Cell culture, X-Radiation treatment
HepG2, SK-Hep1 was purchased from the American Type Culture Collection (Rockville, MD) (ATCC HTB-177). Hep3B was purchased by Korea Cellular Bank (seoul, Korea). It was cultured in DMEM (Corning) with 10% fetal bovine serum, 4.5 g/L glucose, L-glutamine, sodium pyruvate, and 100 U/mL of penicillin and streptomycin, and maintained at 37°C in a humidified chamber containing 5% CO2. Radiation was exposed with 2Gray, 4Gray a using X-RAD320 (1Gray/min at 320KV, 12.5mA, 50cm SSD (HVL≈ 4mm Cu)) equipment.
Radiation and non-radiation exosomes purified using ultracentrifugation methods. 1 × 106 cells were seeded in 100 mm dishes and allowed to recover for overnight. Then, the cells were washed twice with pre-warmed PBS, and the culture medium was replaced with exosome-free medium supplemented with 5% exosome-depleted FBS (SBI). The Radiation exosome is treated 4Gray Radiation using X-RAD 320 (1Gray/min at 320KV, 12.5mA, 50cm SSD (HVL≈ 4mm Cu)) equipment. The medium was gathered and subjected to gradient centrifugation. Briefly, the medium was first centrifuged at 1000g for 10 min, at 3000g for 30 min, then centrifuged in a Beckman Coulter Optima™ L-XP Ultracentrifuge System at 100,000 gavg at 4℃ for 90 minutes with a SW 28 Swinging-Bucket Rotor (k-factor: 71) to pellet exosomes. The supernatant was carefully removed, and crude exosome-containing pellets were resuspended in 1 mL of ice-cold PBS and pooled. A second round of ultracentrifugation [100,000 gavg at 4℃ for 90 minutes with a Type SW 28 Swinging-Bucket Rotor (k-factor: 71)] was carried out, and the resulting exosome pellet resuspended in 500 μl of PBS.
Western blot analysis
The cells were collected, washed with PBS, and lysed in lysis buffer containing protease inhibitors (GenDEPOT, USA). After determining the protein concentration with a Bradford Protein Assay Reagent (Bio-Rad), equal amounts of protein were separated on 10% SDS-PAGE, electrically transferred to nitrocellulose membrane, and blocked with 5% skim milk and 5% BSA. The membranes were incubated with anti-HDAC5 (1:1000; Cell Signaling Technology, USA), anti-p53 (1:2000; Santacruz, USA), anti-p21 (1:2000; Cell Signaling Technology), anti-Puma (1:2000; Cell Signaling Technology), anti-STEAP3 (1:1000; Proteintech, USA), anti-Maspin (1:1000; Cell Signaling Technology), anti-CD63 (1:1000; Santacruz), anti- CD81 (1:1000; Santacruz), anti-HSP90 (1:1000; BD bioscience), anti-BCL2 (1:1000; Cell Signaling Technology), anti-E-cadherin (1:1000; Agilent Dako), anti-Twist1 (1:1000; abcam), N-cadherin (1:1000; Cell Signaling Technology), Vimentin (1:1000; Calbiochem) or anti-beta-actin (1:1000; Santacruz) primary antibody overnight shaking at 4 °C. After washing twice, the membranes were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibody (Jackson ImmunoResearch Inc. USA) at room temperature for 1 hour. Finally, the membranes were incubated with WESTSAVE-UP western blotting substrate (Youngin frontier, Korea), and images were visualized using ChemiDoc imaging system (Bio-Rad, USA) and recorded.
Duolink proximity ligation assay (PLA)
The DuoLink® In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma-Aldrich, Darmstadt, Germany) was used to detect interacting target proteins. Cells were seeded in eight-well chamber removable slides (ibidi GmbH Am Klopferspitz, Germany) and cultured 24hours. Then, HepG2 cells were exposed to 4 g of radiation, and after 12 hours, the slides were washed with cold 1xPBS and fixed in 4% paraformaldehyde for 30 minutes. Then slides were blocked with Duolink Blocking Solution in a pre-heated humidified chamber for 30 min at 37°C. The primary antibody to detect HDAC5 and p53 was added to the slides and incubated overnight at 4°C. Then slides were washed with 1×Wash Buffer A and subsequently incubated with the two PLA probes (1:5 diluted in antibody diluents) for 1 h, then the Ligation-Ligase solution for 30 min, and the Amplification-Polymerase solution for 100 min in a pre-heated humidified chamber at 37°C. Before imaging, slides were washed with 1×Wash Buffer B and mounted with a cover slip using Duolink In Situ Mounting Medium with DAPI. Fluorescence images were acquired using a zeiss LSM 780 confocal microscope. 17
Transmission electron microscopy (TEM)
In order to photograph exosomes, 0.1M of exosome pellets were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate solution (pH 7.0) for 1 hour and then fixed in 2% osmium tetroxide for another hour. (4°C) After dehydration using the graded acetone series, embedding was performed through Spurr's medium (Electron Microscopy Sciences). The resulting section sample was cut at 60 nm with an ultramicrotome (RMC MTXL, USA), followed by double staining using 2% uranyl acetate for 20 minutes and citrate for 10 minutes. Prepared sections were photographed at 80 kV using Hitachi H-7600 TEM (Hitachi, Japan) equipment.
