Cell culture and reagents
An immortalized pancreatic ductal epithelial cell line (HPDE) and pancreatic cancer cell line (Canpan-2) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and the pancreatic cancer cell lines AsPC-1, BxPC-3, and Panc-1 were purchased from the American Type Culture Collection (VA, USA). Cells were cultured in RPMI-1640 medium (GIBCO, NY, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% penicillin/streptomycin (Invitrogen) at 37 °C in a 5% CO2 incubator. The eIF4A inhibitor RocA and c-MYC inhibitor Mycro3 were purchased from MedChemExpress (MCE, USA).
Patient’ follow-up and specimens collection
This study was approved by the Ethics Committee of Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology (TJ-IRB20210927). All patients were completely informed of the possible use of their clinical information/specimens and we obtained full consent for the study. The cohort comprised 53 patients with PDAC who underwent surgical resection from 2009 to 2013 at the Department of Pancreatic and Hepatobiliary Surgery, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). All PDAC specimens were verified by pathologists and met the criteria set by the American Pancreatic Association. None of the patients underwent preoperative neoadjuvant therapy. The patients were evaluated every 3 months during the first 3 years and every 6 months thereafter by physicians who were blinded to the study parameters. Follow-up data were summarized at the end of December 2020. To evaluate the prognostic role of eIF4A1, tissue microarrays of the 53 PDAC samples were collected for further analysis.
IHC staining with the eIF4A1 antibody (Abcam ab31217) was performed to detect the protein expression level. ImageJ (http://imagej.nih.gov/ij) IHC profiler (http://sourceforge.net/projects/ihcprofiler) was used to assess IHC staining of the microarray based on two categories: staining intensity and percentage of staining. The staining intensity was scored as follows: negative (0 points), weak-positive (1 point), positive (2 points), and strong-positive (4 points). The protein expression level was quantified by multiplying the staining intensity and corresponding extents of positive staining (n%: percentage of positive areas to the whole areas). Then we divided the patients into two groups (score < 50, low expression; score > 50, high expression) and performed subsequent survival analysis. The IHC staining results were reassessed by two independent pathologists who were blinded to this study.
Paraformaldehyde fixed samples were washed 3 times with ice-cold PBS for 3 min each time and then incubated in 10% donkey serum in PBS for 20 min. Subsequently, the samples were incubated with the eIF4A1 antibody (Abcam ab31217) in PBS at 4 °C overnight. After washing, fluorochrome-conjugated secondary antibodies (1:400, Alexa Fluor®488 donkey anti-rabbit IgG) were used, and then the samples were treated with DAPI. The fluorescence was visualized under an Olympus microscope.
The lentivirus pLVX-Puro (Addgene) was obtained from DesignGene Biotechnology (Shanghai, China) and used to clone shRNA sequences. The vectors were designated Lv-eIF4A1 (eIF4A1-1, 5′-CACACTGGACTAGTGGATCCCGCCACCATGTCTGCGAGCCAGGATTCCC-3′; eIF4A1-2, 5′-AGTCACTTAAGCTTGGTACGATGAGGTCAGCAACATTGAGG-3′), Lv-sh-c-MYC (c-MYC-1, 5′-GCTTCACCAACAGGAACTATG-3′; c-MYC-2, 5′-GCTTGTACCTGCAGGATCTGA-3′; c-MYC-3, 5’-GGAAACGACGAGAACAGTTGA-3′) and Lv-sh-control (empty vector). The lentivirus plasmid and packaging plasmids were transfected into pancreatic cancer cells with transfection reagent (Lipofectamine®3000, Thermo Fisher Scientific) in OPTI-MEM media (Invitrogen, MA, USA). Lentiviral infection of the target cells was performed in cell culture medium containing 5 μg/ml polybrene (Sigma H9268), and infected cells were selected with 2.5 μg/ml puromycin for follow-up experiments.
Quantitative real-time PCR (RT-qPCR)
cDNA was created with PrimeScriptTMRT reagent Kit Perfect Real Time, (Takara, RR037A) according to the manufacturer’s protocol. Quantitative PCR was performed on a StepOne Real-Time System (Thermo Fisher Scientific) using TB Green® Premix EX Taq™ (Takara, RR820a) according to the manufacturer’s protocol. Melting curve analysis was performed and the amplification plots were evaluated by SDS 1.9.1 software (Applied Biosystems, MA, USA). The 2−ΔΔCt method was used to determine relative fold changes in target gene expression from replicate samples. The bulge-loop primer for miR-9 was provided by RioBIO (Guangzhou, China).
Western blot analysis was performed as described previously . The primary antibodies targeted eIF4A1 (Abcam ab31217), c-MYC (Abcam ab32072), snail (Abcam ab216347), E-cadherin (Abcam ab40772), andβ-actin (Abcam, ab8226). Protein bands were visualized using Beyo ECL Plus and quantified with ImageJ.
