Skip to main content
Download PDF

miR-29b-3p regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically stimulated osteocytes

Cellular & Molecular Biology Letters volume 24, Article number: 11 (2019) Cite this article

Abstract

Background

Mechanical loading is an essential factor for bone formation. A previous study indicated that mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz for 8 h promoted osteogenesis and corresponding mechanoresponsive microRNAs (miRs) were identified in osteoblasts. However, in osteocytes, it has not been identified which miRs respond to the mechanical strain, and it is not fully understood how the mechanoresponsive miRs regulate osteoblast differentiation.

Methods

Mouse MLO-Y4 osteocytes were applied to the same mechanical tensile strain in vitro. Using molecular and biochemical methods, IGF-1 (insulin-like growth factor-1), PGE2 (prostaglandin E2), OPG (osteoprotegerin) and NOS (nitric oxide synthase) activities of the cells were assayed. MiR microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays were applied to select and validate differentially expressed miRs, and the target genes of these miRs were then predicted. MC3T3-E1 osteoblasts were stimulated by the mechanical tensile strain, and the miR-29b-3p expression was detected with miR microarray and RT-qPCR. Additionally, the effect of miR-29b-3p on IFG-1 secretion of osteocytes and the influence of conditioned medium of osteocytes transfected with miR-29b-3p on osteoblast differentiation were investigated.

Results

The mechanical strain increased secretions of IGF-1 and PGE2, elevated OPG expression and NOS activities, and resulted in altered expression of the ten miRs, and possible target genes for these differentially expressed miRs were revealed through bioinformatics. Among the ten miRs, miR-29b-3p were down-regulated, and miR-29b-3p overexpression decreased the IGF-1 secretion of osteocytes. The mechanical strain did not change expression of osteoblasts’ miR-29b-3p. In addition, the conditioned medium of mechanically strained osteocytes promoted osteoblast differentiation, and the conditioned medium of osteocytes transfected with miR-29b-3p mimic inhibited osteoblast differentiation.

Conclusions

In osteocytes (but not osteoblasts), miR-29b-3p was responsive to the mechanical tensile strain and regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically strained osteocytes.

Introduction

Mechanical stimulation plays an essential role in the metabolic balance of bone. Physiological loading can induce bone formation, whereas a lack of loading or excessive loading leads to bone resorption [1,2,3,4].

As the dominant cells in bone tissue, osteocytes respond to mechanical stimulation, sense and integrate mechanical stimuli into biochemical signals to regulate both bone formation and resorption [5]. Previous studies mainly focused on osteocytes’ response to fluid shear stress which inhibits osteocytes apoptosis and promotes survival by modulating the Bcl-2/Bax expression ratio, enhances expression levels of NO and PGE2, and increases COX2 and the OPG/RANKL ratio, playing a dominant role in regulating the activities of both osteoblasts and osteoclasts [6,7,8], thus regulating bone reconstruction and remodeling. However, how osteocytes convert the mechanical stimulation into a biological signal and regulate bone formation (activity of osteoblasts) or resorption (activity of osteoclasts) remains not fully elucidated.

MiRs are small non-coding, single-strand RNAs, which control gene expression by targeting to 3′ untranslated regions of mRNA resulting in translational repression or degradation [9]. It was previously found that miR plays a pivotal role in bone formation [10], and many miRs which regulate bone formation have been identified [10, 11]. Some mechanoresponsive miRs were recently identified, they played significant roles in bone formation. For example, miR-33-5p and miR-132 are responsive to mechanical loading and regulate osteogenesis via targeting Hmga2 and mTOR signaling pathway, respectively [12, 13]. Our previous study confirmed that a mechanical tensile strain of 2500 με at 0.5 Hz for 8 h promoted osteogenesis and mechanoresponsive miRs in osteoblasts were identified [14]. The study urged us to investigate osteocytes’ response to the mechanical tensile strain and to search for mechanoresponsive miRs of osteocytes.

miR-29b regulated osteoblast differentiation (in MC3T3 osteoblasts, miR-29b overexpression promotes osteogenic differentiation) [15], and IGF-1 was confirmed to be a target gene of miR-29b [16, 17]. We speculated that miR-29b was responsive to mechanical strain applied to osteocytes and involved in osteoblast differentiation. However, the mechanism by miR-29b osteocytes convert a mechanical signal into a biological signal and regulate osteoblast differentiation has not been fully elucidated.

