Hasan, Mahadi Kyoto Pharmaceutical University|Tokushima University|JSPS
Hama, Susumu Kyoto Pharmaceutical University
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.
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srep_9_4114.pdf 2.06 MB