MiR-20a: a mechanosensitive microRNA Regulates fluid shear stress-mediated osteogenic differentiation via the BMP2 signaling pathway by target BAMBI and SMAD6

doi:10.21203/rs.3.rs-1502081/v1

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MiR-20a: a mechanosensitive microRNA Regulates fluid shear stress-mediated osteogenic differentiation via the BMP2 signaling pathway by target BAMBI and SMAD6

https://doi.org/10.21203/rs.3.rs-1502081/v1

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Objective: MicroRNAs (miRNAs) are crucial regulators of diverse biological and pathological process. This study aimed to investigate the role of miR-20a in fluid shear stress(FSS)-mediated osteogenic differentiation.

Methods: In the present study, we subject osteoblast MC3T3-E1 cells or mouse bone marrow stromal cells(BMSCs) to single bout short duration fluid shear stress(12 dyn/cm² for 1 hour) by using a parallel plate flow system. The expression of miR-20a was quantified with miRNA array profiling and quantitative RT-PCR (qRT-PCR) during the FSS-mediated osteogenic differentiation. Bioinformatics analysis and a luciferase assay were performed to confirm potential targets of miR-20a.

Results: Osteoblast-expressed miR-20a is sensitive to the mechanical environments of FSS, which is differentially upregulated During Steady FSS-mediated Osteogenic Differentiation. MiR-20a enhances FSS-induced osteoblast differentiation by activating the BMP2 signaling pathway. Both BAMBI and SMAD6 are targets of miR-20a that negatively regulate the BMP2 signaling pathway.

Conclusion: MiR-20a is a novel mechano-sensitive miRNA that can enhance osteoblast differentiation in FFS mechanical environments, implying that this miRNA might be a target for bone tissue engineering and orthodontic bone remodeling for regenerative medicine applications.

Fluid shear stress

MC3T3-E1

mouse BMSCs

miR-20a

osteogenic differentiation

Mechanical stimulation is critical for skeletal development as well as bone remodeling. Mechanical stress-mediated deformation of the mineralized matrix leads a heterogeneous pressure gradients that enhance interstitial fluid flow in the haversian and lacunar-canalicular network systems, generating fluid shear stress (FSS) across bone cell surfaces [1]. FSS is highly used for mechanical stimulation of the bone. Fluid shear stress is a major force involved in bone adaptation. Mechanosensitive cells sense mechanical signals produced by FSS in bone and translate them into biochemical signals[2]. Appropriate FSS has a vital function in modulation of functions of bone cells, including osteoblasts, osteocytes, and human adipose-derived stem cells (ASCs), enhancing cell proliferations and promoting osteogenic differentiations [3–5]. Our previous study indicated that steady fluid shear stress of 12 dyn/cm² for 1 h promoted osteogenic differentiation of murine calvaria-derived preosteoblasts (MC3T3-E1 cells), human periodontal ligament cells (hPDLCs) and mouse bone marrow stromal cells (BMSCs)[6,7].

Bone morphogenetic protein 2 (BMP2) is a key regulatory factor of osteoblast differentiation. Kamiya et al demonstrated that BMP2 signaling pathway has a vital role in transcriptional control of bone formation[8]. BMP2 intracellular receptor Smads, especially Smad5, activates the transcription of osteogenic-associated genes, such as Runt-related transcription factor2 (RUNX2)/ Cbfa-1, SP7/ Osterix, which in turn lead to extracellular matrix (ECM) mineralization to generate osteoids [9]. It was reported that mechanical strain increased phosphorylation of Smad1/5 and activated the BMP2/Smad pathway in osteoblast differentiation[10,11]. Moreover, Liu et al showed that FSS promoted BMP2 secretion and activated the BMP2 signaling pathway, indicating the crucial function of BMP2 signaling pathway in mechanical loading-induced osteogenic differentiation[12]. Similarly, our previous results showed that an association between the BMP2 and integrin β1 pathways have a vital role in FSS-induced osteogenic differentiation. However, the underlying mechanisms require further investigation.

