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.
Mol Med Rep. 2023 Apr;27(4). doi: 10.3892/mmr.2023.12976. Epub 2023 Mar 10.
Fibroblasts are beneficial model cells for 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 () 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 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.
成纤维细胞是研究的有益模型细胞，常用于组织工程。许多转染试剂已被用于将 microRNAs (miRNAs/miRs) 递送入细胞进行遗传操作。本研究旨在建立一种有效的瞬时 miRNA 模拟转染入人真皮成纤维细胞的方法。实验条件包括三种不同的方法：物理/机械转染、两种基于脂质的方法 Viromer Blue 和 INTERFERin。为了评估这些方法的影响，进行了细胞活力和细胞毒性测定。miR-302b-3p 的沉默作用揭示了其靶基因肉碱 O-辛酰基转移酶 () 的表达水平通过逆转录-定量 PCR 改变。本研究表明，所有选择的非病毒瞬时转染系统均表现出良好的效率。还证实了转染，其中观察到 50 nM hsa-miR-302b-3p 转染后 4 小时 基因的表达降低了 21.4 倍，是最有效的方法。然而，这些结果表明，基于脂质的试剂可以在转染后 72 小时保持 miRNA 的沉默效果。总之，这些结果表明，转染可能是小分子 miRNA 模拟物运输的最佳方法。然而，基于脂质的方法允许使用更低浓度的 miRNA 并保持更长时间的效果。