In this study, the osteocytes’ biological response to a mechanical tensile strain of 2500 με at 0.5 Hz for 8 h was investigated, and some novel mechanosensitive miRs were selected. In addition, the involvement of miR-29b in osteocytes’ response to mechanical strain and osteoblast differentiation were studied.

Methods

Cell culture

A mouse MLO-Y4 osteocyte cell line (provided by JENNIO Biological Technology, Guangzhou, China) was cultured in dishes with α-MEM medium (α-MEM, Invitrogen), containing 10% FBS and 1% penicillin. Then the cells were transferred to mechanical loading dishes that were reformed from cell culture dishes (Nalge Nunc International).

Mouse MC3T3-E1 osteoblastic cells (JENNIO Biological Technology, Guangzhou) were cultured with the same medium as mentioned above.

Application of mechanical strain

At confluence, the medium was renewed with FBS-free medium, then the MLO-Y4 cells were stimulated with mechanical tensile strain of 2500 με at 0.5 Hz for 8 h by a four-point bending device, as previously described [18].

Enzyme-linked immunosorbent assay (ELISA)

Following mechanical tensile strain, the expression levels of IGF-1 and PGE2 in the collected culture supernatant were detected using an IGF-1 ELISA kit (Boster Bioengineering Co., Ltd., Wuhan China) and PGE2 EIA kit (Cayman Chemical, Michigan USA), according to the manufacturers’ instructions. An ELISA reader (Thermo Scientific Multiskan FC ELISA Reader, Rockford, IL, USA) was used to measure the absorbances at 450 nm and 420 nm respectively, with the results presented as the content of changes, compared to the unstrained control.

Western blot

Following mechanical tensile strain, cell lysates were prepared in RIPA lysis buffer (Beijing Solarbio Science & Technology, Co. Ltd., Beijing, China). Protein in cell lysates was quantified using the BCA method. Equal amounts of proteins were separated by electrophoresis on a polyacrylamide gel containing 0.15% SDS, then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% skim milk and incubation with primary antibodies overnight at 4 °C, the membranes were incubated with horseradish peroxidase conjugated secondary antibody. The immunoreactive bands were visualized using an ECL detection kit (7 sea biotech Co. Ltd., Shanghai, China). β-actin in cell lysates was used as a loading control. Data were normalized against those of corresponding optical density of β-actin.

Detection of NOS activity

After mechanical tensile strain of MLO-Y4 for 8 h, cells were collected and bathed gently in an ultrasonic processor (UP 400S, Hielscher, Germany) for 2 min. NOS activity was measured using a colorimetric method based on NOS ability to catalyze L-Arg and molecular oxygen to generate NO, and generated NO produces colored compounds with nucleophiles. The optical densities at 530 nm wavelength were obtained using an ELISA reader (Thermo Scientific Multiskan FC ELISA Reader) and activities of NOS were calculated according to the calibration formula provided in the instructions.

Microarray and RT-qPCR validation of miR

The RiboArray miDETECT mouse array (Ribobio Co., Guangzhou, China) and RT-qPCR were used to detect and validate the miR expression levels in MLO-Y4 cells. The miR expression levels of the mechanically strained group were compared with the unstrained group.

Briefly, total RNA extraction and miR enrichment procedures were performed using the Trizol method and an mirVana miR Isolation kit (Ambion Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. Target labeling, hybridization, imaging and data processing were performed, according to the manufacturer’s instructions using a RiboArray miDETECT mouse array (Ribobio Corporation) which contained all mouse miRNAs of Sanger miRBase 19. Data were acquired using Agilent Feature Extraction software version 10.7 (Agilent, Palo Alto, CA, USA) [16, 17]. Further data analyses were performed using GeneSpring GX 10.0 software (Agilent, Palo Alto, CA, USA). Following microarray detection, expression levels of 40 miRs with significant differences were validated by RT-qPCR at Ribobio Co., Ltd. in Guangzhou. The miDETECT A Track Uni-Reverse Primers and miDETECT A Track miRNA Forward Primers (specific primers) for RT-qPCR of these miRs were provided by Ribobio Corporation (Ribobio Co., Ltd. Guangzhou, China). Poly(A) tailing, reverse transcription and qPCR were performed successively using the miDETECT A Track miRNA qRT-PCR Starter Kit (Ribobio Co., Ltd). The reactions were incubated in a 96-well optical plate at 95 °C for 20 s, followed by 40 cycles of 10 s at 95 °C, 20 s at 60 °C, and 10 s at 70 °C. Expression analysis was performed in triplicate for each sample. U6 was used as the normalization control. The miR expression levels were quantified using a CFX 96 system (Bio-Rad Laboratories, Hercules, CA).

