Skip to main content
SpringerLink
Log in

Effect of miR-26a-5p on the Wnt/Ca2+ Pathway and Osteogenic Differentiation of Mouse Adipose-Derived Mesenchymal Stem Cells

  • Original Research
  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Abstract

Elucidation of the molecular mechanisms that regulate the differentiation of adipose-derived mesenchymal stem cells into osteogenic cells may lead to new methods for bone tissue engineering. We examined the role of miR-26a-5p in the regulation of osteogenic differentiation of mouse adipose-derived mesenchymal stem cells (mADSCs) by using mimics and inhibitors of this microRNA. Our results showed that over-expression of miR-26a-5p inhibited osteogenesis and that suppression of endogenous miR-26a-5p promoted osteogenesis. Four bioinformatics algorithms indicated that the 3′UTR of Wnt5a was a potential target of miR-26a-5p. We confirmed this prediction by use of dual-luciferase reporter assay and GFP/RFP assay. We also examined the molecular mechanisms by which miR-26a-5p regulates osteogenesis. Fura-2AM and Western blot assays after transfection indicated that miR-26a-5p repressed WNT5A, inhibited calcium flux and protein kinase C, and suppressed osteogenic differentiation of mADSCs. By contrast, miR-26a-5p inhibition activated these signal proteins and promoted osteogenic differentiation. Taken together, our results suggest that up-regulation of miR-26a-5p inhibits osteogenic differentiation of mADSCs by directly targeting the 3′UTR of Wnt5a, thereby down-regulating the Wnt/Ca2+ signaling pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

ADSCs:

Adipose-derived mesenchymal stem cells

mADSCs:

Mouse adipose-derived mesenchymal stem cells

hADSCs:

Human adipose-derived mesenchymal stem cells

MSCs:

Mesenchymal stem cells

miRNAs:

MicroRNAs

miR-26a:

MicroRNA-26a

miR-NC:

MicroRNA-26a-5p negative control

UTR:

Untranslated regions

CDK6:

Cyclin-dependent kinase 6

HDAC4:

Histone deacetylase 4

USSCs:

Unrestricted somatic stem cells

BMP:

Bone morphogenetic protein

GSK-3β:

Glycogen synthase kinase -3β

PCP:

Planar cell polarity

ROCK:

Rho-associated kinase

PBS:

Phosphate buffer saline

FBS:

Fetal calf serum

qRT-PCR:

Quantitative real-time polymerase chain reaction

GFP:

Green fluorescent protein

RFP:

Red fluorescent protein

MOI:

Multiplicity of infection

WT:

Wild type

Mu:

Mutation

PKC:

Protein kinase C

ALP:

Alkaline phosphatase

OCN:

Osteocalcin

OPN:

Osteopontin

COL1:

Collagen type 1

Runx2:

Runt homology domain transcription factor 2

Osx:

Osterix

CaMKII:

Calmodulin-dependent protein kinase II

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

ARS:

Alizarin red staining

D-HBSS:

D-Hanks balanced salt solution

Dsh:

Dishevelled

RhoA:

Ras homolog gene family, member A

JNK:

c-Jun N-terminal kinase

Fz3:

Frizzled receptor 3

Ror2:

Receptor tyrosine kinase-like orphan receptor 2

P-PDLSCs:

Periodontal ligament stem cells from chronic periodontitis patients

References

  1. Lu CH, Chang YH, Lin SY, Li KC, Hu YC (2013) Recent progresses in gene delivery-based bone tissue engineering. Biotechnol Adv 31:1695–1706

    Article  CAS  PubMed  Google Scholar 

  2. Sriram M, Sainitya R, Kalyanaraman V, Dhivya S, Selvamurugan N (2015) Biomaterials mediated microRNA delivery for bone tissue engineering. Int J Biol Macromol 74:404–412

    Article  CAS  PubMed  Google Scholar 

  3. Konno M, Hamabe A, Hasegawa S, Ogawa H, Fukusumi T, Nishikawa S, Ohta K, Kano Y, Ozaki M, Noguchi Y, Sakai D, Kudoh T, Kawamoto K, Eguchi H, Satoh T, Tanemura M, Nagano H, Doki Y, Mori M, Ishii H (2013) Adipose-derived mesenchymal stem cells and regenerative medicine. Dev Growth Differ 55:309–318

