The FaDu cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS; both Thermo Fisher Scientific, Inc., Waltham, MA USA) depleted of exosomes, 100 U/ml penicillin and 0.1 mg/ml streptomycin. The cells were incubated in an atmosphere of 5% CO₂ at 37°C.

The exosomes were prepared from 293 cells purchased from the American Type Culture Collection. Briefly, 5 ml DMEM with 10% exosome-depleted FBS was added to 60 mm diameter culture dish containing 293 cells (2×10⁶/ml). Following 48 h of incubation, the cells were harvested and the culture medium was centrifuged at 2,000 × g for 30 min at 4°C to eliminate cells and cell debris. The remaining supernatant was mixed with polyethylene glycol (PEG) (16,17). The exosomes were precipitated with an equal volume of PEG buffer (160 g/l PEG and 1 M NaCl) overnight at 4°C and centrifuged at 10,000 × g for 1 h at 4°C. The supernatant was removed, leaving the exosomes in the bottom of the tube. The exosomes were resuspended in 10 µl PBS, and a bicinchoninic acid protein assay kit (BestBio, Shanghai, China) was used to determine the protein concentration. The exosomes were stored at −80°C until use.

The diameters of the exosome/siRNA (5′-AACCUGUUCUGUGUGGUCAGGUUAU-3′; Biomics Biotechnologies Co., Ltd., Jiangsu, China) nanoparticles in water were analyzed at 25°C. The exosome was fixed with 1 ml of 2.5% glutaraldehyde in 0.1 M sodium cacodylate solution (pH 7.0) for 1 h at 4°C. Samples were subsequently embedded in pure low viscosity embedding mixture using Spurr Low Viscosity Embedding kit (cat. no. 01916-1; Polysciences Inc. Warrington, PA, USA) using the embedding mold, according to the manufacturer's protocols, and baked for 24 h at 65°C. Transmission electron microscopy (HT7700; Hitachi, Ltd., Tokyo, Japan) was used to obtain images, operating at an acceleration voltage of 100 kV. The sample solution with TRPP2 siRNA (exosome/siRNA, 4:5 µg/nM) was placed onto a 300-mesh copper grid coated with carbon. Following deposition for 5 min, the surface water was removed with filter paper and air-dried. A 4 wt% aqueous uranyl acetate solution was used for positive staining at room temperature for 20 min.

In the present study, it was assessed whether the exosome/TRPP2 siRNA complex enhanced the stability of TRPP2 siRNA in the presence of RNA nucleases or serum obtained from mice. Following incubation with nucleases for 0, 5, 15, 30, 60 and 120 min at 37°C, or incubation with serum for 120 min, heparin was added to release the TRPP2 siRNA [4:5 ratio of exosomes to TRPP2 siRNA (µg/nM)] in the exosome/TRPP2 siRNA complex groups. Agarose (0.9 g) was added to 100 ml Tris-acetate-EDTA buffer (40 mM Tris, 20 mM NaAc and 1 mM EDTA at pH 8.0) and dissolved by heating to 100°C. An ethidium bromide aqueous solution at a final concentration of 0.5 µg/ml was added to the dissolved gel. As the solution cooled to <50°C, the gel solidified. Once completely solidified, the gel was placed in a tank filled with electrophoresis solution. Electrophoresis was conducted at 30 V for 20 min, and the gel was placed under UV light to observe the siRNA bands. Exosomes (20 or 40 µl) containing proteins (4 µg/µl) were mixed with 200 nM TRPP2 siRNA, with or without serum obtained from mice (cat. no. HQ30078; Hongquan Biotechnology Co., Guangzhou, China).

Target proteins in FaDu cells were examined by western blotting, which was performed as previously described (18). The proteins were extracted from lysates of FaDu cells with a detergent extraction buffer that consisted of 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM disodium salt of EDTA, 0.1% SDS, 1% Triton X-100, and 1% sodium deoxycholate, sodium orthovanadate, sodium fluoride and leupeptin. A total of 30 µg protein per lane was separated via SDS-PAGE on a 10% gel and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with Tris-buffered saline solution containing 10% nonfat milk and Tween-20 (0.1%) for 1 h at room temperature to block nonspecific binding sites. For immunoblotting, the membrane with the transferred proteins was incubated with specific primary antibodies overnight at 4°C (BIOSS, Beijing, China; anti-CD63, cat. no. bs-1523R) (Santa Cruz Biotechnology, Dallas, Texas, USA; anti-TRPP2 cat. no. sc-25749; anti-vimentin cat. no. sc-373717; anti-E-cadherin cat. no. sc-8426; and anti-N-cadherin cat. no. sc-393933), diluted 1:200. Subsequently, the membrane was washed with PBS in triplicate and incubated with the respective horseradish peroxidase-conjugated secondary antibody (dilution, 1:5,000; cat. no. E-AB-1003; Elabscience Biotechnology Co., Ltd, Wuhan City, China) at room temperature for 1 h. The resulting immunosignals were detected using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The optical densities of the protein bands were analyzed using ImageJ 1.50 software (National Institutes of Health, Bethesda, MD, USA). All protein bands were normalized to GAPDH located in the same lane and the results are expressed as the relative optical density.