miRs transfection and preparation of osteocytes’ conditioned medium

MLO-Y4 osteocyte cells, at 70% confluence, were transfected by miR-29b-3p mimic, miR-29b-3p inhibitor and miR control (Ribobio Co., at a final concentration of 50 nM) respectively, using the riboFect CP Transfection Kit (Ribobio Co.) according to the manufacturer’s method.

The cells were carefully washed in serum-free medium to remove proteins from the bovine serum supplement and then incubated in fresh serum-free medium for 24 h (stimulated with mechanical tensile strain or not). After centrifugation at 3000 g for 12 min, the conditioned medium was collected and prepared for the next experiment.

Detection of osteoblastic differentiation

MC3T3-E1 osteoblastic cells at confluence were carefully washed in serum-free medium, then the cells were cultured in osteocytes’ conditioned medium for 24 h. Cells were harvested, washed with a phosphate buffer solution (PBS), and lysed with a lysis buffer (Beijing Solarbio Science & Technology, Co. Ltd). The ALP activity of the lysates was measured with the ALP Activity Assay Kit (Nanjing Jiancheng Biotechnology Co. Ltd., China) at 25 °C according to the provider’s protocol. The bone morphogenetic protein 2 (BMP-2) of the osteoblastic cells was assayed with an ELISA kit (Elabscience Biotechnology Co., Ltd., Wuhan, China), according to the manufacturer’s instructions mentioned above.

cDNA was synthesized using the TIANScript RT kit (Tiangen Biotech Co., Ltd., Beijing, China), then the Runx 2 mRNA was detected using SYBR Green I PCR Mix (Beijing CoWin Biotech Co., Ltd., Beijing, China) according to the manufacturer’s method. The PCR amplification reaction included a denaturation step at 94 °C for 3 min followed by 40 cycles of 94 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. Relative expression was normalized to mRNA levels of GAPDH using the 2-ΔΔCq method.

Bioinformatics analysis

miRWalk2.0 (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/), MicroRNA.

org (www.microrna.org/) and TargetScan (www.targetscan.org/) were applied to predict target genes for these differently expressed miRs. The same target genes for one corresponding miR presented in three online databases were considered as potential targets.

Statistical analysis

All data are presented as the mean ± standard deviation from three separate experiments (n = 5 or 6). Data were tested for normal distribution using the Shapiro-Wilk test and differences between groups were analyzed using one-way analysis of variance and determined by the least significant difference test. Statistical analysis was performed using SPSS software (version 18; SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered to indicate a statistically significant difference.

Results

We first investigated osteocytes’ response to a physiological mechanical tensile strain of 2500 με at 0.5 Hz for 8 h. As shown in Fig. 1, the results of ELISA for IGF-1 and PGE2, western blot for OPG and biochemical method for NOS activity were all changed after applying the mechanical tensile strain to MLO-Y4 cells. All four factors play important roles in bone formation: IGF-1 is widely distributed and plays a significant role in bone development via endocrine, paracrine and autocrine by combining with its ligands [23, 24], PGE2 is a necessary factor in gap junction mediated intercellular communication in osteocytes [25]. OPG has been reported to promote bone formation and its absence caused onset of osteoporosis and arterial calcification in mice [26], and NOS is closely associated with the expression level of NO, which could regulate the activity of osteoblasts [27].

