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MicroRNA Tough Decoy Knockdowns miR-195 and Represses Hypertrophy in Chondrocytes

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Abstract

Cartilage hypertrophy is a condition in which the cells are completely differentiated, and new morphological changes and mineralization prevent proper cellular functions. The occurrence of hypertrophy during differentiation fails current regenerative strategies for treatment. Strategies to minimize hypertrophy in chondrocytes are categorized into two levels of protein and gene. Among these strategies, one way to affect multiple pathways involved in the development of hypertrophy is to manage microRNA activity in cells. Recent miRNA profiling studies have shown that miR-195-5p upregulates through the transition from chondrogenic to hypertrophic state. Bioinformatics assessment of microRNA targets also indicates that several genes repressed by miR-195-5p play important roles in processes related to hypertrophy. The aim of this study was to develop a microRNA Tough Decoy to suppress miR-195-5p and investigate whether it can prevent a hypertrophic state in chondrocytes. The Tough Decoy (TUD) was designed and evaluated bioinformatically and then cloned into the pLVX-Puro plasmid. The TUD function was validated by Dual-Luciferase assay and qRT-PCR. After delivering TUD to C28/I2 chondrocytes cultured in a hypertrophic medium, hypertrophic differentiation was assessed by histochemical staining, quantitative RT-PCR of hypertrophy marker genes, and alkaline phosphatase activity. Results showed that the TUD could inhibit miRNA efficiently and downregulate hypertrophic markers such as RUNX2, alkaline phosphatase, and collagen 10 significantly compared with the control group. Alcian blue and alizarin red staining also demonstrated the optimal effect of gene constructs on tissue properties and mineralization of the TUD group. Delivering the miR-195-5p Tough Decoy to the cartilage cells can prevent the occurrence of hypertrophy in chondrocytes and could be considered as a candidate for the treatment of other diseases such as osteoarthritis.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Funding

This research was financially supported by the deputy of research, Guilan University of Medical Sciences, Iran (grant number: IR-96123).

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Authors and Affiliations

  1. Student Research Committee, Guilan University of Medical Sciences, Rasht, Iran

    Pejman Abbasi Pashaki

  2. Thalassemia and Hemoglobinopathy Research Centre, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

    Fakher Rahim

  3. Department of Medical Biotechnology, School of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran

    Mehryar Habibi Roudkenar & Ammar Ebrahimi

  4. Medical Biotechnology Research Center, School of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran

    SMT Razavi-Toosi & Ammar Ebrahimi

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  1. Pejman Abbasi Pashaki
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  4. SMT Razavi-Toosi
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Contributions

P.A.P.: performed experiments and co-wrote the paper. F.R.: performed experiments. M.H.R.: designed and performed experiments. SMT R.T.: co-wrote the paper. A.E.: designed and performed experiments, performed bioinformatics analyses, analyzed data, and co-wrote the paper.

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Correspondence to Ammar Ebrahimi.

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Abbasi Pashaki, P., Rahim, F., Habibi Roudkenar, M. et al. MicroRNA Tough Decoy Knockdowns miR-195 and Represses Hypertrophy in Chondrocytes. Appl Biochem Biotechnol 191, 1056–1071 (2020). https://doi.org/10.1007/s12010-020-03229-6

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