Preparation of NET-1 siRNA nanoparticles
The NET-1 siRNA duplex was designed according to our previous research(13). The nanoparticles were prepared with DSPG, DSPC, and DSPE-PEG2000, and the weight ratio was 7:2:1. All the phospholipids were purchased from Avanti Polar Lipids (Avanti Polar Lipids, Alabaster, AL, USA. 20 mg of phospholipids were dissolved in a mixed solution of chloroform and methyl alcohol. The mixed solution was subsequently purged by vacuum rotary evaporation to form phospholipid thin film. Then, the thin film was hydrated at 40°C with 5 mL of DEPC treated H2O. This was followed by dissolving the appropriate amounts of NET-1 siRNA duplex in the lipid film solution. The NET-1 siRNA nanoparticles were obtained using an extrusion technology by mini-extruders (Avanti Polar Lipids, Alabaster, AL, USA) through a 400nm membrane for 11 times. The obtained NET-1 siRNA nanoparticles were then transferred into a sealed vial and stored at 4°C for further experiments.
The structure of NET-1 siRNA nanoparticles was detected under transmission electron microscope (TEM, Hitachi TEM system, Japan). The size and zeta potential were investigated by dynamic light scattering (DLS) via the Malvern Zetasizer Nanoseries (Zeta PALS BI-90 Plus, Brookhaven Instruments).
Cell lines and animal tumor inoculation
The human HCC cell line HepG2 cells were a generous gift from the Institute of Cancer Research affiliated with Harbin Medical University (Harbin, China). Cells were cultured in DMEM medium supplemented with10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO2/95% air at 37°C.
BALB/c nude female mice (6–8week, 10–25 g) were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). All the animals were housed in an environment with a temperature of 22 ± 1°C, a relative humidity of 50 ± 1%, and a light/dark cycle of 12:12 h. All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Harbin Medical University institutional animal care and conducted according to the Association for Assessment and Accreditation of Laboratory Animal Care and the Institutional Animal Care and Use Committee guidelines. Mice were anesthetized with 3% isoflurane inhalation and 1L/min 100% oxygen. A total of 5x106 HepG2 cells were suspended in 50 ml PBS and50 ml Matrigel (BD Biosciences, San Jose, CA, USA). Tumor cell–Matrigel mixture (100 ul) was subcutaneously injected in the right back position of the mice.
In vivo studies
Once the tumor diameter reached 0.5 cm, the mice bearing tumors were randomly divided into 3 groups (8 mice/group): group A, blank (PBS); group B, NET-1 siRNA nanoparticles without LIFU irradiation; group C, NET-1 siRNA nanoparticles with LIFU irradiation. All the NET-1 siRNA nanoparticles were intravenously administered via the tail vein at a dose of 5ml/kg body weight.
At 30 min postinjection, all groups were irradiated with LIFU using a CGZZ Low-Frequency Ultrasound Treatment Instrument (Institute of Ultrasound Imaging, Second Affiliated Hospital of Chongqing Medical University, Chongqing, China). LIFU parameters were as follows: frequency of 1 MHz, pulse repetition frequency of 1 kHz, duty cycle yield of 50%, intensity of 1.0 W/cm2, and duration of irradiation of 5 min per mouse. The nude mice were treated twice a week for a total of 60 d. The survival end point was a tumor diameter of 20 mm in any direction (according to the Guidelines for Tumor Induction in Mice and Rats, American Association for Laboratory Animal Science, Memphis, TN, USA); the maximum tumor diameter was measured twice every week using Aixplorer US system with high-frequency probe (Super Linear TM SL15-4, Super Sonic Imagine, Aix-en-Provence, France). The HCC xenograft samples were harvested after euthanasia and formalin-fixed for long-stem storage and proteomic analysis.
Proteomic Analysis
Total Protein Extraction
Inspired by the survival result, FFPE HCC xenograft samples in group A and group C were selected for proteomic analysis. Three pieces of the FFPE samples in each groups were dewaxed with octane and then hydrated with graded ethanol. After hydration, the sample was washed twice with PBS. After removing the PBS solution, an appropriate amount of protein lysate (4% SDS, 100 mM Tris, pH = 8.5) was added and incubated at 95℃ for 10 minutes at room temperature, mixed by shaking, and sonicated in an ice-water bath for 5 minutes. The samples were de-crosslinked with a refractive index at 95℃ for 60 min, then reduced by adding an appropriate amount of TCEP and carboxyamidomethylated in CAA at 95℃ for 5min. The samples were sequentially centrifuged at 12000 g at 4℃ for 15 min. Collecting the supernatant and adding four times volume of pre-cooling acetone at -20℃, and precipitated it at -20℃ for at least 4h. Centrifuging at 12000 g for 15 min at 4℃. Collect the precipitate and air drying. An appropriate amount of protein solution (6 M urea, 100 mM TEAB, pH = 8.5) was added to dissolve the protein pellet.