Fig. 1
figure1

MLO-Y4 cells responded to a mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz for 8 h. a/b After MLO-Y4 cells were stimulated by the mechanical tensile strain, the contents of IGF-1 and PGE2 in the supernatant of cell culture medium were increased (n = 5). c The mechanical tensile strain increased protein levels of OPG (osteoprotegerin) of MLO-Y4 cells (n = 6). d Following mechanical tensile strain, the activities of NOS in the cells were up-regulated (n = 5). *P < 0.05 and **P < 0.01, compared with the unstrained control group

Full size image

Next, we screened and validated 10 miRs’ response to the mechanical tensile strain applied to MLO-Y4 cells. A total of 40 miRs were differentially expressed through a miR microarray screen (Fig. 2). Then, using qPCR, the 40 miRs were validated further. The results of miR microarray and qRT-PCR indentified 10 miRs that were mechanoresponsive miRs: miR-713, miR-706, miR-703, miR-574-3p, miR-467b-3p, miR-466i-5p, miR-466f-5p and miR -208a-3p were up-regulated and miR-29b-3p and miR-361-3p were down-regulated, compared to the unstrained group (Fig. 2, Fig. 3a).

Fig. 2
figure2

A total of forty differentially expressed miRNAs were selected by microarrays. a: up-regulated miRs, b: down-regulated miRs

Full size image
Fig. 3
figure3

In MLO-Y4 osteocytes, miR-29b-3p was one of the ten mechanoresponsive miRs which were confirmed by RT-qPCR. a Based on microarray assay of miRs, the result of RT-qPCR showed that ten miRs were responsive to the mechanical tensile strain, and miR-29b-3p was one of the ten miRs (n = 5). b The miR-29b-3p mimic elevated miR-29b-3p level of osteocyte and the miR -29b-3p inhibitor decreased the miR level (n = 5). c In MC3T3-E1 osteoblastic cells, the mechanical tensile strain did not change the expression of miR-29b-3p, which was demonstrated with microarray and RT-qPCR. Contrarily, in MLO-Y4 osteocytes, the mechanical strain decreased miR-29b-3p expression (n = 5). *P < 0.05 and **P < 0.01, compared with the unstrained control group, or between the indicated groups

Full size image

Bioinformatics was applied to predict target genes for these differently expressed miRs. The results indicated that several target genes were in correlation with bone formation (Table 1). In all of the putative target genes, we found 29 genes that correlated with bone formation and might play important roles in bone formation. Of all the target genes, IGF-1 attracted our attention the most: IGF-1 was a target gene of miR-29b-3p, confirmed by previous studies [16, 17]. In our study, the expression level of IGF-1 was increased (Fig. 1) while the expression level of miR-29b-3p was decreased in mechanically strained osteocytes (Fig. 3a), indicating that miR-29b-3p was likely to regulate bone formation via targeting IGF-1.

Table 1 Predicted target genes of the miRs
Full size table

Finally, we went on study to investigate the involvement of miR-29b-3p in osteocytes’ response to mechanical strain and the effect of the miR on osteoblast differentiation. We found that mechanical tensile strain down-regulated miR-29b-3p expression of MLO-Y4 cells, not MC3T3-E1 cells, and miR-29b-3p inhibited IGF-1 secretion of osteocytes (Fig. 3c). The miR-29b-3p mimic transfection resulted in over-expression of the miR in MLO-Y4 cells, which caused a reduction of IGF-1 levels in osteocytes’ culture supernatant, and miR-29b-3p inhibitor transfection, which resulted in low miR-29b-3p expression, and increased the IGF-1 secretion (Fig. 3b, Fig. 4a). In addition, the conditioned medium of MLO-Y4 cells transfected with miR-29b-3p mimic decreased ALP activity, and reduced expression of BMP-2 and Runx 2 in MC3T3-E1 cells (Fig. 4 b-d). In contrast, the conditioned medium of MLO-Y4 cells transfected with miR-29b-3p inhibitor increased ALP activity and enhanced expression of BMP-2 and Runx 2 in MC3T3-E1 cells (Fig. 4 b-d). These results indicated that miR-29b-3p regulated osteoblast differentiation via osteocyte secretion.

