Patient samples and cell lines
A total of 190 ESCC tissue samples were obtained from patients who underwent cholecystectomy without prior radiotherapy or chemotherapy between 2015 and 2020 at Shantou University Medical College. Informed consent was obtained from all patients participating in the study. This study was approved by the Institutional Ethics Board of the Cancer Hospital of Shantou University Medical College (2021117). ESCC cell lines TE1, 81 T, KYSE410, KYSE180, KYSE450, KYSE150, KYSE510, and KYSE520 were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Shanghai, China) or Roswell Park Memorial Institute (RPMI, Gibco) 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco). All cells were maintained in a humidified incubator (5% CO2) at 37°C. Immortalized esophageal epithelial NE1 cells were cultured in Eagle’s minimum essential medium (Gibco) supplemented with 10% FBS (Gibco). Cell lines were authenticated by short tandem repeat (STR) profiling and confirmed to be mycoplasma negative.
Antibodies and western blotting
Cells were lysed in RIPA buffer (Beyotime, Jiangsu, China) containing a protease inhibitor cocktail (Sigma, St. Louis, CA, USA). Total protein was assessed using a BCA Protein Assay Kit (Beyotime). Western blot analysis was performed as previously described [18]. Antibodies against the following were used in this study, PABPC1 (1:1000, Abcam, SF, USA), IFI27, HA tag, Flag tag, TSG101, CD63, CD9, PCNA, Alix, Ki67, caspase 3, CXCL10, CD34, cleaved-PARP, PARP, eIF4G, cleaved caspase 9, caspase 9, ERK, p-ERK, IFI27, STAT3, p-STAT3, STAT1, p-STAT1, NF-kB, p-NF-kB, β-actin and GAPDH (all 1:1000, Cell Signaling Technology, MA, USA), EXOSC2 and EXOSC4 (both 1:1000, Santa Cruz, MA, USA).
RNA extraction, RNA-sequencing, and quantitative real-time PCR (qRT-PCR)
Using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA), total cellular RNA was extracted from cells. qRT-PCR was performed according to a previously published procedure using the SYBR Green system (Takara, Dalian, China) [19]. The primer sequences are shown in Table S1. For RNA-seq, the mRNA was amplified by PCR and sequenced using an Illumina NovaSeq 6000 (Gene Denovo Biotechnology Co., Guangzhou, China).
Immunohistochemistry (IHC) and immunofluorescence (IF)
IHC staining was performed using the Envision Labeled Peroxidase System (Dako, Carpinteria, CA, USA) as described previously [19]. PABPC1 and IFI27 expression were analyzed based on the proportion and intensity of positively-stained tumor cells. The scores for intensity and fraction of positive cells were multiplied; scores of 0–4 were defined as low expression, and scores of 6–12 were defined as high expression. Immunofluorescence was performed as previously described [19]. Briefly, cells were first fixed in 3% paraformaldehyde, and then permeabilized with 0.5% Triton X-100. The cells were blocked with 5% FBS in PBS, followed by incubation with primary antibody, fluorescent secondary antibody (Invitrogen) and counterstained with DAPI (Beyotime) were then incubated with cells to visualize the targeted proteins and nuclei. The data were then analyzed by fluorescence microscopy.
Drugs, plasmids, siRNA, and stable cell lines
Carbobenzoxy-Leu-Leu-leucinal (MG132), actinomycin D, and cycloheximide (CHX) were purchased from MCE (St. Louis, MO, USA). 5-Ethynyl-uridine (5-EU) and HDAC inhibitor sodium butyrate (NaBu) were purchased from Selleck (St. Louis, MO, USA). The siRNAs were designed and synthesized by GenePharma Company (Shanghai, China). Full-length PABPC1 plasmid, five truncated PABPC1 RRM1-RRM4 and MLLE plasmids, mutant PABPC1 M161A/D165K and RRM1 deletion PABPC1 plasmids, eIF4G, and IFI27 plasmids were purchased from Vigene (Shanghai, China). ESCC cell transfection was carried out using Lipofectamine 3000, Lipofectamine IMAX, and Opti-MEM (Invitrogen, Shanghai, China). For stable expression of PABPC1 or IFI27 in OS cell lines, lentivirus or short hairpin(sh) -RNA transfected cells were selected with puromycin (5 μg/mL, MCE) for 2 weeks.
Co-immunoprecipitation (Co-IP)
Co-IP was performed using the relevant antibodies and protein A/G-conjugated Dynabeads (Beyotime) according to the manufacturer's instructions. In brief, cell lysates were incubated with antibodies overnight at 4 °C. Protein A/G-conjugated beads were added into the lysate at 4 °C for 2 h. Then, the beads were washed with lysis buffer or PBS and boiled in SDS loading buffer. Western blotting was used to detect the immunoprecipitated proteins.
Cell proliferation, apoptosis, invasion, and migration assays
Cell proliferation was examined with a Cell Counting Kit-8 (CCK-8) according to the manufacturer’s instructions (Beyotime). Apoptosis was determined by flow cytometry using Annexin V/PI staining according to the manufacturer’s protocol, and data analysis was performed using BD Accuri C6 software. Cell migration was evaluated by scratch wound healing assay where wounds were scratched on the monolayer of cells using a 200 μL pipette tip. After the cells had been cultured for 48 h, plates were washed once with fresh medium to remove non-adherent cells, and the plates were then photographed. Cell invasion and migration was tested using a transwell assay. Briefly, 100 μL Matrigel (Corning, CA, USA) was first added onto the bottom of the transwell chamber (Corning), and then 1 × 105 cells in serum free medium were placed on the coated membrane in the top chamber and the bottom chamber was filled with DMEM with 10% FBS, then incubated for 24 h. The membrane was then fixed and stained with crystal violet. The cell imagines were taken and counted in 3 random fields with microscope.
