ABSTRACT
Studies have shown the inhibitory effect of microRNA-34a on proliferation, migration, and invasion of oral squamous cell carcinoma. However, the lack of a safe and effective delivery system limits the clinical application of microRNA-34a in oral cancer treatment. An exosome is a small extracellular vesicle that mediates intercellular communication by delivering proteins, nucleic acids, and other contents, and functions as a natural drug delivery carrier. Here, we aimed to explore whether exosomes could be used to load microRNA-34a via co-incubation and further used to treat OSCC. Ultracentrifugation was used to obtain exosomes derived from HEK293T cells and the extracted exosomes were analyzed via transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Subsequently, we loaded cholesterol-modified microRNA-34a into HEK293T cell exosomes by co-incubation. Then, PKH67 and Cy3 co-labeled exo-microRNA-34a were co-incubated with HN6 cells and exosome entry into the HN6 cells was observed using a confocal laser scanning microscope. The cell proliferation, migration, and invasion were assessed by CCK-8 and Transwell assay analysis. SATB2 expression in HN6 cells was analyzed via western blotting. In this study, cholesterol-modified microRNA-34a was loaded into exosomes of HEK293T cells by co-incubation. The microRNA-34a-loaded exosomes were secreted from HEK293T cells and were absorbed by HN6 oral squamous carcinoma cells. Further, microRNA-34a-loaded exosomes led to a significant inhibition of HN6 cell proliferation, migration, and invasion by down regulating SATB2 expression. These results report a new delivery method for microRNA-34a, providing a new approach for the treatment of oral cancer.
KEYWORDS: microRNA-34a, exosomes, oral squamous cell carcinoma, HEK293T cells, HN6 cells
Introduction
Oral squamous cell carcinoma (OSCC) is a highly prevalent malignant human tumor with an ever-increasing incidence [1,2] and has developed as an important public health concern worldwide [3]. OSCC is characterized by aggressive growth, local invasion and distant metastasis, high postoperative recurrence rate, and poor prognosis because it is usually diagnosed in the intermediate or advanced stage [4,5]. The treatment of OSCC can be divided into early and late treatments. Early treatment includes surgery or radiotherapy alone, whereas late treatment requires chemotherapy combined with surgery and/or radiotherapy [6,7]. Current treatment modalities for advanced disease, including surgery, radiotherapy, and chemotherapy, can result in severe dysfunction and toxicity, thus the development of new treatment strategies is urgently needed [8].
MicroRNA-34a, as a member of the microRNA-34 (miR-34) family, regulates cell cycle and apoptosis and has an inhibitory effect on tumor growth [9]. Ge et al. have shown that microRNA-34a expression levels are low in OSCC tissues and four human OSCC cell lines (HN4, HN6, SCC‐9 and CAL27) compared with those in normal oral tissues or human oral keratinocytes (HOKs). The study showed that microRNA-34a inhibits the proliferation, migration, and invasion of OSCC by directly targeting special AT-rich sequence-binding protein 2 (SATB2) [10]. Therefore, microRNA-34a presents as a potential therapeutic target for OSCC. Exosomes are extracellular vesicles sized 40 ~ 150 nm, which can be produced by many types of cells, and have a double-layer lipid membrane structure [11,12]. Studies have shown that when cells secrete exosomes into the extracellular environment, they can transfer various types of functional effectors including RNA, miRNA, and proteins to recipient cells [13,14]. Therefore, exosomes have the potential to act as nanoparticles to deliver drugs or nucleic acid-based molecules for treating tumors. Didiot et al [15]. used a co-incubation method to load hydrophobically modified small interfering RNAs (hsiRNAs) into extracellular vesicles, and found that co-incubating hsiRNAs with exosomes provides a robust, efficient, and highly reproducible method for loading exosomes with chemically synthesized oligonucleotides.
According to a previous study, exosomes derived from HEK293T cells can be engineered as drug delivery vectors [16,17]. Here, we aimed to explore whether exosomes secreted from HEK293T cells can be used to load microRNA-34a via co-incubation and further used to treat OSCC. Our in vitro results verify whether the use of exosomes as vectors to deliver microRNA-34a provides a promising approach for the inhibition of the proliferation, migration, and invasion of OSCC.
