MiR-154-5p regulates osteogenic differentiation of adipose-derived mesenchymal stem cells under tensile stress through the Wnt/PCP pathway by targeting Wnt11

doi:10.1016/j.bone.2015.05.003

Bone

Volume 78, September 2015, Pages 130-141

https://doi.org/10.1016/j.bone.2015.05.003 Get rights and content

Highlights

•
We investigate the mechanism of mechanotransduction from the miRNA perspective.
•
Mechanical tension down-regulates the expression of miR-154-5p.
•
MiR-154-5p inhibition promotes osteogenic differentiation of ADSCs.
•
Wnt11 is a new direct target of miR-154-5p.
•
MiR-154-5p-Wnt11-Wnt/PCP may be an important mechanotransduction pathway.

Abstract

Mechanical stress is a well-acknowledged positive regulatory factor for osteogenic differentiation of adipose- derived mesenchymal stem cells (ADSCs). However, the molecular mechanisms associated with micro-RNAs (miRNAs) whereby ADSCs respond to mechanical stimuli remain elusive. We investigated the mechanism of mechanotransduction from the miRNA perspective in the osteogenic differentiation of ADSCs under tensile stress. Microarray analysis showed that miR-154-5p was remarkably downregulated when ADSCs were subjected to mechanical tension. Bioinformatics analysis with luciferase reporter assays demonstrated that Wnt11 3′UTR was a new direct target of miR-154-5p. Under tensile stress, lentivirus-mediated gain- or loss-of-function studies revealed that forced expression of miR-154-5p inhibited osteogenic differentiation of ADSCs, whereas inhibition of endogenous miR-154-5p with its antisense oligonucleotide (ASO-154-5p) obviously promoted osteogenic differentiation. Furthermore, miR-154-5p overexpression decreased activity of the non-canonical Wnt/PCP (RhoA-ROCK) pathway, as indicated by lower expression of Wnt11, active RhoA and ROCKII in miR-154-5p-treated ADSCs. By contrast, miR-154-5p inhibition activated the Wnt/PCP signals. Taken together, these results demonstrate that, under tensile stress, miR-154-5p negatively regulates ADSCs osteogenic differentiation through the Wnt/PCP pathway by directly targeting Wnt11. This novel regulatory pathway provides new insights into the molecular mechanism of mechanotransduction in osteogenic differentiation of ADSCs.

Introduction

Adipose-derived mesenchymal stem cells (ADSCs) are self-renewing and multipotent cells which hold great promise in potential treatments for tissue regeneration [1], [2]. These cells differentiate along several committed phenotypes, including osteogenic lineages, in response to multiple environmental stimuli, using complex pathways regulated at both the transcriptional and posttranscriptional levels [3], [4], [5]. However, the regulation of cellular osteogenic pathways is not fully elucidated. Investigation of the molecular mechanism of ADSC osteogenic differentiation could help explain the pathogenesis of such skeletal diseases as osteoporosis and lead to the development of possible regenerative therapies.

Mechanical stress is an important factor controlling bone remodeling, a process which maintains bone morphology and function [6], [7]. In previous research, we subjected ADSCs to a cyclic uniaxial tensile strain to achieve optimal differentiation of ADSCs towards the osteogenic lineage; these cells showed an enhanced osteogenic capability [8]. Mechanical stress, converted into the biochemical responses known as mechanotransduction, can promote the osteogenic differentiation of mesenchymal stem cells (MSCs) [9], [10]. Significant recent attention has focused on the molecular mechanisms underlying the enhanced osteogenic properties of cells under mechanical stress [11], [12], [13]. However, the exact mechanisms of mechanotransduction are far from understood and still present a challenge.