MicroRNAs (miRNAs), which form a class of noncoding small (18 to 25 nucleotides) RNAs, are key negative regulators of various biological as well as pathological processes, including apoptosis, development, organogenesis, cell proliferation as well as differentiation[13,14]. Mature miRNAs post-transcriptionally suppress gene expression levels by binding their regulatory targets in 3’-untranslated regions (3’-UTRs) of mRNAs, which leads to mRNA degradation or translational suppression[13,15]. Various miRNAs play significant roles in regulating chemical stimulation-mediated osteogenic differentiation as well as bone formation in vitro. For example, miR-20a, miR-10a, miR-96 and miR-21 promote osteogenic differentiation[16–19], while miR-200a, miR-138, miR-141, as well as miR-133a-5p inhibit osteogenic differentiation[20–22]. Thus, it has been strongly postulated that regulation of osteogenic differentiation by miRNAs is an important aspect of the regulatory machinery. Furthermore, several studies have evaluated mechanical force-mediated changes in expressions of miRNAs associated with bone metabolism. For example, miR-103-a was suppressed during cyclical mechanical stretching-mediated osteoblast differentiation, leading to elevated RUNX2 protein levels by targeting pank3[23]. Additionally, miR-503-5p functions a mechanosensitive miRNA and suppresses osteogenic differentiation of BMSCs in mechanically stretched bones and bone formation in OTM tension sides[24]. PDLSCs exposures to stretching force suppressed activin receptor type IIB (ACVR2B) levels via direct interactions between miR-21 and 3-untranslated repeat sequence of the ACVR2B mRNA[25]. Moreover, FSS-induced osteoblast differentiation is negatively regulated by miR-33-5p as well as its target gene (Hmga2) by regulating stem cells and MSCs proliferations [26]. These findings imply that miRNAs play vital roles in sensing of mechanical loads by osteoblasts. Nevertheless, the impacts of FSS mechanical stimuli on miRNA expressions as well as their roles in mechanotransduction in osteoblasts have yet to be fully identified. Therefore, further studies should confirm the associations between miRNA expression levels and mechanical force. To establish miRNA functions in mechanotransduction of FSS, we screened for mechanosensitive miRNA using the miRNA microarray strategy and recognized miR-20a to be distinctly changed in MC3T3-E1 cells as a response to FSS[27].

We identified differentially expressed miRNAs putatively related to BMP2 signaling pathway and performed miR-20a spatial-temporal up-regulated pattern during FSS-mediated osteogenic differentiation of MC3T3-E1 pre-osteobalstic cells or mouse BMSCs. MiR-20a was shown to be a positive regulator of FSS-induced osteogenic differentiation by activating BMP2 signaling pathway via directly down-regulating BAMBI and SMAD6, negative factors of BMP2 signaling pathway. These results imply that miR-20a has a significant function in FSS-induced osteogenic differentiation.

2.1. Cell culture

Culture of MC3T3-E1 cells, a murine pre-osteoblastic cell line (ATCC, Manassas, VA, USA) was done in α-MEM media (Life Technologies, Grand Island, NY, USA) with 1% penicillin/streptomycin (Life Technologies) and fetal bovine serum (FBS; 10%, Life Technologies). Incubation was done in a 5% CO₂ humidified environment at 37°C. Medium replacement was done after every 3 days. Isolation of BMSCs was done as described[28] and preserved in DMEM with 10% FBS. Multipotential differentiation ability was established (Supplementary Figure S2).

