Lipofectamine 2000™ at transfection dose promotes EphA2 transcription in an HDAC4-dependent manner to reduce its cytotoxicity

The cationic liposome is well-known as an efficient nucleic acid delivery tool; however, the stress responses induced by liposome per se have been rarely revealed. In this study, we found that Lipofectamine™ 2000 (lipo2000), a commonly used commercial cationic liposome transfection, could upregulate EphA2 mRNA expression in multiple cells at transfection dose. Furthermore, lipo2000 treatment could increase the level of EphA2 hnRNA (heterogeneous nuclear RNA). Lipo2000-induced EphA2 upregulation could be depleted upon global transcription inhibition, proving that lipo2000 upregulates EphA2 expression via activating its transcription. Moreover, HDAC4 depletion, a known EphA2 trans-acting regulatory factor, could eliminate the lipo2000-induced EphA2 upregulation, demonstrating that lipo2000 promotes EphA2 transcription in an HDAC4 dependent manner. Functionally, EphA2 knockdown did not affect GFP expression level and the interfering efficacy of siGAPDH, suggesting that EphA2 is unrelated to the nucleic acid delivery capacity of lipo2000. Nevertheless, EphA2 depletion significantly activated autophagy and apoptosis, increasing the cytotoxic effects of lipo2000, which could be rescued by EphA2 restoration, indicating that EphA2 is essential to overcome liposome-related cytotoxicity. Finally, we found that lipo2000 could activate EphA2 transcription in an HDAC4-dependent manner. EphA2 is not associated with the transfection efficiency of lipo2000, but it is vital to reduce lipo2000 cytotoxicity, suggesting that when conducting liposome-mediated gene function studies, especially for EphA2, the stress response of liposomes should be considered to obtain objective results.