Aberrant microRNA (miRNA/miR) expression plays an important role in the pathogenesis of nasopharyngeal carcinoma (NPC). In the present study, the role and underlying molecular mechanism of miR-301a-3p in NPC cells were determined. It was observed that miR-301a-3p upregulation promoted NPC cell proliferation, migration, invasion and epithelial-mesenchymal transition
Nasopharyngeal carcinoma (NPC), a highly malignant tumor originating from the nasopharyngeal epithelium, is among the most prevalent malignancies in China (
MicroRNAs (miRNAs/miRs) act as tumor suppressor genes or oncogenes to modulate tumor progression, and may provide a new approach to explore the molecular mechanisms of the pathogenesis of NPC (
The present study demonstrated that miR-301a-3p upregulation promotes NPC cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT)
Two NPC cell lines, C666-1 [Epstein-Barr virus (EBV+)] and 5–8F (EBV-), and an immortalized nasopharyngeal epithelial cell line (NP69) were procured from the Central South University (Hunan, China). C666-1 and 5–8F cells were maintained in RPMI-1640 medium and supplemented with 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc.). NP-69 cells were cultured in keratinocyte serum-free medium supplemented with human recombinant epidermal growth factor and bovine pituitary extract (Sigma-Aldrich; Merck KGaA). All cells were cultured in a humidified chamber with 5% CO2 at 37°C.
Total RNA was extracted from the cells and subjected to reverse transcription for 60 min at 37°C using the NCode miRNA First-Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). RT-qPCR was performed using the ABI 7900HT system with SYBR-Green PCR Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the following thermocycling parameters: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 35 sec.
miR-301a-3p mimic (5′-CAGUGCAAUAGUAUUGUCAAAGC-3′) and negative control (NC mimic, 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′) were purchased from Guangzhou RiboBio Co., Ltd. miR-301a-3p inhibitor (5′-GCUUUGACAATCTATTGCACTG-3′) and negative control (NC-in, 5′-ACCGCUAAUCAUACGAAUACAC-3′) were purchased from Guangzhou RiboBio Co., Ltd. Plasmids (pcDNA3.1) expressing
To determine the effect of miR-301a-3p on cell proliferation, Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) was used. Transfected cells were plated at 2×103 cells per well in 96-well plates in triplicate. At various time intervals, the number of cells per well was calculated as absorbance units at a wavelength of 450 nm.
The EdU assay was performed using the EdU-594 Cell Proliferation kit (Beyotime Institute of Biotechnology). Transfected cells were plated at 3×105 cells per well in 6-well plates. After 12 h, cells were then washed with PBS and fresh medium containing 10 µM EdU was added. Cells were subsequently incubated for 2 h at 37°C in 5% CO2 and washed with PBS to remove the free EdU probe and medium. Cells were then fixed in 4% paraformaldehyde at room temperature for 15 min and stained with DAPI at room temperature for 5 min. Images were captured using a fluorescence microscope (magnification, ×200).
For the colony formation assay, 300 cells were seeded into individual wells of a 6-well plate and cultured at 37°C for 12 days. The colony counts per well were determined after staining the cells with 0.1% crystal violet at room temperature for 30 min. Images were captured using a Nikon camera (Nikon Corporation). Only unambiguous colonies (diameter >40 µm) in the wells were analyzed using ImageJ software (version 1.49p; National Institutes of Health).
Transwell assays were performed as previously described (
Western blotting was performed as previously described (
Targets of miR-301a-3p were searched for using different computational methods, such as miRanda (
The pMIR-REPORT vector (Promega Corporation) was used to generate luciferase reporters carrying either the wild-type (Wt) or mutated (Mut) 3′-untranslated region (UTR) of
Exosomes were separated from the cell culture medium using a previously described method (
Isolated exosomes were diluted with PBS, and 10 µl sample was absorbed to a copper mesh. Then, the solution on the surface of the copper mesh was absorbed with filter paper after being placed for 5 min. The copper mesh was cleaned once and dyed for 45 sec with 2% 10 µl uranyl acetate. The image was developed with 80–120 kV projection electron microscope (JEM-1230; JEOL). Digital images were collected with a Gatan model 830 ORIUS SC200 CCD camera using Gatan Digital Micrograph software version 3.11 (Gatan, Inc.).