2.2. Application of Fluid Flow Stress

When cells grew to a 80–85% confluence on polylysine (10 µg/ml; Sigma-aldrich) coated glass slides, cell starvation was done for 12 h in a serum-free media after which they were treated with FSS (12 dyn/cm²) for 1 h using a parallel plate flow system that generated a laminar unidirectional flow across cells as previously described[6]. Flow system maintenance was done at 37°C after it was filled with 5% CO₂ serum-supplemented medium (10%). Control cells were cultured at a static station. To conduct the control experiments, cells were placed in identical conditions as those of equivalent FSS experiments, but devoid of FSS stimulus application. After FSS stimulation, cell growth was done in identical osteogenic conditions (β-glycerophosphate (10 mM) and ascorbic acid (50 µg/ml) for MC3T3-E1; β-glycerophosphate (10 mM), ascorbic acid (50 µg/mL) and dexamethasone (10 nM) for BMSCs).

2.3. RNA Isolation, Reverse Transcription and Quantitative Real-Time PCR

The Trizol reagent (Life Technologies) was used for total RNA extraction. Expression levels of mature miRNA were evaluated by stem-loop primer SYBR Green quantitative real time-PCR (qRT-PCR) and were normalized to U6. Gene transcriptional levels were evaluated by qRT-PCR. Normalization of expression levels was done to GAPDH levels. qRT-PCR was done by a 7500 Real Time PCR system (Applied Biosystems, USA) using Platinum SYBR Green qPCR SuperMix-UDG (Life Technologies). mRNA expression level were calculated by the CT method (^△△CT) using GAPDH as an internal reference. The qRT-PCR procedure comprised of 40 cycles (15 s at 94°C, 60.5°C for 15 s, 15 s at 72°C) which followed an initial denaturation step (2 min at 94°C). Primers for this experiment are presented in supplementary table 2.

2.4. Immunoblotting

Quantification was done by a BCA protein assay kit (Pierce, Rockford, IL). Then, SDS-PAGE (12%) was used to separate the cell lysates followed by their transfer to PVDF membranes. Membranes blocking was done using non-fat dry milk (5%) for 1 h followed by overnight incubation in the presence of primary antibodies against BAMBI (1:1000; R&D, USA), SMAD6 (1:500; AVIVA, USA), PPARγ (1:1000; Cell Signaling Technology, USA), BMP2 (1:400; Bioss company, China), or RUNX2 (1:400; Bioss company, China). After washing, membranes were incubated with a HRP-conjugated secondary antibody (Santa Cruz Biotechnology). Detection was done by chemiluminescence. GAPDH protein was the internal loading control. Intensities of the protein fragments were quantified by Image Lab software version 4.0 (BIO-RAD, USA).

2.5. ALP Activity Assessment

Cellular ALP activities were assessed by the PNPP method. P-nitrophenylphosphate was used as the substrate (LabAssay ^TM ALP, Wako, Japan). Enzymatic activities (units/mg protein) were equal to p-nitrophenol (nmol/mL) concentrations released by the sample within 15 min, after background exclusion. ALP activities of every sample were normalized to protein concentrations measured using a BCA protein assay kit (Thermo Scientific Pierce, USA).

2.6. Alizarin Red S staining

To evaluate ECM mineralization as a terminal marker of differentiation, the cells were washed, fixed in paraformaldehyde (4%) and stained using Alizarin Red S solution (1%; Sigma-Aldrich). Imaging was randomly done by light microscopy (400×, Olympus). Then, Alizarin Red S stain quantification was performed by extraction using cetyl-pyridinium chloride monohydrate (TCI, Japan). Measurement of absorbance was done at 560 nm.

2.7. Oligonucleotide Transfection

Transfection of miR-20a mimics, miR-20a inhibitor (anti-miR-20a), negative control (NC), BAMBI silencing oligonucleotides (si-BAMBI), inhibitor-NC and SMAD6 silencing oligonucleotides (si-SMAD6) (RiboBio, China) into cells was done using Lipofectamine RNAiMAX (Invitrogen) as instructed by the manufacturer. Efficiency of transfection was assessed using Cy3-labeled oligonucleotide RNA mimics(Supplementary Figure S3).

