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Evaluation of osteogenic induction potency of miR-27a-3p in adipose tissue-derived human mesenchymal stem cells (AD-hMSCs)

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Abstract

Background

Bone tissue as a dynamic tissue is able to repair its minor injuries, however, sometimes the repair cannot be completed by itself due to the size of lesion. In such cases, the best treatment could be bone tissue engineering. The use of stem cells in skeletal disorders to repair bone defects has created bright prospects. On the other hand, changes in the expression level of microRNAs (miRs) can lead to the commitment of mesenchymal stem cells (MSCs) to cell lineage. Many studies reported that post-transcriptional regulations by miRNAs are involved in all stages of osteoblast differentiation.

Method

After the preparing adipose tissue-derived mesenchymal stem cells, the target cells from the third passage were cultured in two groups, transfected MSCs with miR-27a-3p (DM.C + P) and control group. In different times, 7 and 14 days after culture, differentiation of these cells into osteoblast were measured using various techniques including the ALP test and calcium content test, Alizarin Red staining, Immunocytochemistry technique (ICC). Also, the relative expression of bone differentiation marker genes including Osteonectin (ON), Osteocalcin (OC), RUNX Family Transcription Factor 2 (RUNX2), Collagen type I alpha 1 (COL1) was investigated by real-time RT PCR.

Results

In comparison with control groups, overexpression of miR-27a-3p in transfected cells resulted in a significant increase in the expression of bone markers genes (ON, OC, RUNX2, COL1), alkaline phosphatase (ALP) activity, and calcium content (p < 0.05). In addition, the results obtained from ICC technique showed that osteocalcin protein is expressed at the surface of bone cells. Furthermore, the expression of APC, as a target of miR-27a-3p, decreased in transfected cells.

Conclusion

Our data suggest that miR-27a-3p may positively regulates adipose tissue-derived mesenchymal stem cell differentiation into bone by targeting APC and activating the Wnt/b-catenin pathway.

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Abbreviations

WNT:

Wnt/β-catenin pathway

TGF-β:

TGF-beta signaling

BMP:

Bone morphogenetic proteins

FGF :

Fibroblast growth factors

OC :

Osteocalcin

ON :

Osteonectin

COL1 :

Collagen type I alpha 1

RUNX2 :

RUNX Family Transcription Factor 2

MiRs:

MicroRNAs

MSC:

Mesenchymal Stem Cells

ADMSCs:

Adipose-derived mesenchymal stem cells

APC :

Adenomatous polyposis coli

hBMSCs:

Human bone marrow stem cells

DMEM:

Dulbecco's Modified Eagle Medium

ICC:

Immunocytochemistry

ALP :

Alkaline phosphatase

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Funding

The authors would like to appreciate Research Deputy of Mashhad University of Medical Sciences for supporting this study (IR.MUMS.REC.1400.329).

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Authors and Affiliations

  1. Department of Medical Genetics and Molecular Medicine Department, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

    Fatemeh Arab, Alireza Pasdar & Ehsan Saburi

  2. Assistant Professor of Medical Biotechnology, Department of Medical Biotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

    Seyed Hamid Aghaee Bakhtiari

  3. Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

    Alireza Pasdar & Ehsan Saburi

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  1. Fatemeh Arab
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Correspondence to Alireza Pasdar or Ehsan Saburi.

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Arab, F., Aghaee Bakhtiari, S.H., Pasdar, A. et al. Evaluation of osteogenic induction potency of miR-27a-3p in adipose tissue-derived human mesenchymal stem cells (AD-hMSCs). Mol Biol Rep 50, 1281–1291 (2023). https://doi.org/10.1007/s11033-022-08084-8

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