elevated by miR-3425p inhibition, and these elevations were abrogated when Bmp7 was knocked down. Results implied that miR-342-5p silence might activate the MEK/ERK pathway through up-regulating Bmp7 expression.
3.6. MiR-342-5p inhibition promoted proliferation, migration and differentiation via up-regulating Bmp7 expression
4. Discussion
Subsequent experiments were performed to explore whether Bmp7 was a downstream factor of miR-342-5p. Results in Fig. 7A showed Bmp7 mRNA and protein expressions were significantly down-regulated by miR-342-5p overexpression while were markedly up-regulated by miR-342-5p silence (P < 0.05), suggesting that Bmp7 expression
This study reported for the first time the effects and mechanism of miR-342-5p on osteoblasts, suggesting the possible therapeutic function for fracture. Before investigations, we figured out that miR-342-5p level was arrestingly decreased. Then, after aberrant expression of miR-3425p in MC3T3-E1 cells or hMSCs utilizing miRNAs, we interestingly 255
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Fig. 6. MicroRNA (miR)-342-5p suppresses differentiation of MC3T3-E1 cells. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR342-5p inhibitor or its negative control (NC). Osteogenic differentiation of transfected and un-transfected cells was induced by incubation in complete culture medium containing 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid. Non-treated cells were served as control. mRNA expressions A. and protein expressions B. of Runx2 and Ocn were determined by quantitative reverse transcription PCR and Western blot analysis, respectively. *, P < 0.05. Runx2, runt-related transcription factor-2; Ocn, osteocalcin.
study, miR-342-5p silence increased the levels of those proteins associated with proliferation, which suggested that miR-342-5p silence could promote MC3T3-E1 cells or hMSCs proliferation. Osteoblast migration plays pivotal roles in modeling phase of fracture healing, and elevated osteoblast migration leads to acceleration of bone formation and repair (Fukuyama et al., 2004). After fracture, preosteoblasts are recruited and migrated to the site of bone formation (Thiel et al., 2018). Lack of osteoblast migration has been proved to repress fracture repair (Aryal et al., 2015). Therefore, the effects of miR342-5p on MC3T3-E1 cell migration were next explored. Transwell assay showed miR-342-5p repressed MC3T3-E1 cells or hMSCs migration, which was consistent with a previous literature by Yang et al. (Yang et al., 2016). Mature osteoblasts are crucial for manufacture and mineralization of the osteoid matrix (Khosla et al., 2008; Maes et al., 2007). Maintenance of osteoblast differentiation and activity is pivotal for restoration of skeletal function (Claes et al., 2012). In addition, osteoblasts must proliferate and differentiate, and thereby induce mineralization. Therefore, we next determined the effects of miR-342-5p on osteoblast differentiation. Ocn is the most abundant non-collagenous protein in the bone extracellular, which acts as a late phenotypic osteoblast marker (Fang and Yamaza, 2016; Otsuki et al., 2018). It is synthesized by osteoblasts around the time bone mineralization begins (Karsenty and Olson, 2016). As an essential regulator of osteoblast differentiation and bone formation, Runx2 is reported to trigger Ocn expression via binding to the promoter region of Ocn (Vimalraj et al., 2015). In our study, to confirm the effect of miR-342-5p on osteoblast differentiation, we examined ALP activity in MC3T3-E1 cells after transfection with miR-342-5p mimic and miR-342-5p inhibitor, and results revealed that miR-342-5p overexpression significantly inhibited ALP activity in MC3T3-E1 cells or hMSCs. Further, we found that miR-342-5p overexpression-induced down-regulations of Runx2 and Ocn. These data illustrated that miR-342-5p could repress osteoblast differentiation. Bmp7 (35 kDa) belongs to the transforming growth factor (TGF)-β superfamily, which is a direct inducer of osteoblast differentiation
found that miR-342-5p could inhibit proliferation, migration, differentiation and induced apoptosis. After that, Bmp7 expression was identified to be negatively regulated by miR-342-5p expression, and Bmp7 inhibition could abrogate the alterations of proliferation, apoptosis, migration and differentiation, induced by miR-342-5p inhibition. Additionally, COL4A6 and Bmp2 were predicated as target genes of miR-342-5p. The final Western blot results illustrated that the MEK/ ERK pathway was activated by miR-342-5p silence through up-regulating Bmp7. Several miRNAs are dysregulated at post-fracture days in a literature by Waki et al., and these miRNAs are supposed to play important roles in fracture healing (Waki et al., 2015). Another study also detected the expression of several miRNAs and concluded that these miRNAs might be targets to optimize fracture healing on the basis of the dysregulation after fracture (Seeliger et al., 2014). Therefore, in our study, we measured the expression of miR-342-5p in differentiating osteoblast MC3T3-E1 cells or hMSCs prior to detailed investigations. Results of qRT-PCR showed observable down-regulation of miR-342-5p, strongly indicating that miR-342-5p might be involved in the process of fracture repair. Rapid bone matrix synthesis and mineralization by osteoblasts are observed during fracture healing, indicating osteoblast activity is of great importance for bone repair (Tu et al., 2017). Therefore, we measured the effects of miR-342-5p on MC3T3-E1 cells or hMSCs viability and proliferation. After transfection, cell viability was reduced by miR-342-5p overexpression but was enhanced by miR-342-5p silence, illustrating the helpful effects of miR-342-5p silence on fracture healing. PCNA is a marker of proliferating cells and acts as an auxiliary protein for DNA polymerases (Tsai et al., 2017). CyclinA and CyclinE1 are subunits of CDK2, and binding of CyclinA and CyclinE1 to CDK2 can phosphorylate proteins associated with DNA synthesis, resulting in enhanced proliferation (Arooz et al., 2000). Likewise, cyclinD1 activates the CDK4/6 and thereby phosphorylates tumor suppressor gene retinoblastoma protein, allowing expressions of genes required for transition through the G1 restriction point (Gelbert et al., 2014). In our 256
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Fig. 7. MicroRNA (miR)-342-5p inhibition affects MC3T3-E1 cells through up-regulating bone morphogenetic protein 7 (Bmp7). A. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR-342-5p inhibitor or its negative control (NC), and the expression of Bmp7 was determined by quantitative reverse transcription PCR and Western blot analysis. Non-treated cells were served as control. B–E. MC3T3-E1 cells were transfected with NC, miR-342-5p inhibitor or miR-342-5p inhibitor plus si-Bmp7. Non-treated cells were served as control. Cell viability (B), expression of proteins associated with proliferation (C), apoptosis (D), and migration (E) were assessed by Cell Counting Kit-8 assay, Western blot analysis, flow cytometry and Transwell assay, respectively. F. MC3T3-E1 cells were transfected with NC, miR-342-5p inhibitor or miR-342-5p inhibitor plus si-Bmp7. Differentiation was induced by incubation in complete culture medium containing 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid. Non-treated cells were served as control. Protein expressions of Runx2 and Ocn at 14 days post-induction were detected by Western blot analysis. *, P < 0.05; **, P < 0.01. PCNA, proliferating cell nuclear antigen; CDK, cyclin-dependent kinase; Runx2, runt-related transcription factor-2; Ocn, osteocalcin; si-Bmp7, small interfering RNA targeting Bmp7.