2.8. Luciferase Assay

Possible binding sites for miR-20a within BAMBI or SMAD6 3’-UTR were determined by PicTar and TargetScan. Annealed synthetic oligonucleotides and predicted binding sites were cloned into the XhoI/NotI site of psiCHECK-2 (Promega). Transient transfection of Cos-7 cells with a mutant (MUT) reporter vector or wild-type (WT) and microRNA was done using Lipofectmine 2000 (Invitrogen) at various concentrations. Forty eight hours post-transfection, luciferase activities were assessed by the Dual-Luciferase Reporter Assay System (Promega) as described by the manufacturer. Normalization of the renilla luciferase activities were done to the equivalent firefly luciferase activities and plotted as percentages of the control.

2.9. RNAi

Designing and synthesis of double-stranded siRNAs was done by RiboBio (Guangzhou, China). Transfection of siRNAs (50 nM) was done using Lipofectamine RNAiMAX (Life Technologies) as described by the manufacturer. Double-stranded scrambled RNA was the negative control (NC). The transfection of cells that had been seeded on glass slides was done for 12 h, after which they were subjected to additional treatments.

2.10. Statistical Analysis

Data are presented as mean ± SD for n = 3. One-way ANOVA was used for comparisons of means among groups. Significance was attained at p < 0.05 (*, P < 0.05; **, P < 0.01; ***, P < 0.001.).

3.1 miR-20a is Differentially upregulated During Steady FSS-mediated Osteogenic Differentiation.

Osteogenic differentiation in MC3T3-E1 pre-osteoblastic cells was initiated by a 12 dyn/cm² single load of FSS for 1 h and confirmed by elevated expression levels of osteogenic differentiation biomarker genes, including Runt-related transcription factor-2 (RUNX2), alkaline phosphates (ALP) and SP7 transcription factor 7 (SP7) at 12 h after FSS treatment (pf.)(Fig. 1A). Confirmation of osteoblast phenotypes was done by detection of enhanced ALP activities (Fig. 1B) and Alizarin Red S staining for extracellular matrix mineralization (Fig. 1C). Elevated expression levels of osteoblast-related genes as well as the observed osteoblast phenotypes were consistent with previously reported observations of FSS-promoted osteogenic MC3T3-E1 cell differentiation.

To identify candidate miRNAs that regulate FSS-induced osteogenic differentiation, miRNA array profiling was conducted after treating MC3T3-E1 cells with FSS (12 dyn/cm2) using Agilent miRNA chips. There were marked changes in expression levels of 69 miRNAs after FSS-treatment as described previously[27]. Functional analysis of these miRNAs revealed that they were closely related to the BMP2 signaling pathway, in which 11 miRNAs are involved (Table S1). Quantitative RT-PCR (qRT-PCR) analysis revealed spatiotemporal expression pattern of miR-20a, -19b and 34a of 11 miRNAs during FSS-induced osteogenic MC3T3-E1 differentiation, which were reduced immediately for a short time, but quickly elevated at 6 h, then peaked at 12 h and remained elevated than control until 72 h (Fig. 1D, Figure S1). To ascertain this outcome, we used primary mouse BMSCs. Similarly, the expression of miR-20a decreased immediately for a short time, but consistently increased at 6h post-FSS treatment, then peaked at 24h and remained higher than control until day 7 (Fig. 1E), indicating that miR-20a was an early response gene for FSS in osteoblasts.

3.2. Overexpression of miR-20a Activates Chemical Stimulation-Mediated Osteogenic MC3T3-E1 cell Differentiation.

Overexpression studies were performed to determine biological functions of miR-20a in osteogenic differentiation (Fig. 2). Overexpressed miR-20a elevated mRNA expression of ALP at 48h and increased ALP activity at day 3 (Fig. 2A and 2B). To ascertain osteogenic effects of miR-20a, an ARS staining experiment was conducted. Figure 2C shows that overexpressed miR-20a enhanced matrix mineralization at day 14. Therefore, overexpression of miR-20a increases osteogenic MC3T3-E1 cell differentiation induced by standard osteoblast-induction medium.

3.3 miR-20a Promotes FSS-mediated Osteogenic Differentiation by promoting the BMP2 Signaling Pathway.

