Public data collection and analysis
A TCGA dataset containing gene expression profiles and HNSCC patients’ survival probability was leveraged from https://tcga-data.nci.nih.gov/tcga/. All those HNSCC cohort include 130 patients with high RAB25 expression and 389 patients with low RAB25 expression, which was divided based on a criteria with a false discovery rate (FDR) adjusted p < 0.01, the log(fold-change) > 2.0 defined as downregulated, while the log(fold-change) < − 2.0 was defined as upregulated, respectively. The survival endpoints of all patients were retrieved.
Patients And Tissues Collection
A total of 31 patients with HNSCC enrolled in the present study were first diagnosed at the First Affiliated Hospital of Zhengzhou University on the basis of histopathological evaluation, and the clinicopathological and follow-up information were detailed recorded. None of the patients received preoperative radiotherapy, chemotherapy, or immunotherapy before the surgery. The tumor tissues and the paired-adjacent normal tissues were collected underwent the surgery. All tissue samples were immediately frozen in liquid nitrogen and stored at − 80 °C until required.
All procedures performed in this study were in accordance with the ethical standards of the Ethics Committee of the First Affiliated Hospital of Zhengzhou University and with the 2008 Helsinki declaration. The written informed consent was obtained from all participants prior to the samples collected.
Real-time Pcr (qrt-pcr)
To quantify RAB25, ALIX, and CD63 expression, total RNA was extracted from cells or tissues using TRIzol reagent (Invitrogen), and NanoDrop-1000 spectrophotometer was used to determine RNA concentration. The isolated RNA was reverse transcribed into cDNA using high-capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad, CA, USA). Real-time PCR using SYBR Green I technology was then performed based on the ABI Prism 7500 System (Life Technologies) following the manufacturer's instructions.
For miRNA expression, exosomal miRNAs were isolated by using the SeraMir Exosome RNA Purification Kit (System Biosciences, Mountain View, USA), and cDNA for miRNAs was synthesized using 1 µg of total RNA treated with DNase I and TaqMan microRNA assay kit (Applied Biosystems, Foster City, USA) as described in the manufacturer's protocol. The qRT-PCR was conducted with the ABI Prism 7500 System using microRNA detection kit (RiboBio, Guangzhou, China).
All experiments were undertaken in triplicate and normalized against β-actin. Relative mRNA and miRNA expression were calculated using 2−ΔΔCt method.
Microarray And Data Analysis
For microarrays assay, the tumor tissues from 31 HNSCC patients and the randomly selected 10 paired-adjacent normal tissues were collected underwent uvulopalatopharyngoplasty (UPPP) surgery and prepared as described previously (20). Total RNA isolated from the tissues was used for mRNA microarray at Beijing Genomics Institute (BGI, Shenzhen, China). Microarray data process and analysis were conducted using Illumina BeadStudio software. To understand the biological functions of those proteins, gene ontology (GO) analysis was performed based on the differentially expressed proteins using DAVID 6.7. A p-value < 0.01 denoted significant enrichments in the GO pathways.
Cell Culture
Normal nasopharyngeal epithelial cells NP69, Nasopharyngeal carcinoma cell line CNE-1, Human oral squamous cell carcinoma cell lines HN4, and HN6, and Human Embryonic Kidney (HEK-293) were purchased from Cell Bank of the Chinese Scientific Academy. All cells were routinely grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) in a humidified incubator at 37 °C with 5% CO2 and 95% air.
Cell Transfection
For cell transfection, the mimics and the inhibitors of miR-488, and the negative control, siRNAs and pcDNA of RAB25, and their negative control were purchased from GenePharma (Shanghai, China). The transfection was conducted using Lipofectamine™ 2000 (Thermo Fisher Scientific, US) according to the manufacturer’s instructions. After 24 h, cells were collected for detection of transfection efficiency.
Western Blot
For determine protein expression of RAB25, CD63, TSG101, and their negative control GAPDH. Cells were lysed using RIPA buffer, and after quantified by BCA protein assay kit (Beyotime, China), total protein was loaded onto a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) for electrophoresis and subsequently transferred onto a polyvinylidene difluoride membrane. The membrane was incubated with primary antibody at 4 °C overnight, followed by incubation with peroxidase-linked secondary antibodies (1:10,000) for 1 h. The primary antibodies included in anti-CD63, anti-TSG101, anti-RAB25 and anti-GAPDH, which were all purchased from Abcam. The protein bands were visualized using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, USA). The relative protein expression was normalized to GAPDH.
