Inhibitory effect of HSP27 siRNA complexes with exosomes on growth and differentiation of human SH-SY5Y neuroblastoma cell line

Abstract

Heat shock proteins participate in the regulation of different cell activities in response to stimuli. By applying different strategies, the modulation of heat shock proteins is at the center of attention. Conventional delivery approaches are not fully encouraged due to cytotoxicity and immunogenicity issues. Exosomes are touted as bio-shuttles for delivery of distinct biomolecules inside the cells. Here, we aimed to HSP27 siRNA-tagged exosomes for the inhibition of HSP27 in human neuroblastoma cell line SH-SY5Y and explored differentiation into neuron-like cells. Methods: Exosomes were isolated, characterized by SEM and CD63 then enriched with siRNA against HSP27. Neuroblastoma cells were incubated with exosomes carrying siRNA for 48 hours. Exosome uptake was monitored by immunofluorescence assay. Cell viability and proliferation were analyzed using MTT and BrdU incorporation assays. The ability of cells to form colonies was evaluated by clonogenic assay. Cell potential to express NeuN, a mature neuron factor, was studied by flow cytometry analysis. Results: SEM showed the nano-sized particles and a high level of CD63 after enrichment. Immunofluorescence imaging revealed an appropriate transfection rate in cell exposed to HSP27 siRNA tagged exosomes. Cell viability and proliferation were reduced compared to cells received nude exosomes (p<0.05). Clonogenic activity of cells was diminished by the inhibition of HSP27. Flow cytometry analysis revealed that the inhibition of HSP27 prohibited NeuN content, showing the maturation of SH-SY5Y cells to mature cells compared to control.