Materials and methods
Cell culture
Human HN6 OSCC cells (American Type Culture Collection) were grown in DMEM/F-12 (Gibco, MA) containing 10% fetal bovine serum (FBS) (Sciencell, MA). HEK293T cells (ATCC) were grown in DMEM containing 10% FBS, 1% glutamine, and penicillin/streptomycin. Bovine exosomes were removed from FBS by centrifugation at 200,000 × g for 18 h. Cells were grown in a humidified incubator at 37°C with 5% CO2.
Exosome isolation
The harvested culture supernatant of HEK293T cells was collected to isolate exosomes. Exosome-containing conditioned culture media was centrifuged for 10 min at 300 × g, after which supernatants were centrifuged for 30 min at 1000 × g and for 30 min at 10,000 × g at 4°C to eliminate all remaining debris. A final 90 min spin at 140,000 × g at 4°C was then conducted with an L-80XP ultracentrifuge (Beckman). Exosomes were then resuspended in PBS and the ultracentrifugation step was repeated, followed by resuspension in PBS for downstream use. A Micro BCA Protein Assay kit (Pierce) was used to analyze these exosomes.
Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA)
The prepared exosomal suspension was aspirated with a capillary pipette and dropped onto a copper mesh. Following 1 min incubation, excess liquid was removed from the edge of the drop bead using a filter paper, and 3% tungsten phosphate solution was dripped onto the copper mesh and left for 3 min. The exosomes were observed with Tecnai G2 TEM (FEI). Nanoparticle tracking analysis (NTA) was performed with the ZetaView system (Particle Metrix) to assess exosomal Brownian motion in PBS, with the Stokes–Einstein equation being used to assess size distributions.
Western blotting
Total proteins were extracted from exosomes, HEK293T cells, and HN6 cells. Equal amounts of protein samples were isolated by SDS-PAGE and transferred onto PVDF membranes. Blots were blocked using 5% nonfat milk for 1 h followed by incubation with primary antibodies overnight at 4°C. Protein samples of HEK293T cells and exosomes were detected using antibodies against TSG101 (1:1000, GeneTex, GTX70255), CD9 (1:1000, Abcam, ab2215), or Calnexin (1:1000, Abcam, ab22595). Exosome markers CD9 and TSG101 were determined by Western blotting. The protein samples of HN6 cells were detected using antibodies against SATB2 (1:1500, Proteintech,21,307-1-Ap) or GAPDH (1:4000,Proteintech,10,494-1-AP). Following multiple washings, the membranes were incubated with secondary antibodies coupled to HRP for 1 h. The ECL method was followed to detect the immunoreactive bands.
Loading of exosomes with microRNA-34a
Exosomes (100 μL) were diluted with 250 μL of Diluent C solution. Next, 1 μL of PKH67 dye was added and incubated for 4 min. Excessive staining was prevented by the addition of 500 μL of PBS with 1% BSA. Subsequently, the exosomes were isolated by ultracentrifugation at 140,000 × g and resuspended in PBS. MicroRNA-34a was conjugated at the 3ʹ terminus with cholesterol and modified with 2’-O-methylation (GenePharma). Sequences were as follows: 5ʹ-UGGCAGUGUCUUAGCUGGUUGU AACCAGCUAAGACACUGCCAUU-3ʹ. Cholesterol-conjugated microRNA-34a (200 nM) and 100 μg PKH67-exo were combined for 1 h in 200 μL of PBS at 37°C. The modified microRNA-34a was inserted into the exosomal membrane via hydrophobic interactions, and subsequently, the samples were centrifuged three times using 100-kDa molecular weight cutoff ultrafiltration tubes (Millipore) to remove unincorporated microRNA-34a. The samples were then resuspended in PBS, and these modified exosomes were stored at −80°C. After purification, the microRNA-34a-loaded exosomes (exo-microRNA-34a) were observed under an inverted fluorescence microscope. The co-localization percentage was calculated by the equation % = Cy3 containing exosomes/total exosomes labeled by PKH67 dye. We estimated the microRNA-34a-loading efficiency by calculating the co-localization percentage.
Exosome uptake experiment
HN6 cells were seeded on cell slides in 12-well plates at 5 × 104 cells/well and grown until the growth phase was logarithmic. Next, 200 μL of exo-microRNA-34a (1 mg/mL) was added to wells, and cells were cultured for 3, 6, and 12 h at 37°C with 5% CO2 and saturated humidity. Subsequently, the supernatant was aspirated, paraformaldehyde was used in the presence of Triton X-100 to fix the cells for 3 min, and the cells were washed three washes with PBS. Next, the nuclei were stained with a DAPI staining solution for 2 min, and the cells were rinsed twice with PBS. The excess liquid was then removed using an absorbent paper, and the entry of exosomes into the cells was observed using a confocal laser scanning microscope.