Fig. 4
figure4

miR-29b-3p regulated IGF-1 secretion of MLO-Y4 osteocytes, and after the osteocytes were transfected with miR-29b-3p mimic or miR-29b-3p inhibitor, the osteocytes’ conditioned culture medium influenced osteoblastic differentiation of MC3T3-E1 cells. a miR-29b-3p mimic decreased IGF-1 secretion of osteocytes, and the miR inhibitor increased the IGF-1 secretion (n = 6). The conditioned medium of osteocytes treated with miR-29b-3p mimic reduced ALP activity of osteoblasts (b), BMP-2 protein (c) and Runx 2 mRNA (d) levels of osteoblasts (n = 6). In contrast, the conditioned medium of osteocytes treated with miR-29b-3p inhibitor enhanced ALP activity of osteoblasts (b), BMP-2 protein (c) and Runx 2 mRNA (d) levels of osteoblasts (n = 6). *P < 0.05 and **P < 0.01, compared with the unstrained control group, or between the indicated groups

Full size image

Discussion

In this study, the results indicated that osteocytes responded to a mechanical tensile strain of 2500 με at 0.5 Hz for 8 h. Following osteocytes’ response to this mechanical loading, 10 mechanoresponsive miRs in MLO-Y4 osteocytes were identified, bioinformatics analysis revealed the possible target genes of these miRs, and 29 target genes were correlated with bone formation.

Among the mechanoresponsive miRs and their target genes, the relationship between miR-29b-3p (1 of 10 mechanoresponsive miRNAs) and its target gene, IGF-1, promoted us to find the regulatory relation of osteocytes to osteoblasts under the same mechanical stimulation. In this study, in MC3T3-E1 osteoblastic cells, miR-29b-3p was not involved in osteoblasts’ response to the mechanical strain, and the conditioned medium of the mechanically strained osteocytes promoted osteoblast differentiation of MC3T3-E1 cells. Additionally, the over-expression of miR-29b-3p inhibited osteoblast differentiation via reducing the IGF-1 level in the conditioned culture medium of MLO-Y4 osteocytes which was used to culture MC3T3-E1 cells. During the response to mechanical strain, miR-29b-3p had no direct effect on osteoblasts, and the miR regulated osteoblast differentiation via a mediator: osteocytes, because the mechanical strain up-regulated the cells’ miR-29b-3p, which reduced IGF-1 secretion of MLO-Y4 osteocytes.

Osteocytes in the lacunar-canalicular system of the bone are mainly mechanosensory cells in bone tissue. They transduce mechanical stimulation into biomechanical signals and regulate bone remodeling by regulating the activities of osteoblasts and osteoclasts [19, 20]. In this study, during the response to mechanical strain, miR-29b-3p had no direct effect on osteoblasts, and the miR regulated osteoblast differentiation via a mediator: osteocytes, because mechanical strain up-regulated the cells’ miR-29b-3p, which reduced IGF-1 secretion of MLO-Y4 osteocytes.

It has been reported that osteocytes are responsive to fluid shear stress, microgravity and fluid flow [21, 22]. The results of our study suggested that osteocytes could also respond to mechanical tensile strain and regulate osteoblast differentiation via miR-29b-3p regulating of IGF-1 secretion, which will shed some light on how osteocytes regulate osteogenesis.

Conclusions

Osteocytes responded to a cyclic mechanical tensile strain of 2500 με at 0.5 Hz for 8 h, mechanoresponsive miRs were discovered in osteocytes, miR-29b-3p was responsive to the mechanical tensile strain and the miR regulated osteoblast differentiation via regulating IGF-1 secretion of osteocytes.

Abbreviations

Bcl-2/Bax:

B-cell lymphoma 2/Bax

BMP-2:

Bone morphogenetic protein 2

COX2:

Cyclooxygenase 2

ECL:

Electrochemiluminescence

ELISA:

Enzyme-linked immunosorbent assay

IGF-1:

Insulin-like growth factor-1

miR:

microRNA

NO:

Nitric oxide

NOS:

Nitric oxide synthase

OPG:

Osteoprotegerin

PGE2:

Prostaglandin E2

PVDF:

Polyvinylidene fluoride membrane

RANKL:

Receptor activator of NF-kB ligand

SDS:

Sodium dodecyl sulfate

με:

Microstrain

References

  1. 1.

