Role of autophagy in cell-penetrating peptide transfection model

Abstract

Cell-penetrating peptides (CPPs) uptake mechanism is still in need of more clarification to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by PepFect14 (PF14), with or without oligonucleotide cargo on gene expression, in HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed by qPCR analysis. The gene regulations were then related to the biological processes by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection efficiency of splice correction oligonucleotide complexed with PepFect14, proving that the autophagy process is induced upon the uptake of complexes. Finally, the autophagy induction and colocalization with autophagosomes have been confirmed by confocal microscopy and transmission electron microscopy. We conclude that autophagy, an inherent cellular response process, is triggered by the cellular uptake of CPP-based transfection system. This finding opens novel possibilities to use autophagy modifiers in future gene therapy.

Figure 1

Autophagy pathway induced by transfection with PF14.

Figure 2

PF14 induced changes in the transcriptome of HeLa cells. The most relevant changes in gene expression are expressed as log₂ of fold change as estimated by qPCR and RNA sequencing. RNA sequencing and qPCR analysis on RNA extracted from HeLa cells after 1 h treatment with PF14 (a) or PF14 and splice correction oligo (PF14/SCO) nano-complexes (b). The cycle threshold obtained from qPCR has been transformed into fold change by using a set of references genes (q < 0.01).

Figure 3

Downregulation of splice correction activity in a concentration-dependent manner by using small molecule ligands. (a,b) HeLa pLuc/705 cells treated for 24 h with PF14 and splice correction oligo (PF14/SCO) nano-complexes and splice correction suppressing ligands [CCG 203971 (CTGF inhibitor), PF 573228 (focal adhesion kinase (FAK) inhibitor), Atorvastatin LDL (HMG-CoA reductase inhibitor), Prostaglandin E2]. Splice correction activity is based on luminescence signal from splice corrected luciferase. Splice correction activity was normalized to the baseline signal of untreated cells, and 100% corresponds to treatment with PF14/SCO complexes (P < 0.0001).

Figure 4

Upregulation of splice correction activity in concentration-dependent manner by using small molecule ligands. (a,b) HeLa pLuc/705 cells treated for 24 h with PF14 and splice correction oligo (PF14/SCO) nano-complexes and splice correction activating ligands [Pifithrin-μ (selective inhibitor of heat shock protein 70 (HSP70)), Alprenolol hydrochloride (β-adrenoceptor antagonist), CH-223191 (aryl hydrocarbon receptor (AHR) antagonist), Importazole (nuclear transport receptor importin-β inhibitor), TLR-4-IN-C34 (TLR4 inhibitor), MHY1485 (mTOR activator)]. Splice correction activity is based on luminescence signal from splice corrected luciferase. Splice correction activity was normalized to the baseline signal of untreated cells, and 100% corresponds to PF14/SCO complexes treatment (P < 0.0001).

Figure 5

Induction of autophagy by PF14 and its complexes with SCO. (a) Epifluorescence micrographs of HeLa pLuc/705 cells treated for 24 h with Rapamycin (RAP) 500 nM, or 2 μM PF14, or PF14 and splice correction oligo labelled with Alexa 568 nano-complexes (PF14/SCOAlexa568) (red, formed at molar ratio 10/1) with or without 6 µM focal adhesion kinase inhibitor (PF573228), 10 µM Alprenolol hydrochloride (β-adrenoceptor antagonist), 10 nM mTOR activator (MHY1485), 500 nM RAP, 76 nM TLR-4-IN-C34 (TLR4 inhibitor). Immunofluorescence of LC3B as autophagy marker protein is shown (green), arrows point to SCO colocalized with the nucleus. (b) Quantification of green immunofluorescence signal of LC3B is with the same treatments as in (a). Data were normalized to the baseline green signal of untreated cells, and the 100% as a signal from treatment with RAP alone without PF14/SCO complexes. (c) Quantification of the red signal from PF14/SCO(Alexa568) complexes in the nucleus in the treatments of (a) which have the complexes, data was normalized to the baseline red signal of untreated cells, and the 100% is PF14/SCO(Alexa568) complexes. (d) HeLa pLuc/705 cells treated with PF14/SCO complexes with or without starvation for 4 h with EPSS medium, 500 nM RAP, 500 nM Bafilomycin A (BAFA), 500 nM Wortmannin for 24 h in full DMEM medium. Splice correction activity is based on luminescence signal from splice corrected luciferase. Splice correction activity was normalized to the baseline signal of untreated cells, and the 100% is PF14/SCO complexes treatment. (e) Western blot of LC3B for HeLa pLuc/705 cells lysates, the cells were untreated or treated with 500 nM RAP, PF14, PF14/SCO nano-complexes with or without 500 nM BAFA. LC3BII/LC3BI protein ratios were calculated from band intensities normalized to β-actin bands intensities. Scale bar is 10 µm (*P < 0.0351,**P < 0.0033, ****P < 0.0001).

Figure 6

Analysis of the cellular uptake of PF14/pDNA complexes and induction of autophagy using transmission electron microscopy. HeLa cells were incubated with PF14/pDNA nano-complexes (0.1 μg/ml CR 2) tagged with 10 nm colloidal gold particle (black dots) for 1 h (a,b) 4 h (c–f), 8 h (g,h) or 24 h (i,j). Localization of PF14/pDNA complexes (pointed by arrows) in early (a,b) and late endosomal organelles (c–f); in autophagosomes (g,h) and autolysosomes (i,j). The control cells were treated with 0.5 μM rapamycin for 24 h to promote the formation of autophagosomes and autolysosomes (k,l). Arrows point to PF14/pDNA nano-complexes in the particular organelles, and arrowheads show the fragmentary regions in the limiting membrane of endosomes. Scale bar is 200 nm.

Figure 7

Molecular docking analysis of possible PF14 interactions with HSP70 protein. PF14 peptide is shown in orange, HSP70 is shown in green and hydrogen bonds are shown in blue dashes.