Previous reports indicated that the canonical BMP2 signaling pathway has crucial roles in FSS-mediatd osteogenic differentiation. The function of miR-20a in FSS-mediated osteogenic differentiation was further determined by investigating the effects of the loss (anti-miR-20a) or overexpression of miR-20a on osteogenic MC3T3-E1 cell differentiation in vitro DMSO. Overexpression of miR-20a significantly increased the transcriptional levels of RUNX2, BMP2, and SP7, the main regulators of the BMP2/RUNX2 pathway (Fig. 3A-C). However, a notable decrease in the transcriptional levels of BMP2, RUNX2, and SP7 was observed pf., although higher than the without FSS-treatment groups (Fig. 3D-F). Accordingly, BMP2 as well as RUNX2 protein levels were suppressed in the anti-miR-20a group, although overexpression of miR-20a just slightly increased their protein levels during FSS-induced osteogenic differentiation (Fig. 3G and 3H). Furthermore, we observed enhanced ALP activity and nodule formation with overexpression of miR-20a (Fig. 4A and C), but were attenuated remarkably with anti-miR-20a during FSS-induced osteogenic differentiation (Fig. 4B and 4C).

These results suggest that up-regulated miR-20a promotes FSS-induced osteogenic differentiation as well as bone formation by activating the BMP2 signaling pathway.

3.4. miR-20a activates BMP2 signaling pathway via Direct Targeting to BAMBI and SMAD6.

To understand the functional roles of miR-20a during FSS-promoted osteogenic differentiation, the microRNA target prediction software TargetScan and PicTar were used to search for potential miR-20a targets (Table S3). Bambi, Smad6, and Pparγ,the antagonists of the BMP2/RUNX2 signaling pathway, were identified as putative target miR-20a genes. To ascertain if they are potential miR-20a targets, we analyzed the expressions of BAMBI, SMAD6, and PPARγ pf.. In both the FSS-treatment and miR-20a overexpression groups, there was a marked decrease in BAMBI and SMAD6 protein levels in the MC3T3-E1 cells (Fig. 5A). However, alterations in mRNA levels of BAMBI and SMAD6 were not significant pf. (Fig. 5B), indicating the effect was post-transcriptional. We also confirmed our findings using primary mouse BMSCs. Consistent with the findings in MC3T3-E1 cells, we observed significantly low protein levels of BAMBI and SMAD6, but not mRNA, in both the FSS-treatment and miR-20a overexpression groups (Fig. 5C and 5D). Though, no significant changes in the protein or mRNA levels of PPARγ were observed both in MC3T3-E1 cells as well as in primary mice BMSCs. (Figure S4).

According to the bioinformatic prediction of TargetScan, BAMBI and SMAD6 have a 7-nucelotide seed match site for miR-20a within their 3’UTR, and the putative target site is highly conserved in vertebrate species (Fig. 5E and 5F). To determine if miR-20a can directly target BAMBI and SMAD6, the 3'-UTR of BAMBI or SMAD6 were amplified, then inserted the luciferase reporter vector psiCHECK2 into the 3'-UTR. Reporter vectors exhibiting mutated putative target sites were also inserted. The mutated sites of the 3’UTR of BAMBI or SMAD6 is shown in Fig. 5E and 5F. Ectopic miR-20a was co-transfected with BAMBI-WT, SMAD6-WT, BAMBI-MUT, or SMAD6-MUT vectors into cos-7 cells and luciferase activity was then assessed. Compared to negative control (NC), the luciferase activity of the BAMBI-WT sequence decreased 47.7% by miR-20a. However, luciferase activities were not affected by miR-20a in the BAMBI-MUT sequence (Fig. 5G). Similarly, relative to NC, luciferase activities of the SMAD6-WT sequence decreased 40.3% by miR-20a and was not affected by miR-20a in the SMAD6-MUT sequence (Fig. 5H). In conclusion, BAMBI and SMAD6 are direct targets for miR-20a.