    Carter DR, Van Der Meulen MC, et al. Mechanical factors in bone growth and development. Bone. 1996;18(1 Suppl):5S–10S.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Bonnet N, Ferrari SL. Exercise and the skeleton: How it works and what it really does. IBMS BoneKEy. 2010;7:235–48.

    Article  Google Scholar 

  3. 3.

    Boerckel JD, Dupont KM, Kolambkar YM, et al. In Vivo Model for Evaluating the Effects of Mechanical Stimulation on Tissue-Engineered Bone Repair. J Biomech Eng. 2009;131:084502.

    Article  PubMed  Google Scholar 

  4. 4.

    Nagaraja MP, Jo H. The Role of Mechanical Stimulation in Recovery of Bone Loss-High versus Low Magnitude and Frequency of Force. Life. 2014;4:117–30.

    Article  PubMed  Google Scholar 

  5. 5.

    Schaffler MB, Cheung W, Majeska RJ, et al. Osteocytes: Master Orchestrators of Bone. Calcif Tissue Int. 2013;94:5–24.

    Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Bakker A, Klein-Nulend J, Burger E. Shear stress inhibits while disuse promotes osteocyte apoptosis. Biochem Biophys Res Commun. 2004;320:1163–8.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Bakker AD, Soejima K, Klein-Nulend J, et al. The production of nitric oxide and prostaglandin E2 by primary bone cells is shear stress dependent. J Biomech. 2001;34:671–7.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Bonewald LF, Johnson ML. Osteocytes, mechanosensing and Wnt signaling. Bone. 2008;42:606–15.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Bartel DP. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell. 2004;116:281–97.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Kapinas K, Delany AM. MicroRNA biogenesis and regulation of bone remodeling. Arthritis Res Ther. 2011;13:220.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Qi Z, Liu W, Lu J. The mechanisms underlying the beneficial effects of exercise on bone remodeling: Roles of bone-derived cytokines and microRNAs. Prog Biophys Mol Biol. 2016;122:131–9.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Wang H, Sun Z, Wang Y, et al. miR-33-5p, a novel mechano-sensitive microRNA promotes osteoblast differentiation by targeting Hmga2. Sci Rep. 2016;6:23170.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Qi L, Zhang Y. The microRNA 132 Regulates Fluid Shear Stress-Induced Differentiation in Periodontal Ligament Cells through mTOR Signaling Pathway. Cell Physiol Biochem. 2014;33:433–45.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Guo Y, Wang Y, Liu Y, et al. MicroRNA-218, microRNA-191*, microRNA-3070a and microRNA-33 are responsive to mechanical strain exerted on osteoblastic cells. Mol Med Report. 2015;12:3033–8.

    CAS  Article  Google Scholar 

  15. 15.

    Li Z, Hassan MQ, Jafferji M, et al. Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem. 2009;284:15676–84.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Yuan L, Zhou C, Lu Y, et al. IFN-γ-mediated IRF1/miR-29b feedback loop suppresses colorectal cancer cell growth and metastasis by repressing IGF1. Cancer Lett. 2015;359(1):136–47.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Abonnenc M, Nabeebaccus AA, Mayr U, et al. Extracellular matrix secretion by cardiac fibroblasts: role of microRNA-29b and microRNA-30c. Circ Res. 2013;113(10):1138–47.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Liu L, Guo Y, Wan Z, et al. Effects of Phytoestrogen α-ZAL and Mechanical Stimulation on Proliferation, Osteoblastic Differentiation, and OPG/RANKL Expression in MC3T3-E1 Pre-Osteoblasts. Cell Mol Bioeng. 2012;5:427–39.

    CAS  Article  Google Scholar 

  19. 19.

    Thi MM, Suadicani SO, Schaffler MB, et al. Mechanosensory responses of osteocytes to physiological forces occur along processes and not cell body and require αVβ3 integrin. Proc Natl Acad Sci USA. 2013;110:21012–7.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Pajevic PD. Regulation of bone resorption and mineral homeostasis by osteocytes. Ibms Bonekey. 2009;6:63–70.

    Article  Google Scholar 

  21. 21.

    Yang X, Sun LW, Wu XT, et al. Impact of shear stress and simulated microgravity on osteocytes using a new rotation cell culture device. Acta Astronaut. 2015;116:286–98.