instance, cell adhesion, migration, survival, differentiation, proliferation, and etc. (Roskoski, 2012). The MEK/ERK pathway can affect osteogenesis through regulating Runx2 phosphorylation and transcriptional activity (Jun et al., 2010). A previous study has also proved that Bmp7 stimulates proliferation and cadherin-11 expression through ERK in a murine metanephric mesenchymal cell line (Awazu et al., 2017). Therefore, we finally explored the involvements of the MEK/ERK pathway in the modulation of miR-342-5p in MC3T3-E1 cells. Results in our study illustrated the miR-342-5p inhibition could inhibit the MEK/ ERK pathway through up-regulating Bmp7 expression. 5. Conclusions To summarize, miR-342-5p was identified for the first time to be down-regulated during osteoblast differentiation. Then, we interestingly found miR-342-5p silence could promote proliferation, migration and differentiation of MC3T3-E1 cells through up-regulating Bmp7. Moreover, the MEK/ERK pathway was involved in the regulatory mechanism of miR-342-5p. The distinct effects of miR-342-5p may provide novel therapeutic targets for fracture healing. Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Fig. 8. MicroRNA (miR)-342-5p inhibition might activate the MEK/ERK pathway through up-regulating morphogenetic protein 7 (Bmp7) in MC3T3-E1 cells. MC3T3-E1 cells were transfected with NC, miR-342-5p inhibitor or miR-342-5p inhibitor plus si-Bmp7. Non-treated cells were served as control. Expression of key kinases in the MEK/ERK pathway was measured by Western blot analysis. si-Bmp7, small interfering RNA targeting Bmp7; t-, total; p-, phospho-; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase.
Declaration of Competing Interest Authors declare that there is no conflict of interests. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.molimm.2019.07.027.
(Bragdon et al., 2011). It can up-regulate Runx2 in mice and promote bone formation (Bustos-Valenzuela et al., 2011). Yan et al. have reported that miR-342-5p could regulate TGF-β signaling pathway (Yan et al., 2016). Another literature by Kurrel et al. has reported that miR542-3p can repress proliferation and differentiation of osteoblasts through regulating Bmp7 (Kureel et al., 2014). Therefore, we supposed that Bmp7 might be a downstream molecule of miR-342-5p. Accordingly, we found miR-342-5p expression was negatively correlated with Bmp7 expression. Moreover, subsequent experiments proved that Bmp7 silence could reverse the effects of miR-342-5p inhibition on proliferation, migration and differentiation, verifying that miR-342-5p inhibition might function through up-regulating Bmp7 expression. We then used the bioinformatics software of starbase (http://starbase.sysu. edu.cn/) to seek the complementary seed sequence between 3’UTR of Bmp7 and miR-324. We discovered that there was a targeted binding site between Bmp7 and miR-342-3p, but no targeted binding site between Bmp7 and miR-342-5p. To further seek the target genes, the TargetScan (http://www.targetscan.org/) and dual luciferase reporter assay were carried. Finally, COL4A6 and Bmp2 were predicated as target genes of miR-342-5p. However, whether COL4A6 and Bmp2 joins in affect the functions of miR-342-5p in osteoblasts is still needed further research. The MEK/ERK cascade participates in several cellular processes, for
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
miR-342-5p inhibits expression of Bmp7 to regulate proliferation, differentiation and migration of osteoblasts Xuesen Lia, Kang Lib, Guisheng Yub, Chuandong Yub, Chuanan Liub, a b
T
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Department of Orthopedics, The Affiliated Hospital of Qingdao University, Qingdao 266555, Shandong, China Department of Orthopedics, Heze Municipal Hospital, Heze 274031, Shandong, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fracture healing Osteoblasts microRNA-342-5p Bmp7 MEK/ERK
Background: Fracture healing is a complex process, and patients with fracture will undergo non-union or compromised regeneration. MicroRNA (miR)-342-5p is a Notch downstream molecule, and its roles in fracture healing remain unclear. We aimed to explore the functional roles of miR-342-5p in osteoblasts as well as the underlying mechanisms. Methods: The expression of miR-342-5p in differentiation of MC3T3-E1 cells or hMSCs was examined by quantitative reverse transcription PCR (qRT-PCR). The effects of aberrantly expressed miR-342-5p on cell proliferation, apoptosis, migration, and expressions of proteins associated with proliferation and osteogenic differentiation were determined by Cell Counting Kit-8, trypan blue staining, flow cytometry, Transwell assay, Western blot and qRT-PCR assays, respectively. The downstream factor and the target genes of miR-342-5p as well as the involvements of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were finally assessed. Results: miR-342-5p level was decreased during differentiation of MC3T3-E1 cells or hMSCs. After cell transfection, miR-342-5p overexpression significantly reduced cell viability, induced apoptosis, inhibited proliferation, migration and differentiation, and down-regulated Bmp7 expression. Subsequent experiments showed the effects of miR-342-5p inhibition on MC3T3-E1 cells were abrogated by Bmp7 knockdown. Additionally, COL4A6 and Bmp2 were predicated as target genes of miR-342-5p. Finally, phosphorylated levels of MEK and ERK were increased by miR-342-5p inhibition via up-regulating Bmp7 expression. Conclusion: miR-342-5p inhibition promoted proliferation, migration and differentiation of osteoblasts via regulating Bmp7, along with activation of the MEK/ERK pathway.