Cell proliferation assay
HN6 cells were seeded in 6-well plates at 50%–60% confluence and were cultured overnight. Subsequently, 100 μL of exo-microRNA-34a (1 mg/mL), exosomes, and PBS were added to the wells, and the cells were cultured at 37°C with 5% CO2 and saturated humidity for 48 h. The cells were then digested with 0.25% trypsin (Gibico, US) for proliferation and invasion experiments. Cell proliferation was assessed as follows: HN6 cells (100 µL) were seeded in 96-well plates (1000/well) and precultured at 37°C with 5% CO2 for 24, 48, 72, or 96 h before a CCK-8 solution (10 µL) (Dongren Chemical Technology, Shanghai) was added per well for 2 h. Subsequently, test absorbance at 450 nm was measured using a microplate reader.
Cell migration and invasion assay
The Matrigel matrix (BD, Billerica, MA) was used to simulate a human basement membrane for the invasion assay. DMEM and the Matrigel matrix were mixed in a ratio of nine to one to give a total volume of 120 µL. Following this, the mixture was placed into the chamber to conduct the invasion assay. Migration and invasion assays were performed using a Transwell chamber (Corning, Corning, NY). The chambers were placed into 24‐well plates, and 1 × 104 cells (in 200 µl of DMEM/F‐12) were added to each chamber with 600 µL of DMEM/F‐12 containing 10% FBS per well. After culturing for 24 h, the transmigrated cells were fixed in methyl alcohol, stained with 0.2% (m/v) crystal violet (Sigma-Aldrich Co), and rinsed in PBS. Images of the transmigrated cells were captured under an upright light microscope, and cells in the logarithmic phase were seeded in a 6-well plate. The wound-healing assay was performed with a standard 200-μL pipette tip in cells that were 80% confluent. Subsequently, the cells were further cultivated in a serum-free medium for 12 h. Images of the wound healing process were captured using an inverted light microscope.
Statistical analysis
Statistical analyses of the data were performed using GraphPad Prism software. Comparisons between two groups were performed with Student’s t-test. Significant differences among multiple groups were determined by one-way analysis of variance (ANOVA) or two-way ANOVA followed by Tukey’s post-hoc test. Results are expressed as mean ± SEM. P values < 0.05 were considered statistically significant.
Results
Isolation of exosomes from HEK293T cells
Small vesicles sized 40–150 nm were observed by TEM in conditioned cellular media of HEK293T cells (Figure 1a). The distribution maxima of the particle size of the vesicles was 97.9 nm based on NTA results (Figure 1b), and surface proteins of exosomes, including tumor susceptibility gene 101 protein (TSG101) and CD9, were detected by Western blotting. However, no calnexin (an endoplasmic reticulum marker) was detected in the exosome preparation (Figure 1c), which verified that exosome extraction was successful.
Figure 1.
Secretion of exosomes from HEK293T cells.
(a) Transmission electron micrograph showing the presence of exosomes in the cell culture media. Scale bar: 100 nm. (b) Size distribution of exosomes according to NTA measurements. (c) Western blot analysis for the expression of CD9, TSG101, and calnexin in HEK293T cells. Exosomes isolated from the conditioned medium of HEK293T cells were positive for exosome-specific marker proteins CD9 and TSG101, but negative for calnexin (an endoplasmic reticulum marker).
Loading of microRNA-34a into exosomes
To assess if microRNA-34a-loaded exosomes were internalized by HN6 cells, we first generated fluorescently labeled microRNA-34a-loaded exosomes containing Cy3 (red)-labeled microRNA-34a, with lipophilic PKH67 (green) being used to label exosomes. We then used fluorescence microscopy to show that microRNA-34a modified by Cy3 could effectively co-localize with PKH67-labeled exosomes following co-incubation. The drug loading capacity was found to be approximately 47% (Figure 2a). The integrity of the exosomes was not affected by drug loading (Figure 2b), while the mean particle size of the exosomes had increased (compare Figure 2c with Figure 1b). Exosomal surface proteins including CD9 and TSG101 showed no significant changes (Figure 2d). Taken together, these results show that hydrophobically modified microRNA-34a can enter exosomes without destroying their morphological structure.
Figure 2.