Exosome size distribution and concentration measurement.
To determine exosome size distribution and concentration, nanoparticle tracking-based analyses were performed using a NanoSight (NS500) apparatus (Malvern Instruments Ltd.). Samples were diluted to provide counts within the linear range of the instrument. The videos of 1-minute duration were recorded for each sample, with a frame rate of 30 frames per second. Particle movement was analyzed by NTA software (NTA 2.3; NanoSight Ltd.) according to the manufacturer's protocol. The NTA software was optimized to first identify and then track each particle on a frame-by-frame basis. 18, 19
Small RNA sequencing
HepG2-derived Exosomal small RNA was extracted using a miRNeasy Mini Kit (Qiagen Korea Ltd.) according to the manufacturer’s instructions. RNA quality was assessed by Agilent 2100 bioanalyzer using the RNA 6000 Pico Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using a NanoDrop 2000 Spectrophotometer system (Thermo Fisher Scientific, Waltham, MA, USA).
Library preparation and sequencing
For control and radiation induced HepG2-drived exosomal small RNAs, the construction of the library was performed using NEBNext Multiplex Small RNA Library Prep kit (New England BioLabs, Inc., USA) according to the manufacturer’s instructions. Briefly, for library construction, total RNA from each samples were used 1ug to ligate the adaptors and then cDNA was synthesized using reverse-transcriptase with adaptor-specific primers. PCR was performed for library amplification and libraries were carried out clean-up using QIAquick PCR Purification Kit (Quaigen, Inc, German) and AMPure XP beads (Beckmancoulter, Inc., USA). The yield and size distribution of the small RNA libraries were assessed by the Agilent 2100 Bioanalyzer instrument for the High-sensitivity DNA Assay (Agilent Technologies, Inc., USA). High-throughput sequences were produced by NextSeq500 system as way of single-end 75 sequencing (Illumina, SanDiego, CA., USA).
Sequence reads were mapped by bowtie2 software tool in order to obtain bam file. Mature miRNA sequence is used as a reference for mapping. Read counts mapped on mature miRNA sequence were extracted from the alignment file using bedtools v2.25.0 (Quinlan AR, 2010) and Bioconductor 20 that uses R statistical programming language (R development Core Team, 2016). Read counts were used in order to determine the expression level of miRNAs. Quantile normalization method was used for comparison between samples. For miRNA target study, miRWalk 2.0 21 was performed. Functional gene classification was performed by DIANA 22.
Exosomal small RNA sequencing arrays were prepared, hybridized, and scanned at the local authorized Illumina array service provider (Ebiogen, Seoul, South Korea).
TCGA(The Cancer Genome Atlas) and GEO(Gene Expression Omnibus) Database analysis
The two gene expression profiling data sets (GSE74618, GSE147889) we analyzed were downloaded from the NCBI GEO database.23 The GSE74618 database consists of 218 human HCC tumours samples, 10 adjacent cirrhotic non-tumoral tissue samples, and 10 healthy liver samples. We used adjacent cirrhotic non-tumoral tissue samples and healthy liver samples as controls and compared them with human HCC tumours samples. The GSE1477889 database consists of 97 samples of HCC tumours and 97 samples of the same patient's surrounding chronic hepatitis tissue. Surrounding chronic hepatitis tissue was used as a control and compared with HCC tumour samples. Two data sets were analyzed as volcano plots and mean difference plots, respectively, using the GEO2R analysis tool. TCGA (The Cancer Genome Atlas) data analysis was performed using oncomiR (WashU Pan-Cancer miRNome Atlas) online bioinformatics tool.24 Among the TCGA data, the analysis was conducted using the LIHC (Liver Hepatocellular Carcinoma) database (n=366), and the days' survival rate graph of liver cancer patients was exhibited.
Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses
KEGG is a database that contains genetic information on the human genome, biological cell signalling pathways, and various diseases and chemicals. The KEGG pathway analysis of exosomal miRNA was analyzed using the DIANA-miRPath v3.0 analysis tool. DIANA-miRPath v3.0 is a KEGG-based miRNA pathway prediction tool that actively interacts with the major miRNA analysis tools, DIANA-microT CDS and TargetScan v6.2 database, to analyze the target pathway.22
Cells were seeded in 6-well plates at 1 × 105 cells/mL/well before the transfection and radiation. On the following day, After HepG2 cells were exposed 4gy of radiation using X-RAD 320 and transfection was performed when the cells had reached approximately 80% confluence. miRCURY LNA miR-151a-3p mimic (miRCURY LNA miRNA Mimic, MIMAT0000757) were purchased from QiAGEN (Qiagen Korea Ltd.). And scrambled miRNA control were purchased from BIONEER (Seoul, Korea). The final concentrations of miRNA were 50-100nM Transfections were conducted with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.