1. Introduction When the external force exceeds the bone tolerance, a complete or incomplete break is occurred in the continuity of the bone, resulting in fracture healing (Xiao et al., 2017). Bone fracture, frequently occurred in children, elderly populations and patients with osteoporosis, is one of the most traumatic injuries in human (Lloret et al., 2016). Despite of the non-union which accounts for 10%, many patients with fracture suffers compromised regeneration (Hankenson et al., 2015). Moreover, the possibility of delayed union and non-union is raised with age, metabolic conditions and trauma severity (Hankenson et al., 2015). With this status quo, improving healing process of fracture is of substantial benefit to patients and may significantly lower the social burden. MicroRNAs (miRNAs/miRs), single-stranded non-coding RNAs (approximately 22–24 nucleotides), are identified to participate in diverse
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biological and pathological processes via modulating gene expression at posttranscriptional level (Tsochandaridis et al., 2015). Recently, more and more miRNAs are proved to be involved in the process of fracture repair. For example, miR-92a silence could improve fracture healing through enhancing angiogenesis in young mice (Murata et al., 2014). Bone formation was promoted by miR-503 via suppression of Smurf1 expression (Sun et al., 2017). MiR-342-5p is reported to be included in the imprinted 14q32 miRNA cluster, and acts as an innovative Notch downstream molecule (Snowhite et al., 2017; Yan et al., 2016). The Notch pathway is an important pathway in fracture healing, and it regulates embryological bone development and many process recapitulated in fracture repair (Dishowitz et al., 2013; Matthews et al., 2014). All the observations described above make us hypothesize that miR-342-5p might be involved in the process of fracture healing. The fracture repair is a complex process, in which multiple
Corresponding author at: Department of Orthopedics, Heze Municipal Hospital, No. 2888 Caozhou Road, Heze 274031, Shandong, China. E-mail address: [email protected] (C. Liu).
https://doi.org/10.1016/j.molimm.2019.07.027 Received 10 May 2019; Received in revised form 25 July 2019; Accepted 27 July 2019 0161-5890/ © 2019 Published by Elsevier Ltd.
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Gaithersburg, MD, USA) was used for estimation of MC3T3-E1 or hMSCs cell viability. Briefly, after cell transfection, these transfected cells were collected and transferred to 96-well plates with a density of 5 × 103 cells per well. After incubation for 12, 24, 48 and 96 h at 37 °C, the CCK-8 solution (10 μL) was added into the culture medium in 96well plates. The mixture was incubated at 37 °C for 1 h, and the absorbance at 450 nm was detected using a Microplate Reader (Bio-Rad).
processes are temporally coordinated (Hankenson et al., 2014). Those biological processes include inflammation, intramembranous ossification, chondrogenesis, endochondral ossification and remodeling (Dimitriou et al., 2005). Generally speaking, fracture healing is initiated by inflammation phase, followed by repair phase, and completed by remodeling phase (Sato et al., 2016). As the important cell line in bone tissues, osteoblasts are derived from mesenchymal stem cells and are essential for bone matrix synthesis and bone mineralization (Matic et al., 2016). Osteoblasts are the chief bone-making cells, and reduced osteoblast activity may induce impaired healing of fracture (He et al., 2017). Hence, the alteration of osteoblasts after managements is of great importance for the process of fracture healing. Murine MC3T3-E1 cells are pre-osteoblasts from C57BL/6 mouse calvaria. In our study, we aimed to explore the functional roles of miR342-5p in proliferation, migration and differentiation of MC3T3-E1 cells. As miRNAs are frequently involved in regulation of biological processes through regulating mRNA expression, the downstream molecules of miR-342-5p were studied. Moreover, the involved signaling cascades were also investigated.
