Main materials
Human esophageal squamous carcinoma cell line EC9706 was provided by the Chinese Academy of Sciences Cell Bank (Shanghai, China). RPMI-1640 medium and fetal bovine serum were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the trypsin and MTT kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). The DAB chromogenic reagent kit (PA110) was purchased from Tiangen Biotech Co., Ltd., (Beijing, China), while the RIPA protein lysate (product no. P0013B) and dimethyl sulfoxide (product no. ST038) were manufactured by Biyuntian Biotech Co., Ltd. (Shanghai, China). Rabbit anti-STAT3 polyclonal antibody, mouse anti-p-STAT3 (705-tyrosine phosphorylation site) monoclonal antibody, and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody were purchased from Signalway Antibody LLC (College Park, MD, USA). The ultrasound contrast agent SonoVue (lyophilized preparation) was purchased from Bracco SpA (Milan, Italy), while the Lipofectaine™ 2000 transfection kit for cationic liposomes was obtained from Invitrogen (Thermo Fisher Scientific, Inc.). The ODNs and its mismatch control sequences were designed by Shanghai Shenggong Biological Engineering Co., Ltd. (Shanghai, China). All base sequences were modified by total phosphorothioation. The STAT3 decoy ODN sequence was 5′-CATTTCCCGTAAATC-3′ and 5′-CATTTACGGGAAATG-3′, and was labeled with FITC. The double-stranded mutant ODN control sequence was 5′-CATTTCCTTAAATC-3′ and 5′-GATTTAAGGGAAATG-3′.
The main instruments include an Olympus fluorescence inverted microscope (Olympus Corporation, Tokyo, Japan), an ultrasound therapeutic apparatus (Taizhou People's Hospital, Taizhou, China), an ABI 7500 real-time fluorescence quantitative PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), an Annexin V-FITC/PI apoptosis kit (Hangzhou MultiSciences [Lianke] Biotech Co., Ltd., Hangzhou, China), a CGZZ Ultrasonic gene transfection instrument (Ultrasonographic Image Research Institute, Chongqing Medical University, Chongqing, China; ultrasonic transmitting frequency, 300–1,000 kHz; sound intensity range, 0.25–2.50 W/cm), and a Synergy HT Multi-dection microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).
The study was approved by the Ethics Committee of Ningbo No. 2 Hospital (Ningbo, China).
Cell culture
Cells were cultured in RPMI-1640 medium (100 µg/ml of streptomycin + 100 U/ml of penicillin) containing 10% high-quality fetal bovine serum, and maintained at a specific condition of 37°C with 5% CO2 saturation humidity. Then, cells that adherently grew were closely observed, and the culture fluid was changed every two days. When ~80% confluence was reached, cells were digested with trypsin (0.25%) and passaged. Exponentially growing cells were selected for subsequent experiments.
Preparation of microbubbles
The diameter of the ultrasound contrast agent SonoVue was 2–5 µm, and the average diameter ~2.5 µm. The surface had phospholipids, and was filled with SF6. Before implementation, 5 ml of 0.9% physiological saline was used to dissolute and dilute the SonoVue, and vigorously shaken until the freeze-dried powder completely dissolved into a microbubble suspension. According to previous experiments, the optimal microbubble concentration was 20%.
Groups for the experiment
The present study was divided into three groups: the experimental group, the positive control group and the blank control group. The experimental group included the following sub-groups: Group E, STAT3 Decoy ODN + ultrasound microbubble contrast agent mixed solution + ultrasound irradiation; Group EC, mutant ODN + ultrasonic microbubble contrast agent mixed solution + ultrasonic irradiation. The positive control groups were as follows: Group P, STAT3 decoy ODN + liposomal mixture + ultrasound irradiation; Group PC, mutant ODN + liposomal mixture + ultrasound irradiation. The blank control groups were as follows: Group C, STAT3 decoy ODN + ultrasound irradiation; Group CC, STAT3 decoy ODN + ultrasound microbubble contrast agent mixed solution.
Cell transfection and ultrasonic irradiation
The sense and antisense STAT3 decoy ODN was solubilized with pH 8.0 Tris-HCl EDTA solution (1 mmol/l), respectively. Then, this was annealed at 90°C to form a double strand, keeping the temperature down at 5°C every 15 min, and the reactant mixture was stored at 4°C.
