Establishment of RelB-knockdown cell line
DU145 cell line was bought from ATCC (American Type Culture Collection). The cells passaged 2-5 times were used in our experiments. The cell line was certified using Short Tandem Repeat (STR) profiling (Shanghai Biowing Applied Biotechnology Company). All cell lines were routinely tested for mycoplasma. The RelB silenced DU145 cell line (siRelB) and control cell line (sictrl) had been established in a previous study . Briefly, the shRNA-RelB (targeted gene 275-293: 5'-GCACAGATGAATTGGAGAT-3') was synthesized and cloned into the pSilencer3.1-H1-neo vector (cat no. 5770, Thermo Scientific™, USA). The pSilencer3.1-siRelB and the control plasmids were afterward transduced into cells by Lipofectamine 2000 (cat no. 12566014, Thermo Scientific™, USA) following the instructions. The cells were cultured in the presence of 400 ng/μl neomycin (cat no. E859-5G, Amresco) for about 15 days.
Cell culture and supernatant collection
DU145-siRelB and DU145-sictrl cells were maintained in RPMI-1640 media added with 10% exosome-depleted fetal bovine serum (EXO-FBS-50A-1, SBI, Japan), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 ˚C in a humidified atmosphere containing 5% CO2. After the cell confluence reached 85~95%, the culture medium was collected and stored at -80 ℃ after centrifugation at 500 g for 15 min.
Exosomes extraction and characterization
Exosomes from exo-free FBS supernatant of both siRelB and sictrl were isolated by ultracentrifugation. Firstly, dead cells and large cell debris were deposited by centrifugations at 1200 g for 20 min and 10,000 g for 20 min. Pellets from each centrifugation step were discarded. 100 ml medium was collected and ultracentrifuged at 100,000 g at 4 ℃ for 90 min (Optima XPN-100 ultracentrifuge, Beckman, USA). The sediment was resuspended in 1x PBS for further analysis. The morphological characteristics of exosomes were confirmed by transmission electron microscopy using the method described by Ying et al . Particle size by NanoSight analysis was conducted to verify the uniformity of microvesicle extracted.
Exosome protein extraction, quantification, and immunoblotting
The protein of exosomes was extracted using a 5x RIPA buffer with 1 mM proteinase inhibitor. The mixture was vortex blended and cracked for 20 min on ice. The supernatant was collected after centrifugation at 10,000 g for 12 min. The BCA assay was used for measuring protein concentration (Thermo Fisher Scientific, USA). The absorbance at 562 nm was determined. We drew the standard curve according to the calibrated absorbance value and standard concentration. The concentration of protein samples was calculated based on the standard curve. 20 μg protein of exosomes were loaded on a 10% SDS gel for electrophoresis, followed by transfer to a polyvinylidene fluoride membrane. Primary antibodies used were rabbit monoclonal anti-human flotillin (cat no. 18634, Cell Signaling Technology, Germany), mouse monoclonal anti-human CD81, and mouse monoclonal anti-human CD63 (cat no. NB100-65805 and NBP2-42225, Novus Biologicals, USA). Goat anti-mouse IRDye 800cw secondary antibody and goat anti-rabbit IRDye 680 secondary antibody (cat no. 926-32210 and 926-32221, Li-COR Biosciences, USA) were used as secondary antibodies. The image was scanned by Odyssey’s two-color infrared fluorescence imaging system (Li-COR Biosciences, USA).
Sample Preparation for Mass Spectrometry
The exosome samples were cracked in buffer with 8 M urea, 1% Triton X-100, 65 mM dithiothreitol (DTT, sigma, Germany), and 0.1% proteinase inhibitor (cocktail) using a high-intensity ultrasonic machine (JY99-IIDN, Scientz, China) on ice. Pellets were discarded after centrifugation at 5 000 g for 3 min at 4 °C. The pre-cooled 15% trichloroacetic acid was used to dissolve the protein for 2 h at -20 °C. Protein precipitation was obtained after centrifugation at 15 000 g for 10 min at 4 °C, followed by three washing steps with cold acetone, dried for 5 min and subsequently rehydrated in buffer containing 8 M urea, 100 mM triethylamine-carbonic acid buffer (TEAB, pH 8.0). The BCA assay was used to determine protein concentration. Add 10 mM DTT (Sigma, Germany) into 100 μg protein samples at 37 °C for 1 h, then 20 mM IAA (iodoacetamide, Sigma, Germany) were added for alkylation reduction at room temperature for 45 min. First enzymatic hydrolysis of protein samples was conducted at the present of 1 μg trypsin at 37 °C for 4 h, followed by second enzymatic digestion with another 1 μg trypsin at 37 °C for 12 h. Peptide mixture was acidized with 5% formic acid, demineralized using C18 spin columns, and eluted by 200 μl 50 mM NH4HCO3, then quantified and lyophilized.