    Article  CAS  PubMed  Google Scholar 

  4. Peppo GMD, Marolt D (2013) Modulating the biochemical and biophysical culture environment to enhance osteogenic differentiation and maturation of human pluripotent stem cell-derived mesenchymal progenitors. de Peppo Marolt Stem Cell Res Therapy 4:106

    Google Scholar 

  5. Grottkau BE, Lin Y (2013) Osteogenesis of adipose—derived stem cells. Bone Res 2:133–145

    Article  Google Scholar 

  6. Zhang Z, Wang J, Lu X (2014) An integrated study of natural hydroxyapatite-induced osteogenic differentiation of mesenchymal stem cells using transcriptomics, proteomics and microRNA analyses. Biomed Mater 9:045005

    Article  PubMed  Google Scholar 

  7. Martin EC, Qureshi AT, Dasa V, Freitas MA, Gimble JM, Davis TA (2015) MicroRNA regulation of stem cell differentiation and diseases of the bone and adipose tissue: perspectives on miRNA biogenesis and cellular transcriptome. Biochimie. doi:10.1016/j.biochi.2015.02.012

    Google Scholar 

  8. Zhang J, Tu Q, Bonewald LF, He X, Stein G, Lian J, Chen J (2011) Effects of miR-335-5p in modulating osteogenic differentiation by specifically downregulating Wnt antagonist DKK1. J Bone Mineral Res 26:1953–1963

    Article  CAS  Google Scholar 

  9. Huszar JM, Payne CJ (2014) MIR146A inhibits JMJD3 expression and osteogenic differentiation in human mesenchymal stem cells. FEBS Lett 588:1850–1856

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zeng Y, Qu X, Li H, Huang S, Wang S, Xu Q, Lin R, Han Q, Li J, Zhao RC (2012) MicroRNA-100 regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells by targeting BMPR2. FEBS Lett 586:2375–2381

    Article  CAS  PubMed  Google Scholar 

  11. Li Z, Hassan MQ, Volinia S, van Wijnen AJ, Stein JL, Croce CM, Lian JB, Stein GS (2008) A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci USA 105:13906–13911

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Luzi E, Marini F, Sala SC, Tognarini I, Galli G, Brandi ML (2008) Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Mineral Res 23:287–295

    Article  CAS  Google Scholar 

  13. Trompeter H-I, Dreesen J, Hermann E, Iwaniuk KM, Hafner M, Renwick N, Tuschl T, Wernet P (2013) MicroRNAsmiR-26a, miR-26b, and miR-29b accelerate osteogenic differentiation of unrestricted somatic stem cells from human cord blood. BMC Genomics 14:1471–2164

    Article  Google Scholar 

  14. Luzi E, Marini F, Tognarini I, Galli G, Falchetti A, Brandi ML (2012) The regulatory network menin-microRNA 26a as a possible target for RNA-based therapy of bone diseases. Nucl Acid Ther 22:103–108

    CAS  Google Scholar 

  15. Li Y, Fan L, Hu J, Zhang L, Liao L, Liu S, Wu D, Yang P, Shen L, Chen J, Jin Y (2015) MiR-26a rescues bone regeneration deficiency of mesenchymal stem cells derived from osteoporotic mice. Mol Therapy J Am Soc Gene Therapy 23:1349–1357

    Article  CAS  Google Scholar 

  16. Li Y, Fan L, Liu S, Liu W, Zhang H, Zhou T, Wu D, Yang P, Shen L, Chen J, Jin Y (2013) The promotion of bone regeneration through positive regulation of angiogenic-osteogenic coupling using microRNA-26a. Biomaterials 34:5048–5058

    Article  CAS  PubMed  Google Scholar 

  17. Su X, Liao L, Shuai Y, Jing H, Liu S, Zhou H, Liu Y, Jin Y (2015) MiR-26a functions oppositely in osteogenic differentiation of BMSCs and ADSCs depending on distinct activation and roles of Wnt and BMP signaling pathway. Cell Death Dis 6:e1851

