Non‑viral transfection methods optimized for miRNA delivery to human dermal fibroblasts

Kucharski,Mirosław; Mrowiec,Patrycja; Białka,Szymon; Misiołek,Hanna; Misiołek,Maciej; Sechman,Andrzej; Zięba-Przybylska,Dorota; Ocłoń,Ewa

doi:10.3892/mmr.2023.12976

Non‑viral transfection methods optimized for miRNA delivery to human dermal fibroblasts

Authors:
- Mirosław Kucharski
- Patrycja Mrowiec
- Szymon Białka
- Hanna Misiołek
- Maciej Misiołek
- Andrzej Sechman
- Dorota Zięba-Przybylska
- Ewa Ocłoń
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Affiliations: Department of Animal Physiology and Endocrinology, Anatomy and Genomics, University of Agriculture in Krakow, 30-059 Krakow, Poland, Department of Animal Reproduction, Anatomy and Genomics, University of Agriculture in Krakow, 30-059 Krakow, Poland, Department of Anesthesiology and Intensive Care, Faculty of Medical Sciences in Zabrze, Medical University of Silesia, 40-055 Katowice, Poland, Clinical Department of Otorhinolaryngology and Laryngological Oncology, Faculty of Medical Sciences in Zabrze, Medical University of Silesia, 40-055 Katowice, Poland, Department of Animal Nutrition and Biotechnology, and Fisheries, University of Agriculture in Krakow, 30-248 Krakow, Poland, Centre for Experimental and Innovative Medicine, Laboratory of Recombinant Proteins Production, University of Agriculture in Krakow, 30-248 Krakow, Poland
Published online on: March 7, 2023 https://doi.org/10.3892/mmr.2023.12976
Article Number: 89

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Abstract

Fibroblasts are beneficial model cells for in vitro studies and are frequently used in tissue engineering. A number of transfection reagents have been employed to deliver microRNAs (miRNAs/miRs) into cells for genetic manipulation. The present study aimed to establish an effective method of transient miRNA mimic transfection into human dermal fibroblasts. The experimental conditions included three different methods: Physical/mechanical nucleofection, and two lipid‑based methods, Viromer^® Blue and INTERFERin^®. To evaluate the impact of these methods, cell viability and cytotoxicity assays were performed. The silencing effect of miR‑302b‑3p was revealed to alter the expression levels of its target gene carnitine O‑octanoyltransferase (CROT) by reverse transcription‑quantitative PCR. The present study showed that all selected non‑viral transient transfection systems exhibited good efficiency. It was also confirmed that nucleofection, for which a 21.4‑fold decrease in the expression of the CROT gene was observed 4 h after 50 nM hsa‑miR‑302b‑3p transfection, was the most effective method. However, these results indicated that lipid‑based reagents can maintain the silencing effect of miRNAs up to 72 h after transfection. In summary, these results indicated that nucleofection may be the optimal method for the transport of small miRNA mimics. However, lipid‑based methods allow for the use of lower concentrations of miRNA and maintain longer‑lasting effects.

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