Loading of microRNA-34a into exosomes in HEK293T cells.
(a) Efficient loading of exosomes with microRNA-34a. The arrows point at yellow exosomes, indicating the colocalization of green PKH67-labeled exosomes and red Cy3-labeled miR-34a. Yellow exosomes represent approximately 47% of total exosomes. Scale bar: 10 μm. (b) Transmission electron micrograph of exosomes loaded with microRNA-34a. Scale bar: 100 nm. (c) Size distribution of exo-miR-34a as detected by NTA measurements. (d) Western blot analysis for the expression of CD9, TSG101, and calnexin in HEK293T cells, exosomes, and exo-microRNA-34a, respectively.
Uptake of microRNA-34a-loaded exosomes by HN6
PKH67 and Cy3 co-labeled exo-microRNA-34a were co-incubated with HN6 cells for 3, 6, or 12 h. Cells were fixed and the nucleus was stained with a DAPI staining solution followed by observation for exosome entry into the cells using a confocal laser scanning microscope. The results showed that a small amount of microRNA-34a began to enter HN6 cells at 3 h of co-culture, gradually increasing at 6 and 12 h. Co-localization of PKH67 (exosome marker) and Cy3 (microRNA-34a marker) in the cells was observed; thus, exosomes loaded with microRNA-34a could enter HN6 cells. (Figure 3)
Figure 3.
Uptake of PKH67-labeled Cy3-miRNA-34a-loaded exosomes by HN6 cells.
Kinetics of Cy3-microRNA-34a-loaded exosomes shows co-localization between exosomes and Cy3-microRNA-34a. MicroRNA-34a labeled with Cy3 (red); exosomes labeled with PKH67 (green); and nucleus of HN6 cell labeled with DAPI (blue). Scale bar: 20 μm.
MicroRNA-34a-loaded exosomes suppress the proliferation, migration, and invasion of HN6 cells
HN6 cells were incubated up to 48 h with exo-microRNA-34a, or with exosomes and PBS without microRNA-34a as a control. These cells were then cultured in 96-well plates for 72 or 96 h. The results of the CCK-8 assay showed that the proliferation of HN6 cells treated with exo-microRNA-34a was significantly weakened compared with the control group after 72 or 96 h, indicating that exo-microRNA-34a had a significant inhibitory effect on HN6 proliferation (P< 0.05) (Figure 4a). The role of exo-microRNA-34a in the regulation of cell migration and invasion was evaluated via an in vitro wound‐healing assay and Transwell assay. It was clear that the migration and invasion ability of HN6 cells was significantly suppressed after culturing with exosomes loaded with microRNA-34a (Figure 4b and Figure 4c). Western blotting was performed to test the SATB2 expression level after HN6 cells were co-cultured with exo-microRNA-34a. The results demonstrated that exo-microRNA-34a can downregulate SATB2 in HN6 cells (Figure 4d). These results indicated that the microRNA-34a-loaded exosomes can further influence the migration and invasion ability of HN6 cells.
Figure 4.
Effects of exo-microRNA-34a on the proliferation, migration, and invasion of OSCC HN6 cells.
(a) Exo-microRNA-34a inhibits HN6 cell proliferation. The viability of HN6 cells treated with equal amounts of PBS, exosomes, and exo-microRNA-34a for different time points detected by CCK-8 assay. ***P < 0.001 (n = 3) compared with exosomes and PBS group. (b) HN6 cell migration and invasion ability, evaluated by Transwell assays, was downregulated compared with exosomes and PBS group after culturing with microRNA-34a-loaded exosomes ***P < 0.001. Scale bar: 100 μm. (c) The cell migration ability was tested via wound healing assay. (d) Western blot analysis for SATB2 expression after HN6 cells were cocultured with exo-microRNA-34a.
Discussion
Although miRNAs are promising anticancer therapeutic modalities, the lack of safe and effective delivery systems limits the clinical application of microRNA-34a. In contrast to other drug carrier such as liposomes, exosomes contain transmembrane and membrane-anchored proteins that can enhance endocytosis and thereby facilitate the delivery of their internal material [18]. Moreover, exosomes are characterized by low immunogenicity, low toxicity, the ability to cross the blood–brain barrier, excellent stability in the bloodstream, and certain targeting ability when carrying drugs [19–21]. Therefore, exosomes have great potential in drug delivery and disease treatment and are expected to be the next generation of drug delivery vehicles.