A focus of intense research, micro-RNAs (miRNAs) are small (~ 22 nucleotide long), non-coding, single-stranded RNAs that negatively regulate gene expression posttranscriptionally by imperfect or perfect binding to the 3ˊ untranslated region (3ˊ-UTR) of target mRNAs, leading to inhibition of translation or mRNA degradation [14], [15], [16]. miRNAs are considered key regulators of biologic processes in stem cells, including self-renewal, development, differentiation, growth, and metabolism [17], [18]. Recent microarray analyses have revealed that a cohort of miRNAs are involved in stem cell osteogenic differentiation, and the effects of miRNAs on the osteoblastic differentiation of stem cells have been investigated by modulation of miRNA function [19], [20], [21]. For instance, miR-138 and miR-30e directly target focal adhesion kinase (FAK) and low-density lipoprotein receptor-related protein 6 (LRP6), respectively, and inhibit the differentiation of osteoprogenitors of MSCs [22], [23]. MiR-26a has been reported to inhibit the translation of the osteogenic marker SMAD1 and inhibit the osteogenesis of human ADSCs, whereas miR-346 promotes osteogenic differentiation by inhibiting glycogen synthase kinase 3β (GSK-3β) and activating the Wnt/β-catenin pathway [24], [25]. Despite these studies showing the miRNA regulation of the osteogenic differentiation of stem cells, no reports have investigated the mechanism by which mechanical stimuli is transduced into biological signal from the miRNA perspective during the osteogenic differentiation of ADSCs under tension stress.

The Wnt signal pathway is a complex protein interaction network. Wnt proteins activate cell surface receptor-mediated signal transduction pathways, regulating a variety of cellular activities. At least 19 Wnt proteins have been identified in mammals; Wnt1, 2, 3, 3a, 6 and 10b (the canonical Wnts) and Wnt4, 5a, 5b, 7a and 11 (non-canonical). Three Wnt signal pathways have been identified: the canonical Wnt/β-catenin pathway, the noncanonical Wnt/PCP pathway and the noncanonical Wnt/Ca^2 + pathway [26], [27], [28], [29]. In the last decade, Wnt/β-catenin signaling has been the most studied and many ex vivo and in vivo studies have demonstrated the importance of the canonical Wnt pathway in the osteogenic differentiation of MSCs and in bone mass accrual and maintenance [5], [25], [30], [31]. Recently, the regulatory role of non-canonical Wnts on osteogenic differentiation of MSCs has been of increasing interest. Some reports show that mechanical stimulation can affect the osteogenic differentiation of stem cells through the non-canonical Wnt pathway [32], [33], [34]. Thus, the non-canonical Wnt pathway may be significant in mechanical signal transduction for stem cells under mechanic stimuli, and the non-canonical related miRNA serve as an important challenge.

In this study, we validated that mechanical stress promotes osteogenic differentiation of murine ADSCs, screening for differentially expressed miRNAs induced by mechanical stimulation. Then, combining miRNA microarray with bioinformatics analyses and identifying non-canonical Wnt-related mRNA as a preferential target, we found that non-canonical Wnt11 3′UTR was a potential target gene of the screened miR-154-5p. The dual luciferase reporter assay in 293 T cells and the GFP/RFP reporter assay in ADSCs confirmed that Wnt11 was a direct target of miR-154-5p. Furthermore, our gain- or loss-of-function experiments demonstrated that under mechanical stress miR-154-5p inhibited the Wnt/PCP pathway by repressing Wnt11 mRNA translation in ADSCs and preventing the osteogenic differentiation of ADSCs. To our knowledge, this study is the first to investigate the mechanism by which mechanical stimuli is transduced into biological signal from the miRNA perspective. Understanding the miRNA molecular events involved in osteogenic differentiation under tension stress may lead to novel therapeutic interventions to promote bone formation.