Trypsin treatment
The protein solution was added to flat membrane ultrafiltration (cut off molecular is 10kDa) tube and was centrifuged at 14,000 g at room temperature for 20 minutes, and the flow-through was discarded. 100 μL of 50 mM TEAB was added, and the sample was centrifuged at 14000 g at room temperature for 20 minutes. The washing procedure was repeated four times. 100 μL of 50 mM TEAB and an amount of 1:50 mass ratio of trypsin were added to the protein and incubated at 37℃ overnight. After being centrifuged at 14000 g for 20 min, an equal volume of 2% formic acid was added. After mixing, the solution was centrifuged at 14,000 g for 20 min at room temperature. The supernatant of flow-through was slowly passed through a C18 desalting column, and then 1 mL washing solution (0.1% formic acid, 4% acetonitrile) was added to wash three times in succession, then 0.4 mL of eluent (0.1% formic acid, 75% acetonitrile) was added to elute twice in sequence, the eluent samples were combined and freeze-dried.
LC-MS/MS Analysis
Mobile phase A (100% water, 0.1% formic acid) and B solution (80% acetonitrile, 0.1% formic acid) were prepared. The lyophilized powder was dissolved in 10 μL of solution A, centrifuged at 15,000 rpm for 20 min at four ℃, and 1 μg of the supernatant was injected into a home-made C18 Nano-Trap column (2 cm×75 μm, three μm). Peptides were separated in a home-made analyticalcolumn (15 cm×150 μm, 1.9 μm) using linear gradient elution, as listed in Supplemental Table 1. The isolated peptides were analyzed by the Q Exactive series mass spectrometer (Thermo Fisher), with ion source of Nanospray Flex™(ESI), spray voltage of 2.3 kV, and ion transport capillary temperature of 320°C. Full scan range from m/z 350 to 1500 with resolution of 60000 (at m/z 200), an automatic gain control (AGC) target value was 3×10 6, and a maximum ion injection time was 20 ms. The top 20(40) precursors of the highest abundant in the full scan were selected and fragmented by higher-energy collisional dissociation (HCD) and analyzed in MS/MS, where resolution was 15000 (at m/z 200), the automatic gain control (AGC) target value was 5×104, the maximum ion injection time was 45 ms, a normalized collision energy was set as 27%, and intensity threshold was 2.2×104. The dynamic exclusion parameter was 20 s. The raw data of M.S. detection was named as ".raw".
Data analysis
The identification and quantitation of protein
The resulting spectra from each fraction were searched separately against the homo sapiens uniprot database by the search engines: Proteome Discoverer 2.2 (PD 2.2, Thermo). The search parameters are set as follows: mass tolerance for precursor ion was ten ppm, and mass tolerance for production was 0.02 Da. Carbamidomethyl was specified in PD 2.2 as fixed modifications. Oxidation of methionine (M) and acetylation of the N-terminus was specified in PD 2.2 as variable modifications. A maximum of 2 missed cleavage sites was allowed.
The identified protein contains at least one unique peptide with FDR no more than 1.0%. Proteins containing similar peptides that could not be distinguished by MS/MS analysis were identified as the same protein group. Precursor ion was quantified by a label-free quantification method based on intensity. Mann-Whitney Test statistically analyzed the protein quantitation results for proteins whose quantitation significantly different between experimental and control groups were defined as differentially expressed proteins (DEP).
The functional analysis of protein and DEP
Gene Ontology (GO) and InterPro (IPR) analysis were conducted using the interproscan-5 program against the non-redundant protein database (including Pfam, PRINTS, ProDom, SMART, ProSiteProfiles, PANTHER)(38), the databases of Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the protein family and pathway. The probable protein-protein interactions (PPI) were predicted using the STRING-db server(39) (http://string.embl.de/). The enrichment pipeline was used for enrichment analysis of G.O., IPR, and KEGG(40).
Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed by unpaired, two-tailed Students t-test using the GraphPad Prism 8.0 software (GraphPad Software) if not denoted otherwise. Differences were considered statistically significant at p-value < 0.05. Mann-Whitney Test statistically analyzed the protein quantitation results. Because > 2 groups were compared in this survival study, the log-rank test was used to compare 2 specific groups when the overall values were p < 0.05. Proteins were supposed to be significantly differentially expressed when the p-value < 0.05, Fold Change (FC) ≤0.05 or p-value < 0.05, FC≥2.0.