Background
Esophageal cancer is the sixth most common cause of cancer-related death worldwide and is therefore a major global health challenge. Esophageal squamous cell carcinoma (ESCC) is a highly prevalent cancer in China, which accounts for 90% of esophageal cancer globally [
1]. Angiogenesis provides essential nutrients for tumor cell growth and metastasis, and has been shown to play a role in esophageal cancer [
2]. Tumor cells can induce the formation of new blood vessels via cytokine, exosomes and other molecules [
3,
4]. However, the tumorigenicity and angiogenesis of ESCC is not yet completely understood.
RNA-binding proteins (RBPs) have been identified as modulators of post-transcriptional mechanisms, including RNA splicing, transport, translation, and localization [
5]. Increasing evidence suggests that RBPs have aberrant expression and function in multiple tumor types, such as glioma, hepatocellular carcinoma, and colorectal cancer [
6]. Poly (A) binding protein cytoplasmic 1 (PABPC1), encoded within chromosome region 8q22.2–23, is a cytoplasmic-nuclear shuttling protein expressed in most eukaryotes. PABPC1 is an important RBP for the initiation of protein translation and mRNA decay. Biologically, PABPC1 is necessary for regulating vertebrate oocyte and early embryo translation as well as modulating the protein synthetic capacity of the mammalian heart [
7,
8]. The role of PABPC1in cancer is not without controversy. Abnormal expression and function of PABPC1 have been detected in gliomas and hepatocellular carcinoma, where PABPC1 is highly expressed and enhances tumor cell proliferation [
9,
10], although the combination of PABPC1 and BRCA1 has been suggested to have an anti-cancer function in breast cancer [
11]. PABPC1 is correlated with clinical stage and survival of ESCC patients [
12]. Despite the key role of PABPC1 in cancer progression, the mechanism by which it is regulated and participates in ESCC has not been clearly elucidated.
Interferon alpha (IFN-α) inducible protein 27 (IFI27), encoded within chromosome 14q32, has been reported to be involved in IFN-induced cell apoptosis, proliferation, and immune responses [
13]. Furthermore, it has been shown to be an oncogene that is upregulated in various cancers, such as tongue squamous cell carcinoma, oral squamous cell carcinoma, and cholangiocarcinoma, and is correlated with poor survival [
13‐
16]. Mechanically, IFI27 induces epithelial–mesenchymal transition (EMT) to promote cholangiocarcinoma metastasis [
14]. IFI27 has been reported to be a putative cell proliferation marker and regulate cell cycle proteins, such as p21 and p53 [
17]. Moreover, IFI27 mediated epithelial–mesenchymal transition (EMT) to promote cholangiocarcinoma metastasis [
14]. IFI27 downregulation can inhibit tongue and oral squamous cell carcinoma cell proliferation and migration and invasion, and can promote cell apoptosis [
16]. However, the function of IFI27 in ESCC and how PABPC1 mediates IFI27 have not been elucidated.
In this paper, we characterized PABPC1 expression and its clinical significance in ESCC. PABPC1 is highly expressed in ESCC and is correlated with a lower survival rate. PABPC1 promotes ESCC cell proliferation and invasion. Mechanistically, PABPC1 could increase IFI27 mRNA stability by interacting eIF4G to compete with the RNA exosome complex. PABPC1/IFI27 could increase miR-21-5p expression and promote exosomal miR-21-5p packaging to target human umbilical vein endothelial cells (HUVECs) and increase angiogenesis via inhibiting C-X-C motif chemokine 10 (CXCL10). Taken together, our results demonstrate that PABPC1 plays a tumor promoter role in ESCC and may therefore be a therapeutic target for treating ESCC.
Materials and methods
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).
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 × 10
5 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.
Discussion
In this study, we reported that PABPC1 expression is increased in ESCC, and is associated with histone acetylation in the PABPC1 promoter. We show ESCC patients with high PABPC1 expression have shorter survival compared with patients with low PABPC1 expression. Moreover, PABPC1 promotes ESCC cell growth and invasion via regulating IFI27. The mechanism by which this occurs involves PABPC1 binding eIF4G to block RNA exosome complex-mediated IFI27 mRNA degradation, and elevates miR-21-5p expression to increase miR-21-5p-containing exosomes that can target endothelial cells to increase angiogenesis (Fig.
7H).
The clinical significance of PABPC1 in ESCC was analyzed in samples from our own cohort and a public database (TCGA), which showed PABPC1 is upregulated in ESCC tissues compared with in case-matched normal epithelial tissues, which is consistent with several previous reports focusing on prostate cancer, HCC, and gastric cancer [
24]. However, the mechanisms underlying the increased PABPC1 expression in cancer has not yet been clearly demonstrated. Dong et al. reported that PABPC1 expression could be mediated by SNHG14 by promoting histone acetylation in the SNHG14 promoter [
25]. Histone acetylation is a major histone modification involved in the regulation of chromatin structure and transcription, and this process plays a key role in ESCC tumorigenesis [
26]. Thus, using the ENCODE database, we found that H3K27ac is highly enriched at the PABPC1 promoter. The histone acetyltransferase p300 interacts with transcriptional coactivators to regulate acetyltransferase activities, thus promoting carcinogenesis[
18]. Following experimental verification using ESCC tissues and cells, we identified the Sp1 transcription factor in combination with p300 could elevate histone acetylation at the PABPC1 promoter to elevate PABPC1 expression in ESCC. Sp1 and p300 expression are positively correlated with PABPC1 in ESCC tumors, further supporting our
in vitro findings.
