Introduction
Esophageal cancer, as a global cancer burden, is one of the most aggressive digestive tract malignancies [
1]. According to histological type, esophageal cancer is primarily divided into esophageal adenocarcinoma and esophageal squamous cell carcinoma (ESCC), of which 90% are ESCC in China [
2]. Because esophageal cancer is insidious in its onset, more than 80% of patients lose the opportunity for surgical treatment at the time of presentation, and concurrent chemoradiotherapy is the standard treatment modality for inoperable esophageal cancer [
3]. Despite recent significant advancements in radiotherapy equipment and techniques, the majority of patients still experience local recurrence as a result of radiotherapy resistance, and the 5-year survival rate is less than 30% [
4]. Therefore, it is essential to elucidate the specific mechanism of radioresistance, which can provide new perspectives for the radiosensitization therapy of ESCC.
With the development of biological information technology, non-coding RNAs have attracted much attention as a consequence of their emerging role in cancer, of which microRNAs (miRNAs) are a class of non-coding RNAs composed of approximately 22 nucleotides that are responsible for post-transcriptional modification of genes by binding to the 3′UTR of messenger RNA [
5]. MiRNAs, novel cancer biomarkers, can play regulatory roles by acting as oncogenes or suppressors [
6]. MiRNAs play a pivotal role in radiotherapy, modulating tumor radiosensitivity by taking part in DNA repair, cell cycle analysis, apoptosis, autophagy, and ferroptosis [
7]. As an example, Zheng and coworkers revealed that miR-640 potentiated glioma radiosensitivity by focusing on SLIT1 to repress the Wnt/β-catenin flagging pathway [
8]. Recent work by McGrath and colleagues found that manipulating miR-31 expression altered the radiosensitivity of pancreatic ductal adenocarcinoma cells by controlling oxidative pressure [
9]. In addition, it has been reported that miR-222 focusing on PTEN, and miR-155 focusing on FOXO3a actuated radiation opposition in colorectal malignant growth [
10]. MiRNAs have been found to modulate the esophageal cancer response to radiation therapy. Chen et al. found that miR-450a-5p suppressed autophagy and enhanced radiosensitivity in esophageal squamous cell carcinoma by targeting dual-specificity phosphatase 10 [
11]. It has been reported that miR-196b promotes chemoradiotherapy resistance of esophageal squamous cell carcinoma by inhibiting EPHA7 [
12]. In our previous study, Jin et al. found that upregulation of microRNA-98 increased radiosensitivity in esophageal squamous cell carcinoma [
13]. However, it is still unclear how miRNAs function as latent radiosensitizers in ESCC, and further discovery of miRNAs affecting ESCC radiosensitivity is still needed.
MiR-4443, located on chromosome 3p21.31, is a recently recognized miRNA that has been distinguished as a basic controller of tumorigenesis and metastasis [
14,
15]. The expression levels and regulatory mechanisms of miR-4443 were not completely consistent in different tumor types. For instance, miR-4443 was upregulated in breast cancer [
16] and downregulated in papillary thyroid carcinoma and ovarian cancer [
14,
17]. Intriguingly, it has been discovered that exosomes carrying miR-4443 increase cisplatin resistance in non-small cell lung cancer by modulating ferroptosis caused by FSP1 m6A modification [
18]. Recent work by Wang et al. revealed that miR-4443 was elevated in ESCC [
19]. Nonetheless, there has been little significant understanding of the role of miR-4443 in the ESCC response to radiotherapy, and further elucidation is needed.
Therefore, to explore whether miR-4443 is associated with radiosensitivity and other biological characteristics of ESCC, we established an acquired radioresistant ESCC cell line in which miR-4443 expression was detected. The effect of miR-4443 on radiosensitivity in ESCC was investigated by regulating miR-4443 expression levels up and down by genetic manipulation. In this study, miR-4443 was found to be upregulated in radioresistant ESCC cells. MiR-4443 regulated ESCC radiosensitivity by influencing DNA damage repair, apoptosis, and G2 cycle arrest. Our discoveries reveal that miR-4443 targets PTPRJ to augment radioresistance in ESCC, providing a new basis for developing new therapeutic strategies to improve radiosensitivity in ESCC.