2.5. Trypan blue staining Trypan blue staining was performed to count MC3T3-E1 cells or hMSCs in the supernatant. In brief, 1 × 105 cells were seeded in 60-mm dishes. After transfection for 48 h at 37 °C, these cells were collected, and stained with 0.4% trypan blue (Invitrogen, Carlsbad, CA, USA) for 3 min. The number of the cells was measured by a microscope using a hemocytometer (Hausser Scientific, Horsham, PA). 2.6. Cell apoptosis assay The apoptosis radio of MC3T3-E1 cells or hMSCs was determined using Annexin V-FITC/PI apoptosis detection kit (Beijing Biosea Biotechnology, Beijing, China). In brief, MC3T3-E1 cells or hMSCs were cultured in 6 well-plates, and were transfected with miR-342-5p mimic, miR-342-5p inhibitor and corresponding controls. After transfection for 48 h, these cells were washed twice with phosphate buffered saline (PBS, Sigma-Aldrich) and re-suspended in buffer. Afterward, 10 μL Annexin V-FITC and 5 μL PI were added to stain cells for 15 min at room temperature in the dark. Cell apoptosis was directly assessed by a FACS can (Beckman Coulter, Fullerton, CA, USA) and analyzed by the FlowJo software (Treestar, Ashland, OR, USA).
2. Materials and methods 2.1. Cell culture and differentiation induction Murine MC3T3-E1 cells (China Center for Type Culture Collection, Wuhan, China) were routinely grown in α-Minimum Essential Medium (α-MEM; HyClone, South Logan, UT, USA) containing 10% (v/v) fetal bovine serum (FBS; Gibco) and 1% penicillin and streptomycin (HyClone). Human stromal (skeletal, mesenchymal) stem cells (hMSCs) were procured from human bone marrow aspirate and hMSC-TERT cell line, as well as the cells culture referred to the introduction of Chen et al. (Chen et al., 2014). Cell culture was carried out in a humidified incubator at 37 °C with 5% CO2. Culture media was refreshed thrice a week. At approximately 80% confluence, in vitro differentiation was induced by cell maintaining in osteogenic differentiation media that consisted of α-MEM, 10% FBS, 10 mM β-glycerophosphate, 50 μg/mL ascorbic acid (both Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin and streptomycin. Osteogenic differentiation was lasted for 28 days.
2.7. Cell migration assay in vitro cell migration of MC3T3-E1 cells or hMSCs was examined by using the Tranwell chamber (Costar, Corning, NY, USA) with polycarbonate membrane inserts (8-μm pore size). In brief, 5 × 105 cells suspended in 200 μL FBS-free medium were plated onto the upper chamber. Meantime, the lower chamber was filled up with 600 μL complete medium. The Tranwell chamber containing cells was subject to a humidified incubator at 37 °C for 24 h, and the cells on the upper membrane surface were discarded using a cotton swab. The cells that migrated to the lower side of the insert were fixed by methanol and stained in crystal violet. Migrated cells were counted under a fluorescence microscope (Leica, Wetzlar, Germany) in five randomly chosen fields.
2.2. Clinical sample selection The fracture and healthy individuals were selected from Heze Municipal Hospital, and utilized in this research. The fracture patients include hand fracture (n = 20, aging from 25 to 56, 13 males and 7 females) and intra-articular calcaneal fracture (n = 16, aging from 24 to 59, 10 males and 6 females). The healthy controls (HCs) (n = 20, aging from 24 to 59, 12 males and 8 females) served as control group. The blood samples were achieved from these fracture patients and healthy individuals. Then, RT-qPCR assay was carried out for the determination of miR-342-5p expression after surgery for day 7, 14, 21 and 28. The Medical Ethics Committee of the Heze Municipal Hospital supported this research.
2.8. Detection of alkaline phosphatase (ALP) activity After transfection, MC3T3-E1 cells or hMSCs were cultured into 24well plates, and 200 μL of lysis buffer was added to the culture plate and kept on ice for 5 min. Subsequently, these cells were centrifuged at 1000 × g for 10 min at 4 °C. ALP activity was detected by using a LabAssayTM ALP kit (Wako PureChemical Industries Ltd., Osaka, Japan) according to the kit instruction. The absorbance of each well was measured with a Microplate reader (Bio-Rad, Hercules, CA, USA) at 405 nm.
2.3. Transient cell transfection MiR-342-5p mimic, its control (scramble miRNA), miR-342-5p inhibitor, its negative control (NC), and bone morphogenetic protein 7 (Bmp7)-specific small interfering RNA (si-Bmp7) were all purchased from GenePharma Company (Shanghai, China). Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) was utilized for cell transfection following the manufacturer’s suggestions.
2.9. Dual luciferase reporter assay Luciferase reporter plasmids containing 3′untranslated region (3′UTR) of COL4A6 and Bmp2 and empty luciferase vectors were obtained from Promega (Fitchburg, WI, USA). These vectors were cotransfected with miR-342-5p mimic or its control into cells using Lipofectamine 2000 (Invitrogen). Luciferase activity was analyzed via exploiting Dual-Luciferase Reporter Assay System (Berthold Centro, USA) after 48 h post-transfection following the manufacturer’s
2.4. Cell viability assay A Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, 252
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Fig. 1. MicroRNA (miR)-342-5p is down-regulated during differentiation of MC3T3-E1 cells. A. Osteogenic differentiation of MC3T3-E1 cells was induced by incubation in complete culture medium containing 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid. Expression of miR-342-5p was examined by quantitative reverse transcription PCR at 0, 4, 7, 14, 21 and 28 days post-induction. B. The 36 fracture patients and 20 healthy individuals were selected, and divided into three groups, such as hand fracture (n = 20), intra-articular calcaneal fracture (n = 16) and healthy controls (HCs) (n = 20). The quantitative reverse transcription PCR assay was then executed for the determination of miR-342-5p expression after surgery for day 7, 14, 21 and 28.