Liquid Chromatography-Mass Spectrometry (LC-MS) Procedure
The procedure of LC-MS was referred to the instructions described by Zou et al . The peptides were resuspended in 100 μl solvent A (A: water containing 0.1% formic acid; B: ACN containing 0.1% formic acid), separated by nanoLC and analyzed by on-line electrospray tandem mass spectrometry. The tests were conducted on a set of equipment including an EASY-nLC 1000 system (Thermo Fisher Scientific, Waltham, MA), an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA), and an online nano-electrospray ion source. 2 μl peptide sample was loaded onto the trap column (Thermo Scientific Acclaim PepMap C18, 100 μm x 2 cm), with a flow of 10 μl/min for 3 min and subsequently separated on the analytical column (Acclaim PepMap C18, 75 μm x 25 cm) with a linear gradient, from 2% B to 45% B in 180 min. The column was re-equilibrated at initial conditions for 5 min. The column flow rate was maintained at 300 nl/min. The electrospray voltage of 2.0 kV versus the inlet of the mass spectrometer was used.
The Orbitrap Fusion mass spectrometer adopted a data-dependent mode for data collection by the alternative switch between Mass Scan (MS) and tandem MS (MS/MS) . In MS scan, the ions with 350-1600 m/z and a charge state of >3 were chosen for deeper fragmentation in an orbitrap mass analyzer. The maximum ion introduction times were 50 ms for the full MS scan and 150 ms for MS/MS. The resolution were targeted as 120,000 (m/z, 200) for MS and 15,000 (m/z, 200) for MS/MS. The automatic gain control (AGC) were set as 5 x 105 and 2 x 105 for MS and MS/MS, respectively. Fragmentation was conducted to the ions in +2, +3, +4, or +5 charge state, with a minimum intensity threshold of 20,000. Precursor ions were fragmented in a mode of higher-energy collisional dissociation (HCD) at 30% of normalized energy in the fracture pool. One Microscan was recorded employing a dynamic exclusion of 48 sec all the time.
Identification of differentially expressed proteins (DEPs)
Raw MS data were analyzed by Maxquant database (version 184.108.40.206) and retrieved using the integrated Andromeda search engine with a search strategy of target- decoy. The peptide designation was conducted by running raw data against the SwissProt-Human data set (Release 2017-04-10) (total of 20259 entries). Trypsin (Promega, Madison, WI) was used for specific digestion, and a maximum of 2 missed cleavages was permitted. The mass tolerance for fragment ions and precursor ions were 0.02 Da and ±10 ppm, respectively. Carbamidomethylation of cysteine was set as a fixed modification. Protein N-terminal, O-GlcNAc protein modification, or lysine carbamidomethylation, methionine oxidization were set as variable modifications. False discovery rate (FDR) thresholds set for peptide, protein, and modification sites were all 1%. For protein identification, minimum peptide length was set as 7 amino acids with fewest one match of unique peptide.
The label-free quantification strategy iBAQ (intensity-based absolute quantification) was applied to quantify. The raw data were further processed by Perseus software. Protein group LFQ intensities were log2 transformed. The missing values were replaced by random values in the Gaussian distribution below the median to simulate low abundance LFQ values. Fold change (FC) filtering and Student's t-test were performed to screen DEPs between two groups by using the R software. DEPs were identified if the minimum matched unique peptide >=1, with a threshold p < 0.05, absolute value of FC > 1.25 and the FDR correction <=0.05. Venn diagram, volcano plot, and hierarchical clustering were performed for the DEPs of the two groups.
Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were implemented to understand the exact characters and functions of DEPs. A p-value of less than 0.05 was considered enriched significantly. Three biological repeats were performed in the two groups.
