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

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 transfec- tion, 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.