IgG, ab205718, HRP-conjugated goat anti-mouse IgG, ab6708, Abcam) for 2 h at room temperature. Proteins in the PVDF membranes were detected by an enhanced chemiluminescence (ECL) assay kit (Amersham Biosciences, Pittsburgh, PA, USA).
instructions. 2.10. RNA isolation and quantitative reverse transcription PCR (qRT-PCR) Total cellular RNAs from MC3T3-E1 cells or the blood samples of fracture patients and healthy individuals were extracted using an RNeasy kit (Qiagen, Hilden, Germany) according to the supplier’s instructions. Complementary DNA (cDNA) from miRNAs and total RNAs were respectively synthesized using a TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Scientific, Pittsburgh, PA, USA) and a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific), according to the respective instructions. For quantification of miR-342-5p level, real-time PCR was carried out using the TaqMan Universal Master Mix II (Thermo Scientific). A DyNAmo HS SYBR Green qPCR Kit (Thermo Scientific) was utilized for measurements of mRNA expressions (Runx2, Ocn, Bmp7, COL4A6 and Bmp2) following the supplier’s protocol. The housekeeping genes U6 and GAPDH were used as internal standard for miR-342-5p and mRNAs, respectively. The miR-342-5p primers were 5′-CGG AGG GGT GCT ATC TGT GAT TGA G-3′. The U6 snRNA primers were 5′-GCT TCG GCA GCA CAT ATA CTA AAA T-3′ (sense) and 5′-CGC TTC ACG AAT TTG CGT GTC AT-3′ (antisense). The GAPDH mRNA primers were 5′-GCA CCG TCA CGG CTG AGA AC-3′ (sense) and 5′-TGG TGA AGA CGC CAG TGG A-3′ (antisense). Relative expression was calculated according to the 2−ΔΔCt method (Livak and Schmittgen, 2001).
2.12. Statistical analysis Each experiment was carried out in triplicate. Results were presented as the mean ± standard deviation (SD). Statistical analysis was performed using Graphpad Prism 5 software (GraphPad, San Diego, CA, USA). Statistical significance was determined using analysis of variance (ANOVA) with Bonferroni post-hoc test. A P < 0.05 indicated statistically significant.
3. Results 3.1. Level of miR-342-5p was decreased during differentiation of MC3T3E1 cells Prior to studying the roles of miR-342-5p in MC3T3-E1 cells, alteration of miR-342-5p expression during differentiation of MC3T3-E1 cells was examined. As evidence form Fig. 1, miR-342-5p level was arrestingly decreased during osteogenic differentiation of MC3T3-E1 cells (Fig. 1A). To further elucidate the role of miR-342-5p in fracture healing, we included fracture and healthy individuals. We discovered that miR-342-5p expression level was clearly enhanced in hand fracture and intra-articular calcaneal fracture groups after surgery for day 7. However, miR-342-5p expression was gradually declined in fracture groups after surgery for day 14, 21 and 28 (Fig. 1B). These findings suggested the possible involvements of miR-342-5p in fracture healing.
2.11. Western blot analysis Proteins of MC3T3-E1 cells were extracted using RIPA buffer supplemented with 1% phenylmethylsulfonyl fluoride (both Thermo Scientific). After quantification, protein samples (≃30 μg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane and blocked with nonfat milk powder (5%) in Tris-buffered saline containing 0.05% Tween-20 (TBST). Then, PVDF membranes were incubated with appropriate primary antibody against proliferating cell nuclear antigen (PCNA; ab152112), cyclinA (ab137769), cyclinE1 (ab71535), cyclin-dependent kinase (CDK) 2 (ab32147), cyclinD1 (ab134175), CDK4 (ab137675), runt-related transcription factor-2 (Runx2; ab23981), Bmp7 (ab129156), Bmp2 (ab214821), mitogen-activated protein kinase kinase (MEK; ab178876), phospho (p)-MEK (ab194754), extracellular signal-regulated kinase (ERK; ab115799), pERK (ab214036), GAPDH (ab181603, all Abcam, Cambridge, UK) or osteocalcin (Ocn; orb235048, Biorbyt, Cambridge, UK) and COL4A6 (sc-398655, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at room temperature. After washing with TBST, PVDF membranes were incubated with secondary antibody (HRP-conjugated goat anti-rabbit
3.2. MiR-342-5p was aberrantly expressed in MC3T3-E1 cells or hMSCs MiRNAs were transiently transfected into MC3T3-E1 cells to generate cells aberrantly expressing miR-342-5p. In Fig. 2, miR-342 level in cells transfected with miR-342-5p mimic was significantly higher than that in the Scramble group (P < 0.01). Meanwhile, miR-342-5p level in cells transfected with miR-342-5p inhibitor was markedly lower than that in the NC group in MC3T3-E1 cells (P < 0.01). The same results were presented in hMSCs (P < 0.01 or P < 0.001, Supplementary Fig. 1). Those results observably illustrated that miR-342-5p level was changed after cell transfection.
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CyclinA, CyclinE1, CDK2, CyclinD1 and CDK4 were all observably lowered in cells overexpressing miR-342-5p, and levels of those proteins were all enhanced in cells knocking down miR-342-5p. Additionally, trypan blue staining results showed that the cell numbers of MC3T3-E1 was obviously declined by miR-342-5p overexpression, and increased by miR-342-5p inhibition (P < 0.01, Fig. 3C). Further, flow cytometry assay results displayed that the percentage of apoptotic cells of MC3T3-E1 was significantly induced by miR-342-5p overexpression relative to the Scramble group (P < 0.01, Fig. 3D). In hMSCs, we also discovered that cell viability and cell numbers were both prohibited by miR-342-5p overexpression and accelerated by miR-342-5p inhibition (P < 0.05 or P < 0.01, Supplementary Fig. 2A and B). Further, cell apoptosis was also induced by miR-342-5p overexpression (P < 0.01), and slightly suppressed by miR-342-5p repression (Supplementary Fig. 2C). Those results talked above suggested that miR-342-5p could inhibit MC3T3-E1 and hMSCs cell proliferation. 3.4. MiR-342-5p repressed MC3T3-E1 cells or hMSCs migration
Fig. 2. MicroRNA (miR)-342-5p is aberrantly expressed in MC3T3-E1 cells. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR342-5p inhibitor or its negative control (NC). Non-treated cells were served as control. Expression of miR-342-5p was examined by quantitative reverse transcription PCR. **, P < 0.01.