Dual-luciferase Reporter Assays
The miR-488 and RAB25 interaction information was first predicted using TargetScan database. The experimental verification was conducted based on the dual-luciferase reporter assays. Briefly, the sequences of RAB25 3'-UTR (wild-type 3'-UTR) reporter plasmids was amplified. Site-directed mutagenesis (mut) of RAB25 to the miR-488-binding site was also conducted. The wildtype 3'-UTR or mut 3'-UTR was cloned into the psiCHECK-2 vector for luciferase reporter assays. The vector was co-transfected with miR-488 mimics or the control sequence into the HEK-293 cells using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA), and firefly luciferase activity was measured by Reporter Assay System Kit (Promega) 24 h after transfection.
Cell Colony Formation
Cells were evenly plated in a 6-well plate at a density of 300 cells per well. After overexpression or knockdown of RAB25 or miR-488 for 12 h, the medium was refreshed once a day. After 14 days of culture, cells were fixed using 4% Paraformaldehyde for 15 min, and washed with PBS. The colonies were stained with 0.1% Crystal violet staining solution for 30 min and finally calculated using ImageJ software.
Cell Viability
Cells were planted in a 96-well plate at a density of 2000 cell per well for 24 h to allow cell adherence. Cell viability was measured using MTT assays. Briefly, after incubating with 15 µl MTT solution (5 mg/ ml) (Sigma-Aldrich, MO, USA) in each well for 4 h, the absorbance was measured under 570 nm wavelengths using a microplate reader. Cell growth curves were depicted using GraphPad Prism 7 software.
Exosome Isolation
Exosomes isolation was conducted using ultrafiltration-ultracentrifugation protocol. After cells reached a confluence of approximately 70–80% in the standard medium, the supernatant without of cells and debris was collected for ultrafiltration at 4000 g, 4 °C for 25 min, followed by ultracentrifugation at 120,000 g for 90 min at 4 °C. The exosome pellet was washed in PBS followed by another ultracentrifugation at 120,000 g for 90 min at 4 °C. The collected exosomes were resuspended in PBS. Western blot analysis, electron microscopy analysis (TEM), and nanoparticle tracking analysis (NTA) were conducted to validate the exosome characterization.
In vivo tumorigenesis
For this study, NOD/SCID mice at 6–8 weeks old were purchased from Shanghai Lab. Animal Research Center (Shanghai, China), and housed in the ventilated cage under condition of 25 °C, with 70% humidified air. All mice were kept at a 12 h light/night circle for one week before the experiment. For xenograft culture, 1 × 105 HN4 or HN6 cells that overexpressed miR-488 or its negative control were suspended in the 100 µL PBS and subcutaneously injected into the mice. Tumor volumes were monitored once a week for 4 weeks. In addition, to visualize the role of RAB25 on mice survival, NOD/SCID mice were randomly divided into 10 groups with each group included in 6 mice. The mice were received the subcutaneously injection of HN4 cells with miR-448 knockdown or normal HN4 cells at the number of 1000, 2000, 4000, 8000, and 16000, respectively. Four weeks later, to monitor the survival mice. To observe the role of miR-488 on mice drug responses, NOD/SCID mice were randomly divided into 4 groups, as follows: mice were intravenously injected with 10 mg/kg Docetaxel (2 mg/mL dissolved in 0.9% saline) every second day for 6 days, or 1 × 105 HN4 cells pretreated with miR-488 inhibitor, or intravenously injected with 10 mg/kg Docetaxel + miR-488 inhibitor HN4 cells, or intravenously injected saline with the same volume of Docetaxel. Mice survival was visualized and tumor tissues were obtained for further experiments.
The animal experiments were conducted by the approval of the Ethics Committee at the First Affiliated Hospital of Zhengzhou University. The animals use and care were in accordance with the guidelines of this committee.
Statistical analysis
Data were presented as means ± SD. SPSS 18.0 and GraphPad Prism 7 software was conducted to analyze statistics. Unpaired t-test was performed to analyze the differences between two groups, while one-way ANOVA followed by Holm-Oak’s multiple comparison was conducted to compare the difference among more than two groups. A value of p < 0.05 was considered significant.