Methods
Cell culture
Human ESCC cell lines (Eca-109, KYSE-150, and TE-1) were purchased from the Cell Bank of the Chinese Academy of Sciences Typical Culture Preservation Committee (Shanghai, China). The human normal esophageal cell line (Het-1A) was purchased from ATCC (American Type Culture Collection, Manassas, USA). All ESCC cells were cultured in RPMI 1640 (Gibco, CA, USA) medium with 10% fetal bovine serum (FBS; Gibco, CA, USA) and the Het-1A cell line was cultured in DMEM (Gibco, CA, USA) medium with 10% FBS in a humid atmosphere containing 5% CO2 at 37 °C.
Establishment of radioresistant ESCC cells
The parental KYSE-150 cells were seeded into 6-well plates and cells were initially irradiated with 2 Gy X-ray when the cell density reached 50–60%. After radiation, the culture medium was renewed. When the cells achieved 90% confluence, they were split 1:3 into new 6-well plates and received radiation again. These procedures were repeated 30 times to a total dose of 60 Gy. The surviving cells were characterized as radioresistant cells (termed KYSE-150R cells). The generation of radioresistant cells was validated by performing colony formation assays [
20].
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells using TRIzol (TaKaRa, Dalian, China) and reverse-transcribed into cDNA with a Prime Script™ RT reagent kit (TaKaRa). MicroRNA-specific cDNAs were generated using a Mir-X™ miRNA First Strand Synthesis kit (TaKaRa). qRT-PCR was carried out using TB Green
® Premix Ex Taq™ II (TaKaRa). We normalized the relative miRNA and mRNA expression to U6 and GAPDH, respectively. All experiments were repeated in triplicate. The primer sequences used in this study are listed in Additional file
2: Table S1.
Cell transfection
Lentivirus particles were designed and purchased from GenePhama (Shanghai, China), including LV2N-hsa-miR-4443 mimics, LV2N-hsa-miR-4443 inhibitor sponge, and controls. PTPRJ small interfering RNA (siRNA) (siPTPRJ), siRNA negative control (siNC), PTPRJ overexpression plasmid (PTPRJ), and empty vector (vector) were purchased from Sangon (Shanghai, China). Transfection was performed according to the manufacturer’s instructions. The target sequences are presented in Additional file
3: Table S2.
Clonogenic survival assay
Single-cell suspensions were inoculated into six-well plates at densities of 900–12,000 cells per well (TE-1: 0 Gy: 900 cells; 2 Gy: 1500 cells; 4 Gy: 3000 cells; 6 Gy: 9000 cells; and 8 Gy: 12,000 cells; KYSE150/150R: 0 Gy: 900 cells; 2 Gy: 1200 cells; 4 Gy: 1800 cells; 6 Gy: 3000 cells; and 8 Gy: 6000 cells). After cell adherence, they were subjected to 0, 2, 4, 6, or 8 Gy X-ray irradiation, respectively. After 10–14 days of incubation, during which the cell media was replaced every three days, the cells were rinsed with PBS, fixed with methanol, and stained with crystal violet. Colonies containing more than 50 cells were counted and analysed based on the corresponding numbers of initial inoculated cells. Cell survival curves based on the mean survival fractions of the cell line were fitted to a Single-hit multitarget model: SF = 1 − (1−e(−kD))N.
Cell counting kit-8 (CCK-8) assay
In the exponential growth phase, two thousand cells per well were seeded into 96-well plates (100 μl/well). After adhering to the cells, the cells were treated with a single dose of 6 Gy (day 0). At the specified time point (day 0, day 1, day 2, day 3, day 4, day 5), 10 μl CCK-8 solution (UElandy, Suzhou, Jiangsu, China) was added to each well and then incubated for 1 h. The optical density (OD, 450 nm) values were measured by a microplate reader.
Western blot
Total protein was separated from cell lysates on ice using a radioimmunoprecipitation assay (RIPA) containing protease and phosphatase inhibitors (HEART, Xi’an, Shaanxi, China). We quantified protein levels using an Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein were separated on 6 or 12% SDS-PAGE gels (Beyotime) and transferred to PVDF membranes (Millipore, Billerica, MA, USA), which were incubated with primary antibodies at 4 °C overnight. The primary antibodies used in this study were: γH2AX (1:1000, #80312, Cell Signaling Technology, Danvers, MA, USA), PTPRJ (1:5000, ab181244, Abcam, Cambridge, MA), and β-tubulin (1:10,000, AP0064, Bioworld, USA,). After washing four times with Tris-Buffered Saline and Tween 20 (TBST) buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:2500, bs-0295G-HRP, Bioss, Beijing, China) or goat anti-mouse immunoglobulin (Ig)G (1:2500, bs-0296G-HRP, Bioss, Beijing, China) for 1 h at room temperature was performed. The protein bands were visualized using chemiluminescence kits (Millipore, Billerica, MA, USA).
