Volume 48, Issue 1 (4-2024)
Research in Medicine 2024, 48(1): 28-37 |
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Ethics code: IR.NIGEB.EC.1400.2.26.C
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Khani M, Javeri A, Bazargani A, Karimi G, Fakhr Taha M. Investigating the Effect of miR-302/367 Cluster Overexpression on the Expression of mTOR Signaling Pathway Genes in Human Adipose Tissue- Derived Stem Cells. Research in Medicine 2024; 48 (1) :28-37
URL: http://pejouhesh.sbmu.ac.ir/article-1-3319-en.html
URL: http://pejouhesh.sbmu.ac.ir/article-1-3319-en.html
Department of Stem Cells and Regenerative Medicine, Institute for Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. , mftaha@nigeb.ac.ir
Abstract: (1005 Views)
Background and Aim: The miR-302/367 cluster plays a critical role in reprogramming of somatic cells into pluripotent stem cells and in maintaining pluripotency. Until now, several studies have been conducted on the mechanism of miR-302/367 cluster in reprogramming, while little research has been done specifically on the effect of this cluster on mTOR signaling pathway. The mTOR pathway not only controls various cellular processes, such as proliferation, differentiation and autophagy, but also plays a significant role in the reprogramming process. Therefore, the present study was designed to identify the effect of miR-302/367 cluster on the mTOR pathway in adipose tissue-derived stem cells (ADSCs).
Methods: In this experimental study, the third to fifth- passaged ADSCs were transfected with TDH101PA- GP vector expressing mir-302/367 cluster and the mock vector using a Neon Transfection Kit and Neon Transfection System. One week after transfection, the expression of some mTOR signaling molecules in the ADSCs was assessed by comparative Real- Time PCR. Relative gene expression between the miR-302/367 and mock groups (4 replicates) was calculated by REST 2009 software based on Pair Wise Reallocation Randomization Test. Also, the expression of some mTOR pathway proteins was assessed by western blot analysis.
Results: The overexpression of miR-302/367 cluster significantly reduced the expression of AKT, MTOR, RAPTOR, and RICTOR genes; the expression of these genes in the miR-302/367 transfection group compared to the mock group decreased to 0.44, 0.51, 0.56 and 0.6, respectively (P< 0.05). The expression of genes in the mock group was assumed as one. Also, as revealed by western blot analysis, the expression of phosphorylated AKT and phosphorylated MTOR proteins decreased in the ADSCs transfected with miR- 302/367 cluster compared to the mock group.
Conclusion: Overexpression of the miR-302/367 cluster appears to inhibit the activity of the mTOR signaling pathway in ADSCs and affect the cell reprogramming through this mechanism.
Methods: In this experimental study, the third to fifth- passaged ADSCs were transfected with TDH101PA- GP vector expressing mir-302/367 cluster and the mock vector using a Neon Transfection Kit and Neon Transfection System. One week after transfection, the expression of some mTOR signaling molecules in the ADSCs was assessed by comparative Real- Time PCR. Relative gene expression between the miR-302/367 and mock groups (4 replicates) was calculated by REST 2009 software based on Pair Wise Reallocation Randomization Test. Also, the expression of some mTOR pathway proteins was assessed by western blot analysis.
Results: The overexpression of miR-302/367 cluster significantly reduced the expression of AKT, MTOR, RAPTOR, and RICTOR genes; the expression of these genes in the miR-302/367 transfection group compared to the mock group decreased to 0.44, 0.51, 0.56 and 0.6, respectively (P< 0.05). The expression of genes in the mock group was assumed as one. Also, as revealed by western blot analysis, the expression of phosphorylated AKT and phosphorylated MTOR proteins decreased in the ADSCs transfected with miR- 302/367 cluster compared to the mock group.
Conclusion: Overexpression of the miR-302/367 cluster appears to inhibit the activity of the mTOR signaling pathway in ADSCs and affect the cell reprogramming through this mechanism.
Keywords: miR-302/367 cluster, Transfection, mTOR pathway, ADSC, AKT gene, MTOR gene, RICTOR gene, RAPTOR gene, Phosphorylated AKT, Phosphorylated MTOR
Type of Study: Original |
Subject:
Cellular Sciences (Molecular Cells, Stem Cells)
Received: 2023/08/23 | Accepted: 2023/12/10 | Published: 2024/04/22
Received: 2023/08/23 | Accepted: 2023/12/10 | Published: 2024/04/22
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