Cells were transinfected with scramble siRNA or siRNA targeting eIF4A1 (siRNA1, 5′-GAGTAACTGGAATGAGATT-3′; siRNA-2, 5′-TCCAGCAGCGAGCCATTC-3′; siRNA-3, 5′-CGTGTGTTTGATATGCTTA-3′) and c-MYC (siRNA-1, 5′-GAGGAGACATGGTGAACCA-3′; siRNA-2, 5′-GGGTCAAGTTGGACAGTGT-3′; siRNA-3, 5′-CGACGAGACCTTCATCAAA-3′) with Lipofectamine®3000 (Thermo Fisher Scientific) and OPTI-MEM (Invitrogen, MA, USA) according to the manufacturer’s protocol. eIF4A1-siRNA-1 and c-MYC-siRNA-3 were the most effective in knocking down their respective targets and were used for further analysis. Cells were transfected with CV567 empty vector, pcDNA3.1-eIF4A1 (P1, 5′-CACACTGGACTAGTGGATCCCGCCACCATGTCTGCGAGCCAGGATTCCC-3′) or pcDNA3.2-c-MYC plasmids (P1, 5′-CACACTGGACTAGTGGATCCCGCCACCATGGATTTTTTTCGGGTAGTGG-3′) using the transfection reagent Lipofectamine®3000 (Thermo Fisher Scientific) and OPTI-MEM (Invitrogen, MA, USA) according to the manufacturer’s protocol.
In vitro migration and invasion assay
Cell migration and invasion were analyzed using Transwell chambers (8-μm pore size; Millipore, Billerica, MA, USA) with and without a Matrigel (BD Biosciences, San Jose, CA, USA) matrix in the upper chamber. Four groups of AsPC-1 cells were pretreated in DMSO, RocA (100 nM), Mycro3 (5000 nM), and combination treatment solutions for 4 h. Then the cells were seeded FBS-free culture medium in the upper chamber with 10% FBS culture medium in the lower chamber as a chemoattractant. After 28 h (Migration) or 32 h (Invasion) of incubation, cells on the lower surface of the membrane were washed with PBS, fixed in 4% methanol, and stained with a 0.4% crystal violet solution. Photographs of three randomly selected fields of the fixed cells were captured and cells were counted. The experiments were repeated independently three times.
The sensitivity of cells to RocA and Mycro3 either or in combination was measured by CCK8 assay. Cells were seeded in a 96-well plate at density of 1000 cells per well and then treated with the treatment concentrations (RocA, 100 nM; Mycro3, 5000 nM). After the cells were attached. Then incubated for 4 h, replace fresh 1640 culture medium and cell counting kit-8 (CCK8, promoter, China) to each well according to the manufacturer’s protocol, incubated at 37 °C for 2 h. Absorbance was measured at 450 nm using a microplate reader (Thermo Scientific).
Establishment of a metastatic mouse model
Animal experiments were approved by the Ethics Committee of Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology (TJH-202010007). Four-week-old female severe combined immune deficiency (SCID) mice (Charles River Co., Beijing) were maintained in a specific pathogen-free (SPF) environment. To establish the metastatic mouse model in vivo, 2 × 106 AsPC-1 cells in 200 μl were intravenously injected via caudal vein into each animal. Small animal imaging (Spectral Imaging LagoX) was performed every 3 days after cell injection and the mice were randomly divided into different groups (5/ group). RocA (MCE, USA) was administered daily by intraperitoneal injection (5 mg/kg/d, 3 mg RocA dissolved in 30 μl DMSO, 600 μl PEG300 and 75 μl Tween-80 successively, and then adjusted to a volume of 1.5 ml with normal saline) every day originally , then adjusted to 2.5 mg/kg/d once on an alternate day. Mycro3 was administered intragastrically (100 mg/kg/d, 25 mg mycro3 dissolved in 2.5 ml of 0.5% methylcellulose solution and subject to ultrasonication) . Control mice were treated with intragastric 200 μl of methylcellulose solution daily and 100 μl of methylcellulose solution via intraperitoneal injection once on an alternate day. When the total body weight loss was > 20%, the mice were sacrificed by cervical vertebra dislocation under deep sedation conditions using 70 mg/kg pentobarbital sodium (peritoneal injection).
Subcutaneous xenografts in nude mice
Four-week-old nude mice were obtained from HFK Bioscience Ltd (Beijing, China) and maintained in SPF conditions. AsPC-1 cells (5.0 × 106) suspended in a 100 μl solution comprising equal volumes of medium and matrix gel were subcutaneously implanted into the right flanks of 6-week-old female nude mice. When the tumors had reached a volume of approximately 60–90 mm3, the mice were then randomly divided into two groups. The treatment group received an intraperitoneal injection of RocA (previously adjusted dose), whereas the control group received cosolvent injection alone (n = 4, the treatments were carried out once daily for 28 days. The tumor volumes and total body weight of the animals were measured twice a week. The tumor volumes (mm3) were calculated with the following formula: V = LS2/2 (where L is the longest diameter and S is the shortest diameter). At the end of the experiment, the mice were sacrificed and the tumors were harvested for western blot analysis. The mice were sacrificed 4 weeks after implantation by cervical dislocation under deep sedation conditions using 70 mg/kg pentobarbital sodium (peritoneal injection).
The data are representative of at least three independent experiments or multiple independent mice as indicated. The patient characteristics were summarized as the mean ± standard deviation (SD) for normally distributed continuous variables, median with interquartile range for continuous variables with a skewed distribution, and frequency (percentage) for categorical variables. All analyses were performed using R (http://www.R-project. org, version 3.5.2) with a two-sided significance threshold of P < 0.05.