Immunofluorescence
The cells were seeded on 20 × 20 mm glass-bottom round dishes (Corning, NY, USA) and irradiated with a single dose of 8 Gy after adherence. Then, 24 h after irradiation, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were subsequently permeabilized with 0.1% Triton X-100 before being blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature. The cells were stained overnight with Phospho-Histone H2AX (Ser139) (D7T2V) mouse monoclonal antibody at 4 °C, washed three times in PBS, and further incubated with fitc-coupled second antibody (1:300, Proteintech, China) at room temperature for 1 h. Antifade mounting medium with DAPI (Beyotime Biotechnology, Shanghai, China) was utilized to stain nuclei for 10 min, and then a Leica TC5 SP5 confocal microscope was adopted to visualize and image the cells.
Apoptosis and cell cycle analyses
Cells were first seeded into a 6-well plate at a density of 1 × 105 cells per well and treated with 6-MV X-ray radiation at doses of 0 Gy and 8 Gy. Cells were collected and stained with an Annexin V-APC/7-AAD double staining apoptosis detection kit (LiankeBio, Hangzhou, China) according to the manufacturer's instructions. For cell cycle assays, cells were harvested and stained with PI solution (KeyGen Biotech, Nanjing, China) with RNase A. Flow cytometry was used to evaluate the luciferase intensity at 24 h after treatment.
The GSE43732 miRNA microarray dataset and the GSE53624 gene expression microarray dataset of 119 ESCC patients were downloaded from the Gene Expression Omnibus database (
http://www.ncbi.nlm.nih.gov/geo/) (Additional file
4: Table S3). The maximum expression was considered to be the expression of genes with several probes. The ESCC patients’ transcription profile and clinical data of The Cancer Genome Atlas (TCGA) (
http://cancergenome.nih.gov/) cohort were analyzed as previously described [
21]. Weighted gene co-expression network analysis (WGCNA) based on the FPKM (fragments per kilobase of transcript per million mapped reads) values of TCGA-ESCC data was used to explore the relationships between gene networks and diseases as well as correlations between gene modules and clinical characteristics.
Statistical analysis
GraphPad Prism 8.0 and R 4.2.1 software were used for statistical analyses and data visualization. The data are expressed as the mean ± standard deviation (SD). P < 0.05 indicated statistical significance. Differences between groups were analysed using Student’s t-test, Wilcoxon test, or one-way ANOVA.
Discussion
One of the deadliest cancers in the world is esophageal cancer, and ESCC is the most predominant pathological subgroup. The key factor limiting the effectiveness of radiotherapy for ESCC is radiotherapy resistance. The development of radioresistance is a multigene, multifactor, and multimechanism process. Various factors such as DNA damage repair, cell cycle arrest, tumor microenvironment (including hypoxia and angiogenesis), regulation of autophagy and the presence of tumor stem cells, have been discovered to contribute to tumor radioresistance [
24‐
26]. Notwithstanding, the molecular mechanism of radioresistance in ESCC remains unclear. It is of great scientific value and clinical significance to deeply explore the role of radioresistance and its molecular mechanism to develop radiosensitization strategies targeting key molecules in ESCC.
It has recently been discovered that particular miRNAs are differentially expressed in tumor tissue and normal tissue, which has implications for tumor growth and radiation resistance. MiR-4443 has been identified as a critical regulator of tumorigenesis. By way of illustration, miR-4443 was highly expressed in epirubicin-resistant cell lines, inhibited their apoptosis, and induced malignant progression in breast cancer and non-small cell lung cancer [
16,
27]. However, miR-4443 is also considered to be a tumor suppressor, inhibiting tumor proliferation, migration and invasion, such as in papillary thyroid cancer, glioblastoma and colorectal cancer [
15,
17,
28]. MiR-4443 has already been reported to be highly expressed in ESCC, but whether it affects radiosensitivity in ESCC is unclear. In this study, we established an acquired radioresistant ESCC cell line (KYSE-150R) with a total radiation dose of 60 Gy in reference to the clinical radiotherapy guideline for esophageal squamous cell carcinoma. We demonstrated for the first time that miR-4443 was highly expressed in radioresistant ESCC cells compared to parental cells and that radiation can induce miR-4443 expression. Functional experiments showed that overexpression of miR-4443 significantly enhanced the survival fraction of KYSE-150 cells subjected to different doses of irradiation, while downregulation of miR-4443 inhibited the survival fraction of TE-1 and KYSE-150R cells under different doses of irradiation. This suggested that miR-4443 was a potential therapeutic target for radioresistant ESCC.