Human Umbilical Vein Endothelial Cells (HUVEC) tube formation assay
After co-cultured with different treatment of ESCC cells, 104 HUVECs were cultured in μ-Slide Angiogenesis plate (ibidi, Germany) coated with 10 μL Matrigel (R&D Systems, Minneapolis, MN) for 6 h at 37 °C. The formation of capillary-like structures was captured under a light microscope. The degree of in vitro angiogenesis was represented the formed tube numbers scanned and quantitated in five low-power fields (100x).
Dual reporter luciferase assay
Briefly, HEK293T or tumor cells (3 × 104 cells per well) grown in a 24-well plate were co-transfected, with a luciferase reporter (200 ng per well), miR-21-5p mimic or inhibitor and 10 ng Renilla luciferase vector (pRL-CMV; Genomeditech, China), using Lipofectamine™ 3000 (Invitrogen). After 48 h, a dual reporter luciferase assay was performed according to the manufacturer’s instructions (Promega, Madison, MD, USA). The relative luciferase activity was expressed as the ratio of firefly luciferase activity to Renilla luciferase activity.
RNA stability assay
To measure the stability of IFI27 mRNA, cells were treated with 5 μg/mL actinomycin D (MCE) to block transcription. Cells were collected at 0–24 h after addition of actinomycin D, and the total cellular RNA was isolated. qRT-PCR was performed to measure the half-life of RNA, and GAPDH mRNA was used as an internal control.
Nascent RNA capture assay
To capture the newly synthesized RNA transcripts of IFI27 mRNA, a nascent RNA capture assay was carried out using the Click-IT Nascent RNA Capture Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.
Poly (A) tail-length assay
A poly (A) tail-length assay was performed using a Poly(A) Tail-Length Assay Kit (Invitrogen) according to the manufacturer’ s instructions. Briefly, total RNAs were first added to poly (G/I) and reverse transcribed. Then, the poly (A) length was detected by PCR using both gene-specific primers and the Universal PCR Reverse Primer (Table S1).
Chromatin immunoprecipitation (ChIP) and RNA immunoprecipitation assay (RIP) assays
ChIP assays were performed using an EZ-ChIP kit (Millipore, Beijing, China). DNA was analyzed by qPCR with SYBR Green (Bio-Rad, Shanghai, China) on an ABI-7500 (Applied Biosystems, Shanghai, China) using the primers specified in Table S1. RIP assays were performed using an EZ-Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore). Briefly, cells were collected and lysed in lysis buffer with protease inhibitors. The protein extract (500 μg) was incubated with 3 μg of PABPC1 antibody or IgG overnight at 4 °C. Approximately 30 μL of A/G protein magnetic beads were then added and the mixture was incubated at 4 °C for 4 h. After washing, co-immunoprecipitated RNAs were extracted. RNA fold enrichment is presented as percent input and compared with the IgG control.
Biotin RNA pull-down assay
Biotin RNA pull-down experiments were performed using RNA pulldown kits (Bersin Bio, Guangzhou, China) according to the manufacturer’s instructions. Briefly, the cell lysates were incubated with 100 pmol of synthetic 5’-end biotin-modified IFI27 oligonucleotides overnight at 4 °C. After adding streptavidin agarose beads and incubating at 4 °C for 4 h, precipitates were washed five times and boiled in SDS buffer, followed by western blotting analysis.
Exosome isolation and uptake assay
Exosome isolation was performed as previously described [18]. In brief, cancer cells were cultured with exosome-free FBS-containing media and grown to 70% confluence. Then, cells washed 3 times with PBS, and incubated for 24 h in serum-free media and the supernatant was collected. The supernatant was centrifuged at 3000 × g for 15 min to remove cells and cell debris and it was then mixed with ExoQuick exosome precipitation solution (SBI, Palo Alto, CA) and incubated overnight according to the manufacturer’s protocol. Then, the mixture was centrifuged at 1500 × g for 30 min at 4 °C. The pelleted exosomes were dissolved in PBS and were subsequently divided and transferred to RNase-free tubes to be stored or undergo electron microscopy, protein assays, RNA extraction, and use in in vitro or in vivo treatment. For exosome uptake experiments, exosomes were labeled with a PKH67 Green Fluorescent Cell Linker Kit (Sigma) following the manufacturer’s protocol. Then, 10 µg of exosomes was resuspended in 100 µl PBS and were added to 1 × 105 HUVECs. HUVECs were harvested at 24 h for qRT-PCR and immunofluorescence analysis.
Animal studies
For in vivo assays, each experimental group consisted of six 5-week-old male BALB/c-nu/nu mice. Briefly, 1.0 \(\times\) 107 cells stably overexpressing PABPC1 were suspended in 50 μL serum-free DMEM/Matrigel (1:1) and injected into the oxter of each mouse. The mice were euthanized at 5 weeks after injection, and the weight and size of the tumors were measured. For lung metastasis assays, 1.0 \(\times\) 106 PABPC1-overexpressing cells were suspended in 50 μL serum-free DMEM and injected into the tail veins of mice (n = 6/group). After 6 to 8 weeks, mice were euthanized, lung tissue was excised, and tumor nodules formed in the lung were counted and analyzed by H&E staining. The experimental protocol was approved by the Ethics Committee of Animal Experiments of the Cancer Hospital of Shantou University Medical College (SUMC2021-035).
Statistical analysis
Statistical analyses were performed with SPSS version 22.0. Data are presented as the mean \(\pm\) standard deviation (SD), and statistical significance was determined using unpaired Student’s t-tests. Survival curves were generated by the Kaplan–Meier method. P-values < 0.05 were considered to indicate statistical significance.