Purified exosomes were labeled with PKH26 red, a fluorescent dye, using a linker kit according to the manufacturer's instructions (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) (16). Briefly, 1 µl PKH26 was added to 250 µl diluent C for 5 min at room temperature. An equal volume of exosome-depleted serum was added to terminate the labeling reaction, and PEG precipitation was used to purify the exosomes. The PKH26-labeled exosomes were resuspended in 250 µl PBS. The PKH26-labeled exosomes (5, 10 and 20 µl) were added to FaDu cell culture medium in a 12-well plate with 1.8×10⁶/ml density and incubated at 37°C for 12 h. For the control group, 250 µl dilution buffer was used in place of the exosomes. Following incubation, FaDu cells were washed with PBS. Uptake of the labeled exosomes by FaDu cells was determined using fluorescence microscopy (magnification, ×100).

To determine whether the exosomes were able to deliver TRPP2 siRNA into FaDu cells, 40 µl exosomes (4 µg/µl) was added to 50 ml Opti-MEM (Thermo Fisher Scientific, Inc.) for 5 min at room temperature. In addition, 10 µl fluorescein amidite (FAM)-labeled siRNA (1 µg/µl) was added to 50 ml Opti-MEM for 5 min at room temperature. Following 5 min incubation, the two solutions were mixed together and incubated for 20 min at room temperature, prior to adding to FaDu cells for transfection. For the control group, 40 µl dilution buffer replaced the exosomes. After 24 h from transfection, the fluorescence signal of the FAM-labeled TRPP2 siRNA in the FaDu cells was observed using fluorescence microscopy.

The FaDu cells were cultured in a 6-well plate until 100% confluent, washed with PBS and cultured overnight in low-serum (0.1%) DMEM. The sterile tips of 200 µl pipettes were used to create scratches or ‘wounds’ in the cell layer. The FaDu cells were rinsed with PBS to remove floating cells and were cultured in DMEM supplemented with 0.1% FBS. The lengths of the scratches in each well were recorded by capturing images using a fluorescence microscope (magnification, ×40; Nikon Corporation, Tokyo, Japan) at the same configuration 0, 24 and 48 h following the creation of the scratch. The relative distances that cells migrated through the scratched area were measured, and a percentage of ‘healing’ was calculated. The experiment was repeated four times.

The 8-µm pore polycarbonate membranes (cat no. 3422; Corning Inc., Corning, NY, USA) of 24-well Transwell inserts were coated with Matrigel^® (cat no. BD354277; BD Biosciences, San Jose, CA, USA). In each well, 30 µl of Matrigel^® was added to the upper part of the insert and dried at 37°C to form a thin gel layer for 20 min. Serum-free medium (DMEM; 0.2 ml) was added to the upper chamber containing FaDu cells and medium (0.6 ml) supplemented with 20% FBS was added to the lower chamber. FaDu cells were seeded at a density of 1×10⁵ in the upper part of the chamber. Following 48 h incubation in a 5% CO₂ incubator at 37°C, the Transwell inserts were removed from the plates and the cells that had not migrated from the top of the membrane were wiped away using a cotton swab. Cells passing through the membrane pores were fixed with 4% paraformaldehyde was infiltrated for 5 min at room temperature and stained with DAPI for 3 min at room temperature. A fluorescence microscope (magnification, ×40; Nikon Corporation, Tokyo, Japan) was used to observe and count the number of cells that had successfully migrated through the membrane in four randomly selected areas.

SigmaPlot 13.0 software (Systat Software Inc., San Jose, California, USA) was used to analyze the collected data. All results are presented as the mean ± standard error of the mean. Two-tailed Student's t-tests were used to compare the results of different groups. P<0.05 was considered to indicate a statistically significant difference.

In order to view the morphology of exosome/TRPP2 siRNA, transmission electron microscopy was used to reveal spherical nanoparticles with diameters of 50–100 nm (Fig. 1A). Membranes of exosomes are enriched with numerous types of proteins, including cluster of differentiation (CD)63, CD9, CD37, CD53 and CD81 (19–22); CD63 and CD9 are considered to be specific markers for exosomes (20). In the present study, CD63 was probed in order to determine whether exosomes has been successfully extracted from 293 cells (Fig. 1B) and a gel retardation assay was used in order to determine the TRPP2 siRNA packaging capacity of the exosomes. As presented in Fig. 1C, exosomes encapsulated TRPP2 siRNA in a concentration-dependent manner. As the exosome to TRPP2 siRNA weight/molar (µg/nM) ratio increased, enhanced retardation of the TRPP2 siRNA band was observed, with the retardation of the TRPP2 siRNA band apparent at a 4:5 ratio of exosomes to TRPP2 siRNA (µg/nM) (Fig. 1C). These results indicated that exosomes were capable of encapsulating TRPP2 siRNA.