3.5. Silencing of BAMBI and SMAD6 Mimic the Osteogenic Effects of miR-20a.

To determine whether BAMBI and SMAD6 are functional targets of miR-20a during osteogenic differentiation, we further determined whether inhibition of BAMBI and SMAD6 expressions mimicked the differentiation-enhancing effects of miR-20a. Figure 6A shows that Bambi and Smad6 mRNA expression levels were effectively repressed in MC3T3-E1 cells with RNAi for 24h. Silencing of Bambi and Smad6 resulted in the increased transcription of osteoblast-associated genes Bmp2, Runx2, and Alp (Fig. 5B). Furthermore, silencing of BAMBI and SMAD6 enhanced ALP activity as well as extracellular matrix mineralization (Fig. 5C and 5D). We noticed that the osteogenic effects of BAMBI and SMAD6 knockdown were weaker than those of miR-20a overexpression, indicating that miR-20a may target other genes as well. These results show that knockdown of BAMBI or SMAD6 mimic the osteogenic differentiation of miR-20a.

Mechanical force induced osteoblast differentiation is a basic feature of bone homeostasis, such as orthodontic tooth movement and bone repair. MiRNAs respond to mechanical loading and are involved in osteoblast differentiation as well as bone formation[29]. We found that miR-20a is a novel mechano-sensitive miRNA involved in positive regulation of osteoblast differentiation by activating the BMP2 signaling pathway by repressing BAMBI and SMAD6 expression post-transcriptionally. Thus, miR-20a is a possible therapeutic target for pathological disorders that are associated with skeletal development induced by mechanical environment changes.

FSS, a potent form of bone mechanical stimulation, has a vital role in osteoblast differentiation, function as well as development. Important molecules and pathways involved in FSS-induced osteoblast differentiation have been identified. For example, FSS activates ERK1/2, β-catenin, PGE2 and GSK-3b, which are vital signaling constituents of mechanotransduction pathways in osteoblasts that enhance cellular differentiation[30–32]. It was reported that several signal pathways, including Wnt/β-catenin signaling, Ca2 + signaling, and BMP2 signaling play various roles in FSS-mediated osteoblast differentiation, through which the biochemical process was triggered[30,33,34]. In addition, we previously discovered that the association between integrinβ1 and BMP2 pathways played a vital role in these differentiations[6].

Recent studies demonstrated the mechano-sensitive properties of miRNAs when cells experienced FSS. For example, the up-regulation of miR-132 has a role in FSS-induced differentiation as well as proliferation in the periodontal ligament cells[35]. Similarly, miR-33-5P was elevated and promoted osteoblast MC3T3-E1 cell differentiation under FSS[26]. FSS resulted in the up-regulation of E-Tmod41 via miR-23b-3p inhibition as well as P_E0 promoter activation, thereby contributing to F-actin cytoskeletal remodeling[36]. In rat hind limbs, down-regulation of miR-352 was involved in elevations of FSS-mediated collateral vessel growth [37].The oscillatory shear-sensitive miR-181b regulates aortic valve endothelial matrix degradation[38]. We found that miR-20a expression increased significantly 6 h after FSS in MC3T3-E1 cells (12h in BMSCs). This is the first study to report an miRNA that is involved in activating the BMP2 signaling pathway by FSS. Precisely, in response to FSS, we established spatiotemporal expression pattern of miR-20a in MC3T3-E1 cells or BMSCs. Thus, miR-20a as a mechano-sensitive miRNA is sensitive to FSS.