Quantitative real time reverse transcription polymerase chain reaction(qRT-PCR)
Total RNA was extracted using a miRNeasy Mini Kit (Qiagen Korea Ltd.) in the manufacturers' protocols. For the quantification of matured cellular and exosomal miRNAs, the extracted total RNA was polyadenylated with a poly(A) tailing kit (Ambion, Austin, TX) prior to reverse transcription. And was reverse transcribed to cDNA using PrimeScript™ Reverse Transcriptase (Takara Bio Inc.). The cDNA samples were used for quantitative RT-PCR analysis in triplicate to determine the expression levels of miR-151a-3p using a TB Green® Premix Ex Taq™ (Takara Bio Inc.) and CFX96 Detection System real-time PCR instrument.
The cell proliferation assay was performed using the Chromo-CK Cell Viability Assay KIT (monobio seoul, korea) according to the manufacturer’s instructions. Transfected cells were harvested at the designated times after seeding. Briefly, the reagent (10 μl/well) was added to 100 μl of medium containing cells in each well of a 96-well plate and incubated for 1 hours at 37℃ under humidified 5% CO2 in air. For colorimetric analysis, absorbance at 450 nm was recorded using an ELx800 Absorbance Microplate Reader (SpectraMax 340PC Microplate Reader, Molecular Devices, USA). Each experiment was repeated at least 3 times.
Migration invasion assay
Tumor cell migration and invasion were analyzed in 24-well plates with 8-μm pore size polycarbonate membranes (BD, NJ, USA). For invasion assays, the membranes were coated with diluted Matrigel (BD, NJ, USA) to form matrix barriers. HepG2 were transfected with 151a-3p or negative control. For the migration and invasion assays, the cells (5×10^4 for migration, 1×10^5 for invasion) were resuspended in 200μl of serum-free DMEM at 24 hours post-transfection and added to the upper compartments of the chambers, and the lower compartments were filled with 600μl of DMEM with 10% FBS (different substrates were added accordingly). After incubation at 37°C for 10 hours (migration) or 24 hours (invasion), the cells remaining on the upper surfaces of the membrane were removed. The cells on the lower surfaces of the membrane were fixed, stained with crystal violet and counted under a light microscope. Each experiment was repeated at least 3 times.
Analysis of cell cycle
The Transfected cells were collected and resuspended after radiation exposure. Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining assays were conducted to detect the percentage of apoptotic (FITC-stained) and necrotic (PI-stained) cells in a given population. Analyses were performed by a FACS canto flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The procedures were repeated in triplicate. 25
Analysis of cell apoptosis
The Transfected cells were collected and resuspended with cold PBS after radiation exposure. And fixed in cold 70% ethanol added dropwise to the cell pellet while vortexing. Stored at -20℃ for overnight. Cells were rehydrated with PBS for 10 min at RT and then cells were stained with propidium iodide (PI) staining solution contained with 50 μg/ml PI (BD Biosciences, Franklin Lakes, NJ, USA), 100 μg/ml RNase A, DNase and protease-free (Thermo Scientific) in 1X binding buffer. Analyses were performed by a FACS canto flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The procedures were repeated in triplicate.
Subcutaneous tumor xenograft models
BALB/c nude mice (5-week old male) were purchased from the Charles River Laboratories, Inc (Wilmington, Massachusetts). The fodder was feed to the normal rodent laboratory animal feed. 5x10^6 HepG2 cells in total volume 100ul were injected into the right flank of a 5-week-old nude mouse in a 1:1 ratio with Matrigel (corning 356234). After the average of the transplanted solid cancer size increased by more than 100 mm3, the invivo-jetPEI/target miRNA complex was treated. invivo-jetPEI was used as an efficient carrier for delivering miRNA and was mixed with 5% glucose solution at a ratio of miRNA 10ug / invivo-jetPEI 1.2ul and treated in a total dose of 100 ul. The invivo-jetPEI/target miRNA complex was administered 3 times every 3 days by intratumoral injection. Tumor size was measured using a standard ABS digimatic caliper (CD-15AX) every 3 days after transplantation. Tumor volume was measured using the following formula: ∏ x 4/3 x larger diameter x smaller diameter square. At the time when the volume of the largest tumor of the implanted nude mice in the experimental group exceeded 1000 mm3, the tumor was separated after sacrifice, the weight of the tumor was measured, and expression of the target protein in cancer tissue was analyzed.
Each experiment in our study was repeated three times. Mathematical data are expressed as mean ± SD. Unless indicated, the differences between two groups were analyzed using a Student's t-test (two-tailed). Unless otherwise noted, significant numerical differences between the two groups were analyzed using Student's t-test (two-tailed). The overall survival curve was graphed using the Kaplan-Meier method and compared through the log-rank test. Differences were evaluated statistically significant at P< 0.05. All statistical analysis was performed using SPSS13.0 software (SPSS, Chicago, IL, USA).