It is well-known that DNA is a target of radiation damage and that radiation can damage DNA by acting directly or indirectly [
7]. In DNA damage, cells initiate complex biochemical signal transduction pathways that recognize the type of damage, initiate corresponding repair pathways, repair the damage, or lead to cellular senescence, autophagy, apoptosis, and if not effectively repaired, cell death [
29]. Radiosensitivity is associated with DNA damage repair and miRNAs. It has been reported that miR-146a-5p inhibits the expression of binding replication protein A3 to activate the DNA damage repair pathway, thereby promoting radiosensitivity in hepatocellular carcinoma [
30]. Serine 139 on H2AX is phosphorylated and modified to form phosphorylated H2AX, namely, γH2AX, which is a marker of DNA double-strand breaks and can reflect the degree of DNA damage and repair. Our results demonstrated that the upregulation of miR-4443 decreased γH2AX expression in ESCC cells at 24 h after irradiation, whereas the downregulation of miR-4443 increased γH2AX expression. Another promising finding was that silencing miR-4443 resulted in a greater apoptotic rate in TE-1 or KYSE-150R cells after irradiation than in control cells, indicating that inhibition of miR-4443 could increase apoptosis in irradiated ESCC cells. After the cells were damaged by ionizing radiation, the cells were arrested at various stages of the cell cycle, so that the cells had enough time to repair themselves and evade radiation damage, thus showing radiation resistance. The cell cycle phase affects cell radiosensitivity. Cells in the G2/M phase are the most sensitive to radiation, and the sensitivity decreases as the cell cycle progress from the G1 to the S phase. Our study found that miR-4443 downregulation promoted G2/M arrest in irradiated ESCC cells. Based on the above evidence, we demonstrated for the first time that miR-4443 promoted ESCC radioresistance by enhancing DNA damage repair, inhibiting apoptosis and decreasing the proportion of cells in the G2/M phase.
Direct and negative regulation of downstream targeted mRNAs is one way in which miRNAs exert their biological function. In this study, WGCNA was used to identify genes associated with radiosensitivity and then intersected with the prediction results of the TargetScan and miRDB databases to predict the putative targets of miR-4443. In addition, by bioinformatics methods, we found that PTPRJ expression was low in tumor tissues and that its expression was negatively correlated with miR-4443 expression. The wet experiment further verified that the mRNA and protein expression levels of PTPRJ in miR-4443-overexpressing cells were significantly lower than those in control cells. PTPRJ was therefore identified as a target gene of miR-4443. PTPRJ is located on chromosome 11p11.2 and encodes a receptor-like protein tyrosine phosphatase, whose aberrant expression plays an important role in disrupting the malignant phenotype of tumor cells [
31]. MiRNAs have been reported to regulate the gene expression of PTPRJ and thus affect tumor progression. Zhang et al. found that miR-155 directly bound to the 3′UTR of PTPRJ mRNA and inhibited its expression to regulate the proliferation of colorectal cancer cells [
32]. Luo et al. revealed that PTPRJ was significantly downregulated in hepatocellular carcinoma tissues, and miR-328 significantly promoted the migration and invasion of hepatocellular carcinoma cells by suppressing PTPRJ expression [
33]. Significantly, Shefler and coworkers revealed that the target gene of miR-4443 was PTPRJ, which was fully consistent with our results [
34]. Moreover, the relationship between miR-4443, PTPRJ, and tumor radiotherapy has not been reported. Our findings discovered that PTPRJ reversed miR-4443-induced ESCC cell response to radiation by using rescue studies. In summary, our research demonstrated that miR-4443 reduced the level of apoptosis of ESCC cells after radiation and enhanced the resistance of ESCC cells to radiation by directly inhibiting the expression of PTPRJ.
Nonetheless, this study still has certain restrictions. First, only one type of radioresistant ESCC cell line was established and investigated in this study. Second, these studies were only carried out in vitro, and further verification from animal experiments is lacking. Nevertheless, we preliminarily identified a novel mechanism of miR-4443-mediated tumor radioresistance, offering fresh perspectives for radiosensitization in ESCC. In the future, we will investigate improved techniques to confirm the role of miR-4443 in more radioresistant cell lines and animal trials, and to provide stronger evidence to support the preliminary conclusions of this study.
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