Among the miRNA families, MiR-20a, which is a miR-17-92 cluster member has been widely evaluated. This family has vital functions in tissue as well as organ development. This family of miRNAs is closely related with tumor development [39], autoimmune diseases[40], and osteogenesis[41]. MiR-20a is expressed in mice and is conserved in humans, and especially at elevated levels in osteoblasts and bone tissues [42]. It was reported that the miR-17-92 cluster can suppress TGFB pathway-mediated proliferation inhibition as well as collagen synthesis in PMCs by targeting SMAD4, TGFBR2, and SMAD2 [43]. miR-20a also plays a role in osteogenic differentiation of human mesenchymal stem cells (MSCs) through the BMP2/Runx2 signaling pathway by targeting Crim1, PPARγ and Bambi [16]. In addition, up-regulation of miR-20a promoted osteogenic differentiation of human adipose tissue-derived stem cells (hASCs)[44]. Moreover, a recent study showed that miR20a negatively regulates THP-1 cell proliferation as well as osteoclastogenesis during osteoclast differentiation by down-regulating PPARγ[45]. These studies imply that miR-20a has a vital role in regulation of osteogenic differentiation as well as osteogenesis. Nevertheless, the significance of miR-20a in mechanotransduction was not established. Our findings also demonstrate that miR-20a is involved in regulation of osteoblast differentiations of mouse BMSCs and MC3T3-E1 cells. Our results show that overexpressed miR-20a elevates the expression levels of canonical markers associated with osteoblast differentiation, such as Runx2 and Sp7. Furthermore, abilities of miR-20a to enhance osteoblast differentiations were confirmed in simulated FSS environments. Overexpressed miR-20a promoted osteoblast differentiation induced by simulated FSS. Accordingly, miR-20a knockdown inhibits this differentiation, implying that miR-20a participates in modulation of FSS-induced osteoblast differentiation.

The BMP2 signaling pathway has a vital role in transcriptional regulation of bone formation. The present data demonstrated that miR-20a promotes FSS-mediated osteogenic differentiation by promoting the BMP2 signaling pathway. BMP2 and Runx2 were up-regulated at mRNA as well as protein levels. Their associations may not be direct since miRNAs regulate gene expressions by binding 3'-UTRs of target genes, which leads to translational suppression or mRNA degradation. Consequently, miR-20a may activate the BMP2 signaling pathway by inhibiting its antagonists or negative regulators.

MiRNA has hundreds of target sites and it regulates a majority of protein-coding genes, thereby forming a regulatory network[46]. Based on our findings, Smad6 and Bambi possess high-score miR-20a binding sites in their 3'-UTRs, indicating that they may be targets for miR-20a in BMP2 signaling pathways. Bambi (Bmp and activin membrane-bound inhibitor) is a BMP2 signaling pathway pseudo-receptor whose amino acid sequence is highly to comparable that of BMP2/4 type-I receptors BMPR-IB and BMPR-IA. It has been shown that Bambi inactivates ligand-receptor complexes and antagonizes BMP signaling through associating with BMP ligands as well as functional BMP receptors[47]. Smad6, a Smad family member, functions as a suppressor of certain SMAD family members. Smad6 suppresses Smad1 phosphorylation under the induction of bone morphogenetic protein type IB receptor. Smad6 competes with Smad4 to bind the receptor-activated Smad1/5/8 complex to form an inactive R-SMAD polymer, which interferes with BMP2/Smads signal transduction[48]. In addition, Smad6 promotes SmurF1-mediated Runx2 degradation, thereby inhibiting BMP2/RUNX2 signaling pathway activation [49]. In this study, both Bambi and Smad6 were determined to be regulators of miR-20a that participate in activation of the BMP2/RUNX2 signaling pathway which regulates osteoblast differentiation in FSS simulated environments. We observed that the protein expression of Bambi and Smad6 was suppressed during FSS-induced osteogenic differentiation, similar to the overexpression of miR-20a. We postulated that this decrease of the negative regulator of BMP2/RUNX2 signaling pathway may activate BMP2 signaling, thus enhancing osteogenic differentiation. These results suggest a miR-20a co-regulatory mechanism in osteoblast differentiation, as was demonstrated in our study (Fig. 7).