After cell transfection, migrated cells were counted. As shown in Fig. 4, relative migration of cells overexpressing miR-342-5p was significantly lower than that in the Scramble group, and relative migration of cells knocking down miR-342-5p was dramatically higher than that in the NC group (both P < 0.05). The similar results were showed in Supplementary Fig. 3, which disclosed that cell migration in hMSCs was also prohibited by miR-342-5p overexpression, but expedited by miR342-5p inhibition (P < 0.05 or P < 0.01). Data indicated that miR342-5p could repress MC3T3-E1 cell or hMSCs migration.
3.3. MiR-342-5p inhibited MC3T3-E1 cell or hMSCs proliferation and induced apoptosis After cell transfection, cell viability of MC3T3-E1 cells was measured at 12, 24 48 and 96 h. In Fig. 3A, miR-342-5p overexpression markedly decreased cell viability relative to the Scramble group (P < 0.05 or P < 0.01), and miR-342-5p silenced notably increased cell viability relative to the NC group (P < 0.05 or P < 0.01). Meanwhile, expression of proliferation-related proteins was also examined in cells after cell transfection. In Fig. 3B, levels of PCNA,
3.5. MiR-342-5p suppressed differentiation of MC3T3-E1 cells or hMSCs To investigate the effect of miR-342-5p during differentiation, we measured ALP activity after the transfection with miR-342-5p mimic and miR-342-5p inhibitor. As shown in Fig. 5, ALP activity was
Fig. 3. MicroRNA (miR)-342-5p inhibits MC3T3-E1 cell proliferation and induces apoptosis. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR-342-5p inhibitor or its negative control (NC). Non-treated cells were served as control. A. Cell viability was measured by a Cell Counting Kit-8. B. Expression of proteins associated with proliferation was determined by Western blot analysis. C. Cell numbers were counted by using trypan blue staining. D. Cell apoptosis was examined by flow cytometry. *, P < 0.05; **, P < 0.01; ns, no significance. PCNA, proliferating cell nuclear antigen; CDK, cyclin-dependent kinase. 254
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Fig. 4. MicroRNA (miR)-342-5p represses MC3T3-E1 cell migration. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR-342-5p inhibitor or its negative control (NC). Non-treated cells were served as control. Relative migration was measured by Transwell assay. *, P < 0.05.
might be negatively modulated by miR-342-5p expression. Thus, we hypothesized that miR-342-5p might function through negatively regulating Bmp7. For verification, cells were transfected with NC, miR342-5p inhibitor or miR-342-5p inhibitor plus si-Bmp7. Following results showed miR-342-5p silence-induced increases of cell viability (Fig. 7B), up-regulations of proteins associated with proliferation (Fig. 7C), decrease apoptosis (Fig. 7D), increases migration (Fig. 7E), and osteogenic differentiation (Fig. 7F) were all notably abrogated by Bmp7 silence. Therefore, we concluded that miR-342-5p inhibition might affect MC3T3-E1 cells via up-regulation of Bmp7. Besides of this, we used the bioinformatics software of TargetScan (http://www. targetscan.org/) to find the target genes of miR-342-5p. We found that COL4A6 and Bmp2 3’UTR were both existed targeting sequences (Supplementary Fig. 5A and B). To further confirm whether COL4A6 or Bmp2 is direct target of miR-342-5p, the dual luciferase reporter assay was carried out. Results showed that miR-342-5p overexpression clearly restrained the luciferase activity of reporter genes containing 3’UTR-Wt of COL4A6 or Bmp2 (P < 0.05), but did not affect that of the reporters fused to the mut versions (Supplementary Fig. 5C and D). Further, the mRNA and protein levels of COL4A6 or Bmp2 in miR-324-5p mimic or inhibitor transfected cells were also determined. Results revealed that COL4A6 and Bmp2 mRNA or protein levels were both declined by miR324-5p overexpression (P < 0.05 or P < 0.01), meanwhile enhanced by miR-324-5p suppression (P < 0.01). These relevant data hinted that COL4A6 and Bmp2 were target genes of miR-324-5p.
Fig. 5. MicroRNA (miR)-342-5p inhibits ALP activity in MC3T3-E1 cells. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR342-5p inhibitor or its negative control (NC). Non-treated cells were served as control. ALP activity was detected by using LabAssayTM ALP kit. *, P < 0.05.
significantly suppressed in miR-342-5p mimic-transfecting MC3T3-E1 cells, and promoted in miR-342-5p inhibitor-transfecting MC3T3-E1 cells. Likewise, ALP activity was also restrained in miR-342-5p mimictransfecting hMSCs (P < 0.01), and aggrandized in miR-342-5p inhibitor-transfecting hMSCs (P < 0.05, Supplementary Fig. 4). In addition, the expression of proteins associated with osteogenic differentiation was measured after transfection. When compared with respective controls, expressions of Runx2 and Ocn at mRNA and protein levels were remarkably down-regulated by miR-342-5p overexpression while were notably up-regulated by miR-342-5p silence (all P < 0.05, Fig. 6A–B). Data illustrated that miR-342-5p could suppress differentiation of MC3T3-E1 cells.
