Abstract
Primary hepatocyte cell cultures are widely used for studying hepatic diseases with alterations in hepatic glucose and lipid metabolism, such as diabetes and non-alcoholic fatty liver disease. Therefore, small interfering RNAs (siRNAs) provide a potent and specific tool to elucidate the signaling pathways and gene functions involved in these pathologies. Although RNA interference (RNAi) in vitro is frequently used in these investigations, the metabolic alterations elucidated by different siRNA delivery strategies have hardly been investigated in transfected hepatocytes. To elucidate the influence of the most commonly used lipid-based transfection reagents on cultured primary hepatocytes, we studied the cytotoxic effects and transfection efficiencies of INTERFERin®, Lipofectamine®RNAiMAX, and HiPerFect®. All of these transfection agents displayed low cytotoxicity (5.6–9.0 ± 1.3–3.4 %), normal cell viability, and high transfection efficiency (fold change 0.08–0.13 ± 0.03–0.05), and they also favored the satisfactory down-regulation of target gene expression. However, when effects on the metabolome and lipidome were studied, considerable differences were observed among the transfection reagents. Cellular triacylglycerides levels were either up- or down-regulated [maximum fold change: INTERFERin® (48 h) 2.55 ± 0.34, HiPerFect® (24 h) 0.79 ± 0.08, Lipofectamine®RNAiMAX (48 h) 1.48 ± 0.21], and mRNA levels of genes associated with lipid metabolism were differentially affected. Likewise, metabolic functions such as amino acid utilization from were perturbed (alanine, arginine, glycine, ornithine, and pyruvate). In conclusion, these findings demonstrate that the choice of non-viral siRNA delivery agent is critical in hepatocytes. This should be remembered, especially if RNA silencing is used for studying hepatic lipid homeostasis and its regulation.
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Acknowledgments
We thank Rolf Gebhardt for valuable discussion and Doris Mahn, Fatina Siwczak, Robert Karandi, and Claudia Eser for excellent technical assistance. We also thank Maria Thomas for fruitful discussion at the beginning of the project. This work was founded by the German Federal Ministry of Education and Research (BMBF; VIRTUAL-LIVER network, Grants FKZ 0315735, 0315736, and 0315775). UH was supported by the Robert-Bosch Foundation (Stuttgart, Germany).
Author contributions
MMS realized the transfection experiments. JB realized the metabolome experiments, and UH made the metabolome measurements. SV made the metabolome heat map. MMS and JB contributed the cell culture and cytotoxicity and viability experiments expertise. MMS and CT realized the lipidome experiments, and SS and AS made the lipidome measurements. KA and CR contributed to qPCR experiments. MMS and JB developed the concept and wrote the manuscript.
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Böttger, J., Arnold, K., Thiel, C. et al. RNAi in murine hepatocytes: the agony of choice—a study of the influence of lipid-based transfection reagents on hepatocyte metabolism. Arch Toxicol 89, 1579–1588 (2015). https://doi.org/10.1007/s00204-015-1571-0
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DOI: https://doi.org/10.1007/s00204-015-1571-0