The transfection operations for groups E and EC were as follows: serum-free RPMI-1640 nutrient solution was used to adjust the concentration of ODN and 1 ml of ultrasonic microbubble suspension to 100 pmol. The above two liquids were respectively mixed, gently shaken, and subsequently placed on ice for 20 min. The STAT3 decoy ODN (mutant ODN)-microbubble mixture was mixed with cells at a density of 1×105 while being exposed to ultrasound (frequency, 1 MHz; irradiation intensity, 0.5 W/cm2; duration time, 10 sec; interval, 10 sec; total irradiation time, 1 min). After 6 h of reaction, the sealing membrane was removed, and the medium containing 10% fetal bovine serum was replaced by incubating at 37°C in a 5% CO2 incubator.
For groups P and PC, liposome transfection was performed, according to the kit instructions. Then, 2–4 µg of STAT3 decoy ODN/mutant ODN (100 pmol) and 5 µl of Lipofectamine were respectively dissolved in serum-free and antibiotic-free medium, and mixed were thoroughly. Afterwards, this was allowed to stand for 5 min, and was incubated at room temperature for 20 min. Then, cells were diluted to adjust the density to 1×105. Next, the STAT3 decoy ODN-liposome mixture was mixed with cells and placed at 37°C. Then, ultrasonic irradiation was performed at the same conditions as above, incubated for another 6 h, and the RPMI-1640 medium containing 10% fetal bovine serum was replaced for further culture.
For group C, 100 pmol of ODN was mixed with EC9706 esophageal squamous carcinoma cells at a density of 1×105, and subjected to ultrasound irradiation under the above conditions. After 6 h, the serum that contained the RPMI-1640 medium was replaced. For group CC, the same procedure was performed as that in groups E and EC, except for the ultrasound irradiation.
Detection after transfection
After 48 h of operating, according to the above, the cells were observed and photographed under an inverted fluorescence microscope (magnification, ×400). Then, 10 fields were randomly selected from each slide, and the transfection rate was calculated by the ratio of the number of green fluorescence cells and the number of total cells.
Detection of cell apoptosis by flow cytometry
Cells were collected after digestion, cultured for 24 h, washed with pre-chilled 4°C PBS twice, and the supernatant was discarded. Then, 70% ethanol was used to fix cells. Next, the samples were centrifuged at 5,013 × g for 10 min at 28°C and resuspended in 500 µl of binding buffer to a cell density of 1×10 cells/ml. Subsequently, 5 µl of FITC-labeled Annexin V mixture and 10 µl of propylene iodide (PI; 1 µg/ml) solution were added and carefully mixed. After 15 min of dark reaction at room temperature, 400 µl of 1X binding buffer was added. After the full reactions, BD Accuri™ C6 Flow Cytometer (BD, Loveton Circle, USA) was used to detect 1×104 cells per sample.
Cell survival curve measurement by MTT assay
The log phase growth of EC9706 cells was diluted to 1×108/l of cell suspension, and inoculated on 96-well plates at 200 µl per well. Then, the diluted STAT3 decoy ODN, mutant ODN, liposomes and ultrasonic microbubble mixture was added into the corresponding wells after 24 h of incubation. Each group was set up with six duplicate wells, and 150 µl of culture medium was added. The control group consisted of an equal volume of dimethyl sulfoxide and serum-free medium without any treatment. The change in culture fluid at 12, 24, 48 and 72 h after the start of the culture observed, and 20 µl of MTT (5 mg/ml) was simultaneously added at room temperature for another 4 h. Then, the supernatant was carefully discarded, and 150 µl of DMSO was added. Subsequently, dimethyl sulfoxide (150 µl) was added, and lightly shaken for 10 min until the crystals were dissolved. The absorbance (A) of each well was measured using a microplate reader at 490 nm. The formula was: Cell inhibition rate % = (A control group - A experimental group)/A control group ×100%.
Revese transcription-quantitative PCR (RT-qPCR)
Cells were lysated with TRIzol reagent. Then, the lysate was transferred to a centrifuge tube, chloroform was added, and the supernatant was taken after shaking and centrifugation at 12,000 × g for 15 min at 4°C. Next, the upper RNA was extracted and an equal volume of isopropanol was added. The mixture was centrifuged at 12,000 × g for 10 min at 4°C after mixing well. Then, the precipitate was collected, washed with 75% ethanol, and air-dried and dissolved in DEPC to synthesize the cDNA by reverse transcription. The internal reference gene was β-actin. The reaction conditions were as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 1 min with 35 cycles, and finally, extension at 72°C for 10 min. The Ct values of the internal reference genes and the genes of the respective groups were measured, and the relative expression levels of the target gene were calculated using the formula 2−ΔΔCq (7). The experiment was repeated three times. In the present experiment, the internal reference gene and target gene primer sequences are presented in Table I.