Section snippets

ADSC isolation and culture

Male C57BL/6 mice (6 weeks old) were obtained from the Sichuan University Animal Experimental Center, and all procedures were approved by the Animal Research Committee of Sichuan University. Inguinal fat pads were finely minced and digested with 0.2% collagenase type I in a 37 °C shaking incubator for 45 min. The digested tissue was filtered through sterile 100-um nylon mesh, centrifuged at 1000 rpm for 8 min, resuspended and cultured in regular growth medium, consisting of Minimum Essential Medium

Characterization of mouse ADSCs

Oil-red staining experiments showed that mouse ADSCs differentiated into the adipogenic lineage phenotype after one week (Fig. 1B). Alizarin red staining experiments showed that the cells differentiated into the osteogenic lineages phenotype after two weeks (Fig. 1C). Flow cytometry analysis of passage 3 mouse ADSCs showed that the cells were positive for CD29 (98.75 ± 1.83%), Sca-1 (94.81 ± 1.43%) and CD105 (56.13 ± 0.74%), but negative for the hematopoietic stem cell markers CD45 (1.04 ± 0.42%) and

Discussion

In recent years, cell loading systems in vitro have been widely used to simulate the biomechanical environment of cells in vivo [37], [38]. In our study, we chose tension caused by a four-point bending mechanical loading device as the mechanical stimulation. We used 2,000 με of strain to simulate physiological stimulation as it is widely considered equivalent to physiological loading in vivo [39]. Under mechanical stress, our murine ADSCs showed enhanced osteogenic capability, indicated by

Conclusion

In this, the first investigation into the mechanism of mechanotransduction from the miRNA perspective in osteogenic differentiation of ADSCs under tension stress, we provide a novel mechanism by which mechanical tension is translated into biochemical signals to stimulate the osteogenic response in ADSCs. Mechanical tension down-regulated the expression of miR-154-5p and induced Wnt11 up-regulation, promoting osteogenic differentiation of murine ADSCs. Furthermore, by modulating miR-154-5p

Disclosure of potential conflicts of interest

No conflicts of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 10502037, 31070833), the Science and Technology Foundation of Sichuan Province (No. 2010GZ0225, 2011GZ0335 and 2009SZ0139) and the Cooperation Science Foundation between Sichuan university and Luzhou city (No. 2013CDLZ-S19).

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^☆

Author contributions:

Jianwei Li.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing;

Chen Hu: conception and design, collection and assembly of data;

Lu Han: data analysis and interpretation, manuscript writing;

Lei Liu: Data analysis and interpretation;

Wei Jing: conception and design, Provision of study material;

Wei Tang: Administrative support;

Weidong Tian: conception and design;

Jie Long: conception and design , manuscript writing, final approval of manuscript.

¹: Co-first author: The first three authors equally contributed to this work.

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Original Full Length ArticleMiR-154-5p regulates osteogenic differentiation of adipose-derived mesenchymal stem cells under tensile stress through the Wnt/PCP pathway by targeting Wnt11☆

Highlights

Abstract

Introduction

Section snippets

ADSC isolation and culture

Characterization of mouse ADSCs

Discussion

Conclusion

Disclosure of potential conflicts of interest

Acknowledgments

Trends Biotechnol

Mutat Res

Cell

Trends Genet

Trends Cell Biol

Cell Signal

Dev Cell

J Biol Chem

Bone

Cell Stem Cell

Trends Biotechnol

Semin Cell Dev Biol

Dev Biol

Biomaterials

Dev Cell

Concise review: human adipose-derived stem cells: separating promise from clinical need

Stem Cells

Adipose-derived stem cells for regenerative medicine

Circ Res

Adipose-derived stem cells: isolation, characterization, and differentiation potential

Cell Transplant

Review of signaling pathways governing MSC osteogenic and adipogenic differentiation

Scientifica

Periosteal bone formation stimulated by externally induced bending strains

J Bone Miner Res

Morphologic stages in lamellar bone formation stimulated by a potent mechanical stimulus

J Bone Miner Res

The osteogenic response of undifferentiated human adipose-derived stem cells under mechanical stimulation

Cells Tissues Organs

Primary cilia-mediated mechanotransduction in human mesenchymal stem cells

Stem Cells

Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway

Stem Cells Dev

Mechanical strain inhibits adipogenesis in mesenchymal stem cells by stimulating a durable beta-catenin signal

Endocrinology

Original Full Length Article
MiR-154-5p regulates osteogenic differentiation of adipose-derived mesenchymal stem cells under tensile stress through the Wnt/PCP pathway by targeting Wnt11☆