The SPSS 17.0 software (SPSS, Inc.) was used for statistical analyses. All data are presented as the means ± standard deviations from at least three independent experiments. The differences between groups were analyzed using unpaired Student's t-test. Comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
RT-qPCR was first used to examine the expression levels of miR-301a-3p in two NPC cell lines (C666-1 and 5-8F) and in the NP69 immortalized normal human nasopharynx epithelial cell line. As shown in
To determine the effects of miR-301a-3p on the migration and invasion of NPC cells, the Transwell chamber assay was performed. miR-301a-3p overexpression significantly enhanced the invasive and migratory abilities of both C666-1 and 5–8F cells (
The potential regulatory targets of miR-301a-3p were explored via bioinformatics analysis of the miRanda and TargetScan databases. The analysis revealed
Finally, C666-1 and 5–8F cells were co-cultured with exosomes containing high miR-301a-3p levels, in order to determine whether this miRNA could be delivered via exosomes and regulate the function of recipient cells. Exosomes secreted from C666-1 cells stably overexpressing miR-301a-3p were isolated. TEM revealed that the exosomes had a lipid bilayer membrane structure, and western blotting confirmed the expression of exosomal markers CD63 and TSG101 (
The present study focused on the regulatory functions of miR-301a-3p in NPC cells. It was discovered that miR-301a-3p upregulation promotes the proliferation, migration, invasion and EMT of NPC cells
The aberrant expression of miR-301a-3p is associated with cancer initiation and development. miR-301a-3p has been reported to be upregulated in several types of cancer. For instance, Li
Existing experimental data suggest that miR-301a-3p directly regulates the expression of numerous target mRNAs, including
Finally, it was demonstrated that NPC cells transmit miR-301a-3p to surrounding cancer cells via exosomes and increased miR-301a-exo levels promote cell proliferation and invasion. Exosomes, secreted by various cell types, can be absorbed by the surrounding cells and exert their biological functions in these receptor cells (
In conclusion, the present data indicate that miR-301a-3p promotes the proliferation and invasion of human NPC cells by downregulating BTG1 and that this miRNA participates in cell-cell communication via exosomes. These findings provide a new direction for further investigating the pathogenesis of NPC. However, expression patterns of miR-301a-3p and survival outcomes of patients with NPC were not analyzed in the present study. Thus, these topics remain to be verified in future research.
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No funding was received.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
QC and HJ made substantial contributions to the study conception and design. QC, QL and LX performed data acquisition, analyses and interpretation. QC drafted the manuscript and critically revised it for important intellectual content. QC and HJ confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
miR-301a-3p promotes NPC proliferation
miR-301a-3p promotes cell migration, invasion and EMT of C666-1 and 5–8F cell lines. (A) Transwell migration assay of C666-1 and 5–8F cells transfected with miR-301a-3p mimic or NC. Magnification, ×200. (B) Transwell invasion assay of C666-1 and 5–8F cells transfected with miR-301a-3p mimic or NC. Magnification, ×200. (C) Transwell migration and invasion assays of C666-1 cells transfected with a miR-301a-3p inhibitor or NC. Magnification, ×200. The (D) mRNA and (E) protein expression levels of EMT markers in the indicated cells as detected via reverse transcription-quantitative PCR and western blotting, respectively. **P<0.01 vs. NC group. miR, microRNA; EMT, epithelial-mesenchymal transition; NC, negative control; in, inhibitor.
miR-301a-3p binds to the 3′-UTR of
miR-301a-3p affects the proliferation, migration and invasion of nasopharyngeal carcinoma cell cells via exosomes. (A) Exosome identification via transmission electron microscopy. (B) Detection of the exosome marker proteins CD63 and TSG101 via western blotting. (C) Reverse transcription-quantitative PCR analysis of miR-301a-3p expression in C666-1 and 5–8F cells after treatment with miR-301a-exo or NC-exo. (D) Cell Counting Kit-8 assay to detect the viability of C666-1 and 5–8F cells after treatment with miR-301a-exo or NC-exo. (E) EdU assay to detect the proliferation of C666-1 and 5–8F cells after treatment with miR-301a-exo or NC-exo. Magnification, ×200. (F) Transwell invasion assay. Magnification, ×200. *P<0.05; **P<0.01 vs. NC-exo group. miR, microRNA; BTG1, B-cell translocation; exo, exosome; NC, negative control.