3.7. MiR-342-5p silence activated the MEK/ERK pathway through upregulating Bmp7 MC3T3-E1 cells were transfected with NC, miR-342-5p inhibitor or miR-342-5p inhibitor plus si-Bmp7, and the involvements of the MEK/ ERK pathway in the regulation of miR-342-5p were finally explored. In Fig. 8, expressions of p-MEK and p-ERK were both elevated by miR-3425p inhibition, and these elevations were abrogated when Bmp7 was knocked down. Results implied that miR-342-5p silence might activate the MEK/ERK pathway through up-regulating Bmp7 expression.
3.6. MiR-342-5p inhibition promoted proliferation, migration and differentiation via up-regulating Bmp7 expression
4. Discussion
Subsequent experiments were performed to explore whether Bmp7 was a downstream factor of miR-342-5p. Results in Fig. 7A showed Bmp7 mRNA and protein expressions were significantly down-regulated by miR-342-5p overexpression while were markedly up-regulated by miR-342-5p silence (P < 0.05), suggesting that Bmp7 expression
This study reported for the first time the effects and mechanism of miR-342-5p on osteoblasts, suggesting the possible therapeutic function for fracture. Before investigations, we figured out that miR-342-5p level was arrestingly decreased. Then, after aberrant expression of miR-3425p in MC3T3-E1 cells or hMSCs utilizing miRNAs, we interestingly 255
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Fig. 6. MicroRNA (miR)-342-5p suppresses differentiation of MC3T3-E1 cells. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR342-5p inhibitor or its negative control (NC). Osteogenic differentiation of transfected and un-transfected cells was induced by incubation in complete culture medium containing 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid. Non-treated cells were served as control. mRNA expressions A. and protein expressions B. of Runx2 and Ocn were determined by quantitative reverse transcription PCR and Western blot analysis, respectively. *, P < 0.05. Runx2, runt-related transcription factor-2; Ocn, osteocalcin.
study, miR-342-5p silence increased the levels of those proteins associated with proliferation, which suggested that miR-342-5p silence could promote MC3T3-E1 cells or hMSCs proliferation. Osteoblast migration plays pivotal roles in modeling phase of fracture healing, and elevated osteoblast migration leads to acceleration of bone formation and repair (Fukuyama et al., 2004). After fracture, preosteoblasts are recruited and migrated to the site of bone formation (Thiel et al., 2018). Lack of osteoblast migration has been proved to repress fracture repair (Aryal et al., 2015). Therefore, the effects of miR342-5p on MC3T3-E1 cell migration were next explored. Transwell assay showed miR-342-5p repressed MC3T3-E1 cells or hMSCs migration, which was consistent with a previous literature by Yang et al. (Yang et al., 2016). Mature osteoblasts are crucial for manufacture and mineralization of the osteoid matrix (Khosla et al., 2008; Maes et al., 2007). Maintenance of osteoblast differentiation and activity is pivotal for restoration of skeletal function (Claes et al., 2012). In addition, osteoblasts must proliferate and differentiate, and thereby induce mineralization. Therefore, we next determined the effects of miR-342-5p on osteoblast differentiation. Ocn is the most abundant non-collagenous protein in the bone extracellular, which acts as a late phenotypic osteoblast marker (Fang and Yamaza, 2016; Otsuki et al., 2018). It is synthesized by osteoblasts around the time bone mineralization begins (Karsenty and Olson, 2016). As an essential regulator of osteoblast differentiation and bone formation, Runx2 is reported to trigger Ocn expression via binding to the promoter region of Ocn (Vimalraj et al., 2015). In our study, to confirm the effect of miR-342-5p on osteoblast differentiation, we examined ALP activity in MC3T3-E1 cells after transfection with miR-342-5p mimic and miR-342-5p inhibitor, and results revealed that miR-342-5p overexpression significantly inhibited ALP activity in MC3T3-E1 cells or hMSCs. Further, we found that miR-342-5p overexpression-induced down-regulations of Runx2 and Ocn. These data illustrated that miR-342-5p could repress osteoblast differentiation. Bmp7 (35 kDa) belongs to the transforming growth factor (TGF)-β superfamily, which is a direct inducer of osteoblast differentiation
found that miR-342-5p could inhibit proliferation, migration, differentiation and induced apoptosis. After that, Bmp7 expression was identified to be negatively regulated by miR-342-5p expression, and Bmp7 inhibition could abrogate the alterations of proliferation, apoptosis, migration and differentiation, induced by miR-342-5p inhibition. Additionally, COL4A6 and Bmp2 were predicated as target genes of miR-342-5p. The final Western blot results illustrated that the MEK/ ERK pathway was activated by miR-342-5p silence through up-regulating Bmp7. Several miRNAs are dysregulated at post-fracture days in a literature by Waki et al., and these miRNAs are supposed to play important roles in fracture healing (Waki et al., 2015). Another study also detected the expression of several miRNAs and concluded that these miRNAs might be targets to optimize fracture healing on the basis of the dysregulation after fracture (Seeliger et al., 2014). Therefore, in our study, we measured the expression of miR-342-5p in differentiating osteoblast MC3T3-E1 cells or hMSCs prior to detailed investigations. Results of qRT-PCR showed observable down-regulation of miR-342-5p, strongly indicating that miR-342-5p might be involved in the process of fracture repair. Rapid bone matrix synthesis and mineralization by osteoblasts are observed during fracture healing, indicating