Abstract
Low electric treatment (LET) promotes intracellular delivery of naked siRNA by altering cellular physiology. However, which signaling molecules and cellular events contribute to LET-mediated siRNA uptake are unclear. Here, we used isobaric tags in relative and absolute quantification (iTRAQ) proteomic analysis to identify changes in the levels of phosphorylated proteins that occur during cellular uptake of siRNA promoted by LET. iTRAQ analysis revealed that heat shock protein 90 (Hsp90)α and myristoylated alanine-rich C-kinase substrate (Marcks) were highly phosphorylated following LET of NIH 3T3 cells, but not untreated cells. Furthermore, the levels of phosphorylated Hsp90α and protein kinase C (PKC)γ were increased by LET both with siRNA and liposomes having various physicochemical properties used as model macromolecules, suggesting that PKCγ activated partly by Ca2+ influx as well as Hsp90 chaperone function were involved in LET-mediated cellular siRNA uptake. Furthermore, LET with siRNA induced activation of Rho GTPase via Hsp90 and PKC, which could contribute to cellular siRNA uptake accompanied by actin cytoskeleton remodeling. Collectively, our results suggested that LET-induced Rho GTPase activation via Hsp90 and PKC would participate in actin-dependent cellular uptake of siRNA.
Highlights
Www.nature.com/scientificreports cellular uptake efficacy of nucleic acids between Low electric treatment (LET) and electroporation is needed to be directly compared, LET without cytotoxicity is expected to be more useful drug delivery methods than electroporation
The 15 proteins that had the largest upregulation in phosphorylation following LET were: filamin C (Flnc), isoform 5 of heterogeneous nuclear ribonucleoproteins C1/C2 (Hnrnpc), protein Njmu-R(5730455P16Rik), 28 kDa heat- and acid-stable phosphoprotein (Pdap1), protein LYRIC (Mtdh), high mobility group protein B1 (Hmgb1), myristoylated alanine-rich C-kinase substrate (Marcks), elongation factor 1-β (Eef1b2), AHNAK nucleoprotein isoform 1 (Ahnak), heat shock protein HSP 90-α (Hsp90aa[1] or Hsp90α), stress-70 protein, mitochondrial (Hspa9), poliovirus receptor (Pvr), Isoform 1 of E3 ubiquitin-protein ligase HUWE1 (Huwe1), protein AHNAK2 (Ahnak2) and pumilio 2 (Pum2) (Table 1)
To understand cellular events activated by Hsp90α or PKCγ in LET-mediated cellular uptake of siRNA, we focused on the actin cytoskeleton that is www.nature.com/scientificreports linked to the plasma membrane[38]
Summary
Www.nature.com/scientificreports cellular uptake efficacy of nucleic acids between LET and electroporation is needed to be directly compared, LET without cytotoxicity is expected to be more useful drug delivery methods than electroporation. We previously reported that various functional nucleic acids including siRNA delivered by LET have their functionality in the cells[19,20]. We previously showed that protein kinase C (PKC) is activated through influx of calcium ions into cells during transdermal delivery of cationic liposomes by LET22. To clarify the cellular uptake mechanisms involved in LET, in the current study we used isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to identify proteins that have altered phosphorylation status following LET. In this study we show that the proteins showing up-regulated phosphorylation following LET promote actin cytoskeleton remodeling via activation of Rho GTPase that is associated with cellular siRNA uptake
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