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wang Y, Li Y-P, Paulson C, Shao J-Z, Zhang X, Wu M, Chen W (2014) Wnt and the Wnt signaling pathway in bone development and disease. Front Biosci (Landmark Ed) 19:379–407

    Article  Google Scholar 

  19. Veeman MT, Axelrod JD, Moon RT (2003) A second canon: functions and mechanisms of β-catenin-independent Wnt signaling. Dev Cell 5:367–377

    Article  CAS  PubMed  Google Scholar 

  20. Lin SS, Ueng SW, Niu CC, Yuan LJ, Yang CY, Chen WJ, Lee MS, Chen JK (2014) Hyperbaric oxygen promotes osteogenic differentiation of bone marrow stromal cells by regulating Wnt3a/beta-catenin signaling–an in vitro and in vivo study. Stem Cell Rese 12:260–274

    Article  CAS  Google Scholar 

  21. Egea V, Zahler S, Rieth N, Neth P, Popp T, Kehe K, Jochum M, Ries C (2012) Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/beta-catenin signaling. Proc Natl Acad Sci USA 109:E309–E316

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Liu N, Shi S, Deng M, Tang L, Zhang G, Liu N, Ding B, Liu W, Liu Y, Shi H, Liu L, Jin Y (2011) High levels of beta-catenin signaling reduce osteogenic differentiation of stem cells in inflammatory microenvironments through inhibition of the noncanonical Wnt pathway. J Bone Mineral Res 26:2082–2095

    Article  CAS  Google Scholar 

  23. Tu X, Joeng KS, Nakayama KI, Nakayama K, Rajagopal J, Carroll TJ, McMahon AP, Long F (2007) Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation. Dev Cell 12:113–127

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Li J, Hu C, Han L, Liu L, Jing W, Tang W, Tian W, Long J (2015) MiR-154-5p regulates osteogenic differentiation of adipose-derived mesenchymal stem cells under tensile stress through the Wnt/PCP pathway by targeting Wnt11. Bone 78:130–141

    Article  CAS  PubMed  Google Scholar 

  25. Santos A, Bakker AD, de Blieck-Hogervorst JM, Klein-Nulend J (2010) WNT5A induces osteogenic differentiation of human adipose stem cells via rho-associated kinase ROCK. Cytotherapy 12:924–932

    Article  CAS  PubMed  Google Scholar 

  26. Chakravorty N, Ivanovski S, Prasadam I, Crawford R, Oloyede A, Xiao Y (2012) The microRNA expression signature on modified titanium implant surfaces influences genetic mechanisms leading to osteogenic differentiation. Acta Biomater 8:3516–3523

    Article  CAS  PubMed  Google Scholar 

  27. Lin YF, Jing W, Wu L, Li XY, Wu Y, Liu L, Tang W, Long J, Tian WD, Mo XM (2008) Identification of osteo-adipo progenitor cells in fat tissue. Cell Prolif 41:803–812

    Article  CAS  PubMed  Google Scholar 

  28. Yoshizawa S, Brown A, Barchowsky A, Sfeir C (2014) Magnesium ion stimulation of bone marrow stromal cells enhances osteogenic activity, simulating the effect of magnesium alloy degradation. Acta Biomater 10:2834–2842

    Article  CAS  PubMed  Google Scholar 

  29. Santos A, Bakker AD, Zandieh-Doulabi B, Semeins CM, Klein-Nulend J (2009) Pulsating fluid flow modulates gene expression of proteins involved in Wnt signaling pathways in osteocytes. J Orthop Res 27:1280–1287

    Article  CAS  PubMed  Google Scholar 

  30. Hagenmueller M, Riffel JH, Bernhold E, Fan J, Katus HA, Hardt SE (2014) Dapper-1 is essential for Wnt5a induced cardiomyocyte hypertrophy by regulating the Wnt/PCP pathway. FEBS Lett 588:2230–2237

    Article  CAS  PubMed  Google Scholar 

  31. Vivancos V, Chen P, Spassky N, Qian D, Dabdoub A, Kelley M, Studer M, Guthrie S (2009) Wnt activity guides facial branchiomotor neuron migration, and involves the PCP pathway and JNK and ROCK kinases. Neural Dev 4:7