Western blotting
Each group of cells was washed three times with pre-chilled PBS. The RIPA lysate was added to extract the total tissue protein, according to protein kit instructions. The BCA kit was used to quantitatively analyze the protein concentration. Then, 20 µg of cellular total protein was respectively taken from each group for 10% SDS-PAGE separation. The semi-dry method was used to separate the protein, followed by transmembrane at a constant-voltage electrophoresis of 100 V. Then, 5% skim milk powder was used for blocking for ~1 h, and reacted overnight with p-STAT3 primary antibody (dilution, 1:250; cat. no. 4905) and STAT3 primary antibody (dilution, 1:600; cat. no. BF0374) for 2 h at a temperature of 4°C. Subsequently, the membrane was washed with TBST, and goat anti-mouse IgG secondary antibody (dilution, 1:1,000; cat. no. F0106B) added at 37°C and incubated for 1 h. The color was developed using a DAB kit and analyzed using Image J software. The experiment was repeated three times. The expression level of each detection factor was evaluated using the gray ratio of the target gene bands and β-actin bands.
Nude mouse model of subcutaneous transplantation tumor
Forty BALB/c female nude mice, aged 4–6 weeks with mean body mass 19.35±0.46 g were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Art. no. 401). During the feeding process, the temperature was maintained between 24–26°C with a suitable humidity (55–70%), free access to food and water and a daily 12 h light/dark cycle. Before the experiment, all the mice were adaptively fed for one week. After being anaesthetized via inhalation with 2% isoflurane, the mice were placed in the supine position and were depilated for ultrasound irradiation. The EC9706 single cell suspension was prepared and resuspended with PBS. Then, cell density was adjusted to 5×107 cells/ml. All treatments were performed on a clean bench. Subsequently, 75% alcohol was used to partially sterilize the mice. Under sterile conditions, 0.2 ml of a single cell suspension was subcutaneously injected on the right side. Tumors formed after one week of cell inoculation. When the tumors grew to ~100 mm2, mice with strong tumor growth were selected and sacrificed. Then, the necrotic tissue was removed, washed with physiological saline, cut into 2-mm3 uniform pieces, and inoculated on the right back of the nude mice. The animals were kept under the conditions of SPF with unlimited feeding. Mice were observed after 10 days. A total of 30 nude mice with a tumor size of ~100–200 mm3 were selected and randomly divided into six groups, with five mice in each group. Mice in the experimental group were slowly injected by each mixture (all, 0.25 ml) using a 1-ml syringe through the tail vein, while mice in the control group were injected with an equal volume of saline. After the injection, the ultrasound machine was immediately used three times, except for the CC group (output, 0.5 W/cm2; duration, 20 sec; interval, 20 sec). After the end of irradiation, mice were routinely reared, and the length and width of the transplanted tumor were measured every three days using a vernier caliper. Tumor volume = tumor length × width2 × 1/2. The experiment ended at 15 days after inoculation, and the nude mice were sacrificed by cervical dislocation. The tumors of the nude mice were excised and weighed to calculate for the tumor inhibition rate. The tumor inhibition rate % = (tumor mass of the control group - tumor mass of the experimental group)/tumor mass of the control group ×100%.
Statistical analysis
The data obtained from the experiment were analyzed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). Quantitative data were expressed as mean ± standard deviation (mean ± SD). Variance analysis was used to determine the immunofluorescence and flow cytometry results after transfection in each group. Pairwise comparison was performed using the SNK-q test. Repeated calculation of variance was performed to analyze the difference in the inhibition rate of the different groups of cells at corresponding time-points, and explore the effect of the overexpression by different methods to transfect STAT3 decoy ODN on the proliferation of cancer cells. At the same time, variance analysis was used to evaluate the RT-qPCR detection and western blotting results. Furthermore, the SNK-q test was used to analyze the differences in the expression levels of gene products in each group, thereby exploring the possible mechanism of STAT3 decoy ODN.