    CAS  Article  Google Scholar 

  22. 22.

    Cherian PP, Sillerjackson AJ, Gu S, et al. Mechanical Strain Opens Connexin 43 Hemichannels in Osteocytes: A Novel Mechanism for the Release of Prostaglandin. Mol Biol Cell. 2005;16:3100–6.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Juffer P, Jaspers RT, Lips P, et al. Expression of muscle anabolic and metabolic factors in mechanically loaded MLO-Y4 osteocytes. Am J Physiol Endocrinol Metab. 2012;302:E389–95.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Sheng MH, Zhou XD, Bonewald LF, et al. Disruption of the insulin-like growth factor-1 gene in osteocytes impairs developmental bone growth in mice. Bone. 2013;52(1):133–44.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Cheng B, Kato Y, Zhao S, et al. PGE2 is essential for gap junction-mediated intercellular communication between osteocyte-like MLO-Y4 cells in response to mechanical strain. Endocrinology. 2001;142:3464–73.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Zheng C, Chu P, Wu CC, et al. Association between Increased Serum Osteoprotegerin Levels and Improvement in Bone Mineral Density after Parathyroidectomy in Hemodialysis Patients. Tohoku J Exp Med. 2012;226:19–27.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    van't Hof RJ, Ralston SH. Nitric oxide and bone. Immunology. 2001;103(3):255–61.

    Article  Google Scholar 

  28. 28.

    Skerry TM. Identification of novel signaling pathways during functional adaptation of the skeleton to mechanical loading: the role of glutamate as a paracrine signaling agent in the skeleton. J Bone Miner Metab. 1999;17:66–70.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Harper JA, Yuan JS, Tan JB, et al. Notch signaling in development and disease. Clin Genet. 2003;64:461–72.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Proff P, Römer P. The molecular mechanism behind bone remodelling: a review. Clin Oral Investig. 2009;13:355–62.

    Article  PubMed  Google Scholar 

  31. 31.

    Qiu W, Andersen TE, Bollerslev J, et al. Patients with high bone mass phenotype exhibit enhanced osteoblast differentiation and inhibition of adipogenesis of human mesenchymal stem cells. J Bone Miner Res. 2007;22:1720–31.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Gao J, Cheng TS, Qin A, et al. Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43 in osteocytes. Oncotarget. 2016;7:26966–78.

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Yan B, Li J, Zhang L. Identification of B cells participated in the mechanism of postmenopausal women osteoporosis using microarray analysis. Int. J. Clin. Exp. Med. 2015;8:1027–34.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Schindeler A. Pre-clinical Fracture Repair Studies: Meeting Report from the 31st Annual Meeting of the American Society for Bone and Mineral Research. Ibms Bonekey. 2009;6:481–4.

    Article  Google Scholar 

  35. 35.

    Siddappa R, Martens A, Doorn J, et al. cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo. Proc Natl Acad Sci. 2008;105:7281–6.

    CAS  Article  Google Scholar 

  36. 36.

    Sevetson B, Taylor S, Pan Y. Cbfa1/RUNX2 Directs Specific Expression of the Sclerosteosis Gene (SOST). J Biol Chem. 2004;279:13849–58.

    CAS  Article  Google Scholar 

  37. 37.

    Takaoka K, Koezuka M, Nakahara H. Telopeptide-depleted bovine skin collagen as a carrier for bone morphogenetic protein. J Orthop Res. 1991;9:902–7.

    CAS  Article  Google Scholar 

  38. 38.

    Urist MR. Bone: formation by autoinduction. Science. 1965;150:893–9.

    CAS  Article  Google Scholar 

  39. 39.

    Fukada T, Hojyo S, Furuichi T. Zinc signal: a new player in osteobiology. J Bone Miner Metab. 2013;31:129–35.

    CAS  Article  Google Scholar 

  40. 40.

    Hoffmann A, Gross G. BMP signaling pathways in cartilage and bone formation. Crit Rev Eukaryot Gene Expr. 2001;11:23.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Kobayashi T, Lu J, Cobb BS, et al. Dicer-dependent pathways regulate chondrocyte proliferation,differentiation. Proc Natl Acad Sci USA. 2008; 105:1949–1954.