    Article  PubMed  PubMed Central  Google Scholar 

  32. Nitzki F, Zibat A, Konig S, Wijgerde M, Rosenberger A, Brembeck FH, Carstens PO, Frommhold A, Uhmann A, Klingler S, Reifenberger J, Pukrop T, Aberger F, Schulz-Schaeffer W, Hahn H (2010) Tumor stroma-derived Wnt5a induces differentiation of basal cell carcinoma of Ptch-mutant mice via CaMKII. Cancer Res 70:2739–2748

    Article  CAS  PubMed  Google Scholar 

  33. Hutchins BI, Li L, Kalil K (2011) Wnt/calcium signaling mediates axon growth and guidance in the developing corpus callosum. Dev Neurobiol 71:269–283

    Article  CAS  PubMed  Google Scholar 

  34. Dissanayake SK, Weeraratna AT (2008) Detecting PKC phosphorylation as part of the Wnt/calcium pathway in cutaneous melanoma. Methods Mol Biol 468:157–172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Olivares-Navarrete R, Hyzy SL, Hutton DL, Dunn GR, Appert C, Boyan BD, Schwartz Z (2011) Role of non-canonical Wnt signaling in osteoblast maturation on microstructured titanium surfaces. Acta Biomater 7:2740–2750

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Baksh D, Boland GM, Tuan RS (2007) Cross-talk between Wnt signaling pathways in human mesenchymal stem cells leads to functional antagonism during osteogenic differentiation. J Cell Biochem 101:1109–1124

    Article  CAS  PubMed  Google Scholar 

  37. Green JL, Kuntz SG, Sternberg PW (2008) Ror receptor tyrosine kinases: orphans no more. Trends Cell Biol 18:536–544

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Oishi I, Suzuki H, Onishi N, Takada R, Kani S, Ohkawara B, Koshida I, Suzuki K, Yamada G, Schwabe GC, Mundlos S, Shibuya H, Takada S, Minami Y (2003) The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway. Genes Cells 8:645–654

    Article  CAS  PubMed  Google Scholar 

  39. Mikels AJ, Nusse R (2006) Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context. PLoS Biol 4:e115

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 10502037, 31070833, and 31570950), the Science and Technology Foundation of Sichuan Province (Nos. 2010GZ0225, 2011GZ0335, and 2009SZ0139), and the Cooperation Science Foundation between Sichuan University and Luzhou city (No. 2013CDLZ-S19).

Author information

Author notes

    Authors and Affiliations

    1. The State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, 610041, People’s Republic of China

      Shasha Li, Lei Liu, Wei Jing, Wei Tang, Weidong Tian & Jie Long

    2. Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, Chengdu, 610041, People’s Republic of China

      Jie Long

    3. Department of Oral and Maxillofacial Surgery, General Hospital of Ningxia Medical University, Yinchuan, 750004, People’s Republic of China

      Chen Hu

    4. Department of Oral and Maxillofacial Surgery, Binzhou Medical University Hospital, Binzhou, 256603, People’s Republic of China

      Jianwei Li

    Authors
    1. Shasha Li
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Chen Hu
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Jianwei Li
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Lei Liu
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Wei Jing
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Wei Tang
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Weidong Tian
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Jie Long
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Jie Long.

    Ethics declarations

    Conflict of interest

    Shasha Li, Chen Hu, Jianwei Li, Lei Liu, Wei Jing, Wei Tang, Weidong Tian, and Jie Long have declared no conflict of interest.

    Human and Animal Rights and Informed Consent

    All experiments were approved by the Animal Research Committee of Sichuan University and were conducted in accordance with the guidelines for the management and handling of experimental animals.

    Additional information

    Shasha Li, Chen Hu, and Jianwei Li are co-first author and equally contributed to this work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Li, S., Hu, C., Li, J. et al. Effect of miR-26a-5p on the Wnt/Ca2+ Pathway and Osteogenic Differentiation of Mouse Adipose-Derived Mesenchymal Stem Cells. Calcif Tissue Int 99, 174–186 (2016). https://doi.org/10.1007/s00223-016-0137-3

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s00223-016-0137-3

    Keywords

    Search

    Navigation