Protection of the exosome/TRPP2 siRNA complexes against nucleases and other enzymes in the bloodstream is critical for their use in siRNA-based therapies. Agarose gel electrophoresis was conducted to determine the stability of the exosome/TRPP2 siRNA complex. The agarose gel electrophoresis results indicated that naked (non-complexed; siRNA only) TRPP2 siRNA degraded within 5 min, whereas the TRPP2 siRNA encapsulated within exosomes remained intact, as no decrease in the density of the TRPP2 siRNA band was observed at any time examined (Fig. 1D and E). Hence, exosomes effectively protected TRPP2 siRNA against nuclease degradation and substantially enhanced TRPP2 siRNA stability.

Exosomes/TRPP2 siRNA complexes were used as a tool to deliver TRPP2 siRNA into FaDu cells in order to regulate cellular biological behavior, and eventually develop a therapeutic approach to treat HNC (Fig. 2).

A fluorescence assay was used in order to determine whether FaDu cells were able to effectively uptake exosomes and whether exosomes were able to deliver TRPP2 siRNA into the FaDu cells. As presented in the fluorescence microscopy images in Fig. 3A, red fluorescence signals from the PKH26-labeled exosomes were observed in FaDu cells following transfection (Fig. 3Ad-f), whereas no red fluorescence signals were observed in the control group (Fig. 3Aa-c). Green fluorescence signals from FAM-labeled exosome/TRPP2 siRNA complexes, were observed in FaDu cells and not from controls (Fig. 3B). Together, these results demonstrated that FaDu cells uptake exosomes and that exosomes may have the ability to encapsulate and deliver TRPP2 siRNA into FaDu cells.

TRPP2, a nonselective cation channel encoded by the PKD2 gene, serves an important role in a number of cellular processes (23). A previous study demonstrated that TRPP2 expression levels are increased significantly in human laryngeal squamous cell carcinoma and that TRPP2 enhances metastasis in these cells by regulating EMT (5). The results presented above indicated that exosomes may be capable of delivering TRPP2 siRNA into FaDu cells in vitro; however, it was unknown as to whether TRPP2 siRNA achieved TRPP2-specific gene silencing. Thus, western blot analyses were used in order to assess the expression levels of TRPP2 in FaDu cells following transfection. The results demonstrated that TRPP2 expression levels were significantly decreased following the transfection of TRPP2 siRNA into FaDu cells compared with the control (FaDu cells transfected with scrambled siRNA) (Fig. 4). Thus, these findings indicated that exosomes may successfully deliver TRPP2 siRNA into FaDu cells and that TRPP2 siRNA may significantly suppress TRPP2 expression in FaDu cells.

Previous studies have demonstrated that TRPP2 enhances metastasis and invasion by regulating EMT in human laryngeal squamous cell carcinoma (5). During EMT, E-cadherin expression levels, regarded as a prognostic biomarker for patients with numerous types of cancer, are significantly decreased, and cells produce more vimentin. In the present study, western blotting analyses were used in order to assess vimentin, E-cadherin and N-cadherin expression levels. The results demonstrated that the expression levels of N-cadherin and vimentin were decreased significantly in FaDu cells transfected with exosome/TRPP2 siRNA, compared with FaDu cells transfected with scrambled siRNA. Furthermore, E-cadherin levels were significantly increased in FaDu cells transfected with exosome/TRPP2 siRNA, compared with FaDu cells transfected with scrambled siRNA (Fig. 5), suggesting that exosome/TRPP2 siRNA complexes may reduce metastasis and invasion by inhibiting EMT. Taken together, these findings indicated that exosomes may be ideal vectors to deliver TRPP2 siRNA into FaDu cells, and that the exosome/TRPP2 siRNA complex is a potential siRNA-based therapy for HNC.

Cell migration and invasion are critically involved in the metastasis of laryngeal cancer. Therefore, FaDu cells were treated with exosome/TRPP2 siRNA complexes in order to determine whether this treatment slowed cell migration and invasion in vitro. When FaDu cells were transfected with exosome/TRPP2 siRNA complexes, wound healing data indicated that the migration speed of the FaDu cells was significantly decreased (Fig. 6). To investigate the effect of exosome/TRPP2 siRNA complexes on cell invasion, exosome/TRPP2 siRNA complex-transfected FaDu cells were cultured in Matrigel-coated Transwell inserts to simulate invasion through an extracellular matrix. The results demonstrated that compared with FaDu cells transfected with scrambled control siRNA, fewer of the cells transfected with exosome/TRPP2 siRNA complexes moved through the matrix (Fig. 7). These results indicated that exosome/TRPP2 siRNA complexes may inhibit FaDu cell migration and invasion in vitro.