osteoblast activity is of great importance for bone repair (Tu et al., 2017). Therefore, we measured the effects of miR-342-5p on MC3T3-E1 cells or hMSCs viability and proliferation. After transfection, cell viability was reduced by miR-342-5p overexpression but was enhanced by miR-342-5p silence, illustrating the helpful effects of miR-342-5p silence on fracture healing. PCNA is a marker of proliferating cells and acts as an auxiliary protein for DNA polymerases (Tsai et al., 2017). CyclinA and CyclinE1 are subunits of CDK2, and binding of CyclinA and CyclinE1 to CDK2 can phosphorylate proteins associated with DNA synthesis, resulting in enhanced proliferation (Arooz et al., 2000). Likewise, cyclinD1 activates the CDK4/6 and thereby phosphorylates tumor suppressor gene retinoblastoma protein, allowing expressions of genes required for transition through the G1 restriction point (Gelbert et al., 2014). In our 256
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Fig. 7. MicroRNA (miR)-342-5p inhibition affects MC3T3-E1 cells through up-regulating bone morphogenetic protein 7 (Bmp7). A. MC3T3-E1 cells were transfected with scramble miR, miR-342-5p mimic, miR-342-5p inhibitor or its negative control (NC), and the expression of Bmp7 was determined by quantitative reverse transcription PCR and Western blot analysis. Non-treated cells were served as control. B–E. MC3T3-E1 cells were transfected with NC, miR-342-5p inhibitor or miR-342-5p inhibitor plus si-Bmp7. Non-treated cells were served as control. Cell viability (B), expression of proteins associated with proliferation (C), apoptosis (D), and migration (E) were assessed by Cell Counting Kit-8 assay, Western blot analysis, flow cytometry and Transwell assay, respectively. F. MC3T3-E1 cells were transfected with NC, miR-342-5p inhibitor or miR-342-5p inhibitor plus si-Bmp7. Differentiation was induced by incubation in complete culture medium containing 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid. Non-treated cells were served as control. Protein expressions of Runx2 and Ocn at 14 days post-induction were detected by Western blot analysis. *, P < 0.05; **, P < 0.01. PCNA, proliferating cell nuclear antigen; CDK, cyclin-dependent kinase; Runx2, runt-related transcription factor-2; Ocn, osteocalcin; si-Bmp7, small interfering RNA targeting Bmp7.
instance, cell adhesion, migration, survival, differentiation, proliferation, and etc. (Roskoski, 2012). The MEK/ERK pathway can affect osteogenesis through regulating Runx2 phosphorylation and transcriptional activity (Jun et al., 2010). A previous study has also proved that Bmp7 stimulates proliferation and cadherin-11 expression through ERK in a murine metanephric mesenchymal cell line (Awazu et al., 2017). Therefore, we finally explored the involvements of the MEK/ERK pathway in the modulation of miR-342-5p in MC3T3-E1 cells. Results in our study illustrated the miR-342-5p inhibition could inhibit the MEK/ ERK pathway through up-regulating Bmp7 expression. 5. Conclusions To summarize, miR-342-5p was identified for the first time to be down-regulated during osteoblast differentiation. Then, we interestingly found miR-342-5p silence could promote proliferation, migration and differentiation of MC3T3-E1 cells through up-regulating Bmp7. Moreover, the MEK/ERK pathway was involved in the regulatory mechanism of miR-342-5p. The distinct effects of miR-342-5p may provide novel therapeutic targets for fracture healing. Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Fig. 8. MicroRNA (miR)-342-5p inhibition might activate the MEK/ERK pathway through up-regulating morphogenetic protein 7 (Bmp7) in MC3T3-E1 cells. MC3T3-E1 cells were transfected with NC, miR-342-5p inhibitor or miR-342-5p inhibitor plus si-Bmp7. Non-treated cells were served as control. Expression of key kinases in the MEK/ERK pathway was measured by Western blot analysis. si-Bmp7, small interfering RNA targeting Bmp7; t-, total; p-, phospho-; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase.
Declaration of Competing Interest Authors declare that there is no conflict of interests. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.molimm.2019.07.027.
(Bragdon et al., 2011). It can up-regulate Runx2 in mice and promote bone formation (Bustos-Valenzuela et al., 2011). Yan et al. have reported that miR-342-5p could regulate TGF-β signaling pathway (Yan et al., 2016). Another literature by Kurrel et al. has reported that miR542-3p can repress proliferation and differentiation of osteoblasts through regulating Bmp7 (Kureel et al., 2014). Therefore, we supposed that Bmp7 might be a downstream molecule of miR-342-5p. Accordingly, we found miR-342-5p expression was negatively correlated with Bmp7 expression. Moreover, subsequent experiments proved that Bmp7 silence could reverse the effects of miR-342-5p inhibition on proliferation, migration and differentiation, verifying that miR-342-5p inhibition might function through up-regulating Bmp7 expression. We then used the bioinformatics software of starbase (http://starbase.sysu. edu.cn/) to seek the complementary seed sequence between 3’UTR of Bmp7 and miR-324. We discovered that there was a targeted binding site between Bmp7 and miR-342-3p, but no targeted binding site between Bmp7 and miR-342-5p. To further seek the target genes, the TargetScan (http://www.targetscan.org/) and dual luciferase reporter assay were carried. Finally, COL4A6 and Bmp2 were predicated as target genes of miR-342-5p. However, whether COL4A6 and Bmp2 joins in affect the functions of miR-342-5p in osteoblasts is still needed further research. The MEK/ERK cascade participates in several cellular processes, for
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