Zhang et al documented that miR-20a has a key role in hMSCs osteogenic differentiation. These effect is achieved through the BMP/Runx2 signaling pathway by targeting PPARγ, Bambi as well as Crim1[16]. We also confirmed that miR-20a regulated FSS-induced osteogenic differentiations of MC3T3-E1 cells and mouse BMSCs via the BMP2 signaling pathway by targeting BAMBI and SMAD6. However, PPARγ did not appear to be directly targeted of miR-20a. This indicates that the function of miR-20a is highly conserved and has similar functions across different species. Conversely, it implies that miR-20a targets may differ among species.

A few limitations of our study are worth noting. In accordance with previous investigations, we evaluated the effects of 12 dynes/cm2 single bout FSS for 1 hour on osteoblast differentiations. Nevertheless, the magnitude as well as application time of FSS may have an effect on osteoblast differentiation. The significance of miR-20a in FSS-mediated osteoblast differentiation should be further evaluated in various FSS environments. Furthermore, our in vitro findings were not confirmed in vivo.

In summary, a particular mechano-sensitive miRNA, miR-20a, senses the mechanical environment in osteoblasts and moderates their differentiation. Specifically, miR-20a suppressed several BMP2 signaling pathway-associated genes at post-transcriptional levels to yield mild regulations of individual target genes and thereby ultimately enhance osteoblast MC3T3-E1 cell osteogenesis. These results elucidate on the mechanisms of mechanotransduction in osteoblasts and imply that miRNAs may be targeted in bone tissue engineering and orthodontic bone remodeling for regenerative medicine applications.

FSS: Fluid shear stress; BMP2: Bone morphogenetic protein 2; miRNA: microRNA; BMSCs: bone marrow stromal cells; pf.: post-FSS treatment; RUNX2: Runt-related transcription factor-2; SP7: SP7 transcription factor 7; PPARγ: Peroxisome proliferator-activated receptor gamma; BAMBI: BMP and activin membrane-bound inhibitor; SMAD6: Mothers against decapentaplegic family member6; ALP: Alkaline phosphatase; ARS: Alizarin Red S staining; DMSO: Dimethyl sulfoxide; siRNA: small interfering RNA .

Acknowledgments

Not applicable.

Authors’contributions

ZLP and ZHM designed the study. ZLP, YXW, FX and SSX performed the study. GQL and AH supervised the experiment. All authors approve the final study.

Funding

This work was supported by Grants from National Natural Science Foundation of China (No. 81470766) and Natural Science Foundation of Guangdong Province (No. 81070860). We thank Professor Guangmei Yan (Sun Yat-sen University) and his group for providing technical support.

Availability of data and materials

We declare that the materials described in the manuscript will be freely available to all scientists for non-commercial purposes.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing of interest

There is no conflict of interest that could inappropriately influence the work reported in this manuscript.

Author details

¹ Department of Stomatology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. ²Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University and Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China.

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No competing interests reported.

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MiR-20a: a mechanosensitive microRNA Regulates fluid shear stress-mediated osteogenic differentiation via the BMP2 signaling pathway by target BAMBI and SMAD6

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Abstract

Figures

1. Background

2. Materials And Methods

2.1. Cell culture

2.2. Application of Fluid Flow Stress

2.3. RNA Isolation, Reverse Transcription and Quantitative Real-Time PCR

2.4. Immunoblotting

2.5. ALP Activity Assessment

2.6. Alizarin Red S staining

2.7. Oligonucleotide Transfection

2.8. Luciferase Assay

2.9. RNAi

2.10. Statistical Analysis

3. Results

3.1 miR-20a is Differentially upregulated During Steady FSS-mediated Osteogenic Differentiation.

3.2. Overexpression of miR-20a Activates Chemical Stimulation-Mediated Osteogenic MC3T3-E1 cell Differentiation.

3.3 miR-20a Promotes FSS-mediated Osteogenic Differentiation by promoting the BMP2 Signaling Pathway.

3.4. miR-20a activates BMP2 signaling pathway via Direct Targeting to BAMBI and SMAD6.

3.5. Silencing of BAMBI and SMAD6 Mimic the Osteogenic Effects of miR-20a.

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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