UALCAN database and GeneMANIA analysis
UALCAN is a web portal integrating and analyzing cancer data based on The Cancer Genome Atlas (TCGA) database, providing plots and graphs describing gene expression and clinical prognosis according to sample type, race, age, or other factors . In the present study, we used UALCAN to interrogate the correlation between RelB and ICAM1 and the expression of the ICAM1 gene in PC patients. GeneMANIA is an online tool processing protein-protein interaction (PPI) network, and giving information about physical interaction, gene co-expression, gene co-location, gene enrichment analysis, and website prediction . We predicted the interaction between RelB and ICAM1 and generated the networks through GeneMANIA.
Construction and verification of hICAM1 and hctrl cell lines
The ICAM1 overexpressed cell line (hICAM1) and control cell line (hctrl) were established by transfecting pCMV3-ICAM1-His plasmid (cat no. HG10346-CH, Sino Biological Inc., China) and negative control vector (cat no. CV015, Sino Biological Inc., China) into DU145 cells with Lipofectamine 2000 reagent (cat no. 12566014, Thermo Scientific™, USA). Cells were maintained in the culture medium containing 400 ng/μl hygromycin (Roche Diagnostics GmbH, Germany) for two weeks. RNA was extracted using TRIzol (cat. no. 15596-026, Invitrogen life technologies, USA) method according to the instruction. RT-qPCR was conducted to test the ICAM1 mRNA expression in different clones and exosomes, using the following primers; forward primer, 5‘-TCCGGCGCCCAACGTGATTC-’3, and reverse primer, 5‘-CGGGGGCCATACAGGACACG-’3. The expression was normalized to β-actin (5’-GCTACGAGCTGCCTGACGG-3’ for forward primer, and 5’-TGTTGGCGTACAGGTCTTTGC-3’ for reverse primer). △Ct was the Ct difference between ICAM1 and β-actin of each sample. 2−△Ct was calculated to show the relative expression of ICAM1. Western blotting was applied to detecting the ICAM1 protein expression in cells or exosomes. Rabbit polyclonal anti-human ICAM1 (cat no. 4915, Cell Signaling Technology, Germany) was used as the primary antibody. The concentration of soluble ICAM1 (sICAM1) in exosome was measured using ELISA (cat no. DCD540, R & D systems, USA), and calculated based on the optical density (OD) at 450 nm. GraphPad Prism 6.0 was used to plot.
Description of the biological characters of cell lines
The cell growth, migration, and invasion activities were dynamically monitored by the xCelligence RTCA instrument (Roche) as described before . In the cell growth assay, cells were planted at 7 000 per well. The monitoring was last for 80 h. A CIM 16-plate was used in cell migration assay, equipped with an upper and lower chamber. Cells were seeded at 40, 000 per well suspended in 30 μl pure RPMI-1640 in the upper chamber, while 170 μl complete media was loaded into the wells of the lower chamber. Wells on the upper chamber pre-coated with matrigel were used for the cell invasion assay. 60, 000 cells were seeded. The cell index was continuously monitored for 30 h for migration or invasion assay. CCK-8 assay (cat. no. ab228554, Abcam, England) was used to assess cell proliferation. Briefly, 6000 cells suspended in 100 μl culture medium were planted into 96-well plate with quintuplicate wells per sample. Three wells added with 100 μl culture medium were used as blank control. The 96-well plate was continued to incubate for 2 h after adding 10 μl CCK-8 reagent. The OD values at 450 nm were measured at 24 h, 48 h, and 72 h. AnnexinV and PI dyes were adopted to analyze the early apoptosis using flow cytometry. 1.5×105 cells suspended in 3,000 μl complete medium were planted into a 6-well plate with triplicate wells per sample. Cells were harvested and stained using annexin-V, PI, or both for 15 min in darkness according to the instructions (cat. no. 40302ES50, Yeasen Biotech, China). The early apoptosis proportion was analyzed at 24 h, 48 h, and 72 h. The protein level of metastasis-related proteins integrin β-1, MMP9 (cat no. 4706 and 13667, Cell Signaling Technology, Germany) and uPA (cat no. sc-59727, Santa Cruz Biotechnology, USA) were analyzed by western blotting. The image was scanned by Odyssey. The data were analyzed and plotted using GraphPad Prism 6.0.