Ultracentrifugation technology is the most commonly used method of exosome purification and is considered the “gold standard” for exosome isolation [22]. We used ultracentrifugation (differential centrifugation) to obtain exosomes derived from HEK293T cells and analyzed the extracted exosomes via TEM, NTA, and Western blotting. The three methods complemented each other and proved that exosomes were successfully extracted from the culture supernatant of HEK293T cells.
Several strategies have been proposed and developed for loading microRNA-34a into exosomes, e.g. sonication, electroporation, and combination incubation with permeabilizing agents or RNAs that have been lipophilically modified. Electroporation can induce siRNA precipitation and aggregation, leading to an overestimation of the loading efficiency of vesicles [23]. Furthermore, sonication and incubation with a permeabilizing agent can lead to the reorganization and deformation of the exosome, which disrupts its integrity [24]. Because of loading difficulties, recent attempts have aimed at loading hydrophobic modified RNA onto extracellular vesicles. O’Loughlin et al [25] assessed and optimized the method of loading extracellular vesicles with cholesterol-conjugated siRNA (cc-siRNA), which can promote concentration-dependent silencing of human antigen R (HuR), a therapeutic target of cancer, in cells treated with extracellular vesicles loaded with cc-siRNA. In our study, after co-incubation of cholesterol-modified microRNA-34a with exosomes, fluorescence imaging revealed the successful loading of Cy3-labeled microRNA-34a into exosomes, while no significant change in the morphology of the vesicles was observed under TEM, indicating that exosome integrity was not affected. Meanwhile, NTA results showed that the average particle size of loaded exosomes increased, which was consistent with the report by O’Loughlin et al. [25], indicating that the drug entered the exosome. As was observed using a confocal laser scanning microscope, exo-microRNA-34a was absorbed by HN6 cells. CCK-8 assay, wound‐healing assay, and Transwell assay showed a significant inhibitory effect of exo-microRNA-34a on HN6 cell proliferation, migration, and invasion. Furthermore, the protein levels of SATB2 in HN6 cells significantly decreased. This was consistent with the findings of Ge et al [10]. These findings demonstrated that the constructed drug-loaded exosomes significantly inhibited the proliferation, migration, and invasion of OSCC cells.
Recent studies have shown that microRNA-34a-5p is significantly down-regulated in the derived exosomes of carcinoma-associated fibroblasts (CAFs). These exosomes can metastasize to OSCC cells. Further, the low microRNA-34a-5p-expressing exosomes derived from CAFs can promote the malignant progression of OSCC [26]. By incubating HN6 cells in the presence of exosomes loaded with miRNA34a mimics we successfully inhibited the proliferation, migration, and invasion of HN6 cells. Therefore, exosomes loaded with microRNA-34a are a very promising tool for OSCC treatment. However, we still lack in vivo experimental studies to confirm the inhibitory effect of exo-microRNA-34a on OSCC, which is worthy of further studies.
In conclusion, we have demonstrated that cholesterol-modified microRNA-34a can be loaded into exosomes secreted by HEK293T cells via co-incubation. MicroRNA-34a-loaded exosomes can be taken up by HN6 cells and inhibit their proliferation, migration, and invasion. Although the therapeutic application of microRNA-34a-loaded exosomes requires further in vivo studies, exosomes have great potential for the delivery of miRNAs in miRNA supplementation therapies for OSCC.
Supplementary Material
Acknowledgments
We thank Dr. Xin Ge and Dr. Qiu-Wangyue Sun for their excellent technical support. This research was funded by the National Natural Science Foundation of China: 81371123, Priority Academic Program Development of Jiangsu Higher Education Institutions: PAPD,2018-87, Southeast University‐Nanjing Medical University Cooperative Research Project: 2017DN03.
Funding Statement
This work was supported by the National Natural Science Foundation of China [81371123]; Southeast University‐Nanjing Medical University Cooperative Research Project [2017DN03]; Priority Academic Program Development of Jiangsu Higher Education Institutions [PAPD, 2018- 87].
Authors’ statement
Wei Deng, Ying Meng, Bin Wang contributed to acquisition of data, and draft this manuscript, Chen-xing Wang, Chen-xing Hou, Qing-hai Zhu, Yu-ting Tang performed the data collection and interpretation, Jin-hai Ye contributed to the conception and design of the study, and critical revising of the paper. All authors read and approved the final manuscript.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Supplementary material
Supplemental data for this article can be accessed here
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