Furthermore, we found that PABPC1 promotes ESCC cell proliferation, migration, and invasion, and inhibits apoptosis, indicating that PABPC1 acts as a tumor promoter in ESCC cells. Similar findings have been reported in HCC and gastric cancer [
9,
27]. However, the mechanisms by which PABPC1 promotes tumorigenesis in different cancer types are diverse. In hepatocellular carcinoma, PABPC1 interacts with AGO2 to augment miRNA repression by facilitating RISC binding and enhancing miRNA-mediated deadenylation, leading to the inhibition of tumor suppressor genes [
9]. Zhu et al. found that PABPC1 upregulation plays a carcinogenic role by inhibiting mir-34c expression in gastric cancer cells [
27]. In this study, we used RNA-seq to determine that the IFN pathway is a downstream pathway of PABPC1. The IFN pathway is a key mediator in tumor progression and exerts multiple biological effects, including antiviral and antitumor activities in patients with cancer and viral diseases [
28]. Among these DEGs, we found IFI27, a key member of the IFN-α-induced protein family, to be upregulated by PABPC1. Rescue experiments revealed that PABPC1 promotes ESCC cell proliferation and invasion through upregulating IFI27.
We then explored how PABPC1 mediates IFI27 expression. Previous research has demonstrated that IFI27 protein is stabilized by EGF in keratinocytes [
17]. However, according to our observations, protein stabilization did not contribute to high IFI27 expression in ESCC. In eukaryotic cells, mRNA homeostasis is achieved through a balance between mRNA synthesis and degradation. Since PABPC1 plays critical roles in mRNA translation, stabilization, and degradation, we predicted that PABPC1 could increase the stability of IFI27 mRNA. Indeed, we found that PABPC1 overexpression could enhance IFI27 mRNA stability by prolonging its half-life. Using mutant PABPC1 plasmids, we found that the PABPC1 could interact with eIF4G to protect IFI27 mRNA from degradation. Then, we further explored the detailed mechanisms of PABPC1-mediated IFI27 mRNA stabilization, which leads to ESCC tumorigenesis. In eukaryotic cells, the RNA exosome core contains a barrel-like structure (constituted by EXOSC 4 ~ 9) and a cap (constituted by EXOSC 1 ~ 3) [
21]. The RNA exosome comprises a ring-like structure and two catalytic components, and plays a major role in various RNA processing and degradation pathways [
29,
30]. EXOSCs are noncatalytic but are essential for the degradation and processing of target RNA, and EXOSC2 knockdown severely diminishes RNA exosome function [
29]. In this study, we confirmed that EXOSC knockdown can markedly restore IFI27 mRNA expression, and PABPC1 could compete with EXOSCs to stabilize IFI27 mRNA.
Angiogenesis is defined as the formation of new blood vessels from preexisting vessels and has been characterized as an essential process for tumor cell proliferation and metastasis [
31]. Exosomes derived from cancer cells contain a wide variety of miRNAs, and a large amount of evidence indicates that exosomal miRNAs are primary inducers of angiogenesis through activation of signaling pathways that trigger a network of signaling processes that promote endothelial cell growth, migration, and survival from pre-existing vasculature [
26]. Our study shows that PABPC1 can increase expression of miR-21-5p, in ESCC cells, which is then encapsulated in ESCC cell-derived exosomes to target vessel endothelial cells and induce angiogenesis. Importantly, we further investigated how miR-21-5p is packaged into exosomes, showing that PABPC1 can bind miR-21-5p, through an ACUGAUG sequence, to direct miR-21-5p packaging into exosomes, similar to other studies showing involvement of other RBPs, such as hnRNPQ and hnRNPA2B1, in exosomal miRNA export [
32,
33]. Previous studies showed the pro-angiogenic properties of endogenous miR-21-5p and cancer cell derived-miR-21-5p to be dependent on KRIT1 and Spry1 in vessel endothelial cells [
34‐
36]. We show here that miR-21-5p-mediated suppression of CXCL10, a potent endogenous inhibitor of angiogenesis [
23,
37], in HUVECs is in part responsible for tumor cell-induced angiogenesis and tumor growth in ESCC. The role of CXCL10 in inhibiting angiogenesis (angiostasis) has been extensively studied in various tissues and cancers, including the cornea, skeletal muscle cells and Kaposi sarcoma [
37‐
39]. This newly discovered axis implicates miR-21-5p in multiple regulatory functions in angiogenesis. However, other functional components present in exosomes may also contribute to angiogenesis, and how miR-21-5p is packaged into exosomes has not been fully elucidated. Therefore, further research should be carried out to better illustrate these problems.
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