  42. 42.

    Whitfield JF. The solitary (primary) cilium-a mechanosensory toggle switch in bone and cartilage cells. Cell Signal. 2008;20:1019–24.

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Lian J, Stein GS. Runx2/Cbfa1: a multifunctional regulator of bone formation. Curr Pharm Des. 2003;9:2677–85.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Schatteman GC, Morrison-Graham K, van Koppen A, et al. Regulation and role of PDGF receptor alpha-subunit expression during embryogenesis. Development. 1992;115:123–31.

    CAS  PubMed  Google Scholar 

  45. 45.

    Kilian KA, Bugarija B, Lahn BT, et al. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc Natl Acad Sci. 2010;107:4872–7.

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Hishiya A, Ito M, Aburatani H, et al. Ataxia telangiectasia mutated (Atm) knockout mice as a model of osteopenia due to impaired bone formation. Bone. 2005;37:497–503.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Krishnan V, Moore TL, Ma Y, et al. Parathyroid hormone bone anabolic action requires Cbfa1/Runx2-dependent signaling. Mol Endocrinol. 2003;17:423–35.

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Day TF, Yang Y. Wnt and hedgehog signaling pathways in bone development. J Bone Joint Surg. 2008;90(Supplement 1):19–24.

    Article  PubMed  Google Scholar 

  49. 49.

    Srinivasan S, Ausk BJ, Bain SD, et al. Rest intervals reduce the number of loading bouts required to enhance bone formation. Med Sci Sports Exerc. 2015;47:1095–103.

    Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Lieben L. Bone: The circadian clock controls bone remodelling. Nat Rev Rheumatol. 2016;12:132–3.

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    van der Meulen MC, Boskey AL. Atypical subtrochanteric femoral shaft fractures: role for mechanics and bone quality. Arthritis Res Ther. 2012;14:220.

    Article  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Kwan Tat S, Padrines M, Théoleyre S, et al. IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology. Cytokine Growth Factor Rev. 2004;15:49–60.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

The authors are grateful to Xi Lu and Junchen Li at the College of Biotechnology, Guilin Medical University, for their tremendous support.

Funding

This study was supported by grants from the National Nature Science Foundation of China (No. 31660261, No. 11432016) and the Natural Science Foundation of Guangxi (2018JIA140050, 2016GXNSFAA380322).

Availability of data and materials

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Author information

Author notes

  1. Qiangcheng Zeng and Yang Wang contributed equally to this work.

Affiliations

  1. key laboratory of Functional Bioresource Utilization in University of Shandong, Shandong Key Laboratory of Biophysics, Dezhou University, Dezhou, 253023, China

    Qiangcheng Zeng, Jie Gao, Zhenghua Li & Yong Guo

  2. Department of Biomedical Engineering, College of Biotechnology, Guilin Medical University, No. 1 Zhiyuan Road, Lingui District, Guilin City, 541100, Guangxi, China

    Yang Wang, Zhixiong Yan, Xianqiong Zou, Yanan Li, Jiahui Wang & Yong Guo

  3. Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044, China

    Yang Wang

  4. Medical Department, Secondary Renmin Hospital of Dezhou, Dezhou, 253023, Shangdong, China

    Jie Gao

Authors
  1. Qiangcheng Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jie Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhixiong Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhenghua Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xianqiong Zou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yanan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jiahui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yong Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

QZ designed the study, analyzed data, and prepared conditioned medium; YW generated mechanical tensile strain, performed ELISA and Western blot; ZY performed all RT-qPCR analyses and assisted in the bioinformatics analysis; ZL and JG participated in Western blot and ELISA and performed bioinformatics analysis; YL cultured the cells; JW participated in cell culture and detected NOS activities; YG supervised the experiments and drafted the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Yong Guo.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zeng, Q., Wang, Y., Gao, J. et al. miR-29b-3p regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically stimulated osteocytes. Cell Mol Biol Lett 24, 11 (2019). https://doi.org/10.1186/s11658-019-0136-2

Download citation

Keywords

  • Mechanical tensile strain
  • Osteocyte
  • Osteoblast differentiation
  • miRNA microarray

Cellular & Molecular Biology Letters

ISSN: 1689-1392

Contact us