Introduction
Nasopharyngeal Carcinoma (NPC) is a malignant tumor originating from the nasopharyngeal epithelium [
1]. Its incidence demonstrates distinct geographical variations, with Southeast Asia, South China, and North Africa being high-risk regions [
2,
3]. Genetic susceptibility and environmental factors such as EBV viral infection, contribute jointly to the development of nasopharyngeal carcinoma [
4]. Radiotherapy stands as the primary clinical treatment approach for nasopharyngeal carcinoma due to its relative sensitivity to ionizing radiation. However, despite its effectiveness in achieving local control, some cancer cells exhibit resistance to radiation therapy. This leads to the persistence of cancer cells, local recurrence, distant metastasis, and ultimately treatment failure and patient mortality [
5‐
7].
Radiation therapy induces DNA double-strand breaks (DSBs), which represent one of the most severe forms of DNA damage and can lead to genomic instability, cell cycle arrest, and apoptosis, among other catastrophic cellular events [
8‐
10]. Cells possess multiple DNA damage repair systems, and successful repair of DNA damage enables cell survival, contributing to radiotherapy resistance. Cellular DNA double-strand break repair predominantly engages two main pathways: homologous recombination (HR) and non-homologous end joining (NHEJ) [
11]. HR initiation hinges upon the core protein recombinase RAD51, which is recruited to DNA double-strand break ends by the BRCA1-PALB2-BRCA2 complex to displace RPA. HR utilizes the undamaged homologous sister chromatid as a template for precise repair [
12,
13]. Conversely, NHEJ directly ligates the two ends without template involvement, facilitated by proteins such as Ku70-Ku80 and DNA-PKcs [
14,
15]. Despite advancements, many aspects of the DNA damage repair pathways remain elusive. Understanding the underlying biological mechanisms of nasopharyngeal carcinoma radiotherapy resistance is paramount for identifying biomarkers and optimizing clinical treatment strategies for nasopharyngeal carcinoma diagnosis, prognosis, and therapy.
Circular RNA (circRNA) represents a novel class of non-coding RNA that has garnered significant attention in recent years. CircRNA is widely implicated in the progression of nasopharyngeal carcinoma, functioning either as an oncogene or a tumor suppressor gene. Our team has previously identified numerous circRNAs involved in the pathogenesis and progression of nasopharyngeal carcinoma, exerting influence on invasion, migration, immune evasion, and radiotherapy resistance [
16‐
22]. Moreover, circRNA plays a crucial role in mediating the tumor’s resistance to radiotherapy, making it a potential molecular target for enhancing radiotherapy sensitivity, as well as serving as clinical diagnostic and prognostic indicators [
23,
24]. Till now, the molecular mechanisms underpinning radiotherapy resistance in nasopharyngeal carcinoma remain poorly understood. Notably, the emerging role of circRNAs in radiotherapy resistance warrants extensive exploration.
In this study, we report for the first time that circCDYL2 is highly expressed in nasopharyngeal carcinoma tissues and is closely associated with poor patient prognosis. In vitro and in vivo experiments confirm that circCDYL2 promotes radiotherapy resistance of nasopharyngeal carcinoma. A detailed analysis unravels the mechanism by which circCDYL2 enhances the capacity of nasopharyngeal carcinoma cells to repair DNA damage through homologous recombination following exposure to ionizing radiation. It achieves this by facilitating the translation initiation of RAD51, a key recombinase, ultimately culminating in radiotherapy resistance.
Materials and methods
Cell lines
The nasopharyngeal carcinoma cell lines, HNE2 and CNE2, utilized in this study were obtained from the Cancer Research Institute, Central South University. These cells were cultured in a DMEM medium (Life Technologies, NY, USA) supplemented with 10% fetal bovine serum (Gibco, MA, USA) and 1% penicillin-streptomycin (Life Technologies, NY, USA). All cell cultures were maintained at 37 °C in a 5% CO2 humidified incubator.
Clinical nasopharyngeal carcinoma samples
In this study, two sets of clinical samples were employed. The first set comprised 45 fresh nasopharyngeal carcinoma tissues, while 23 control nasopharyngeal epithelium (NPE) samples were collected from either Xiangya Hospital or the Affiliated Cancer Hospital of Xiangya School of Medicine of Central South University for RT-qPCR detection of circCDYL2 expression. Further clinical information is detailed in Supplemental Table
1. The second set consisted of paraffin-embedded tissue section samples from 203 newly diagnosed nasopharyngeal carcinoma patients, including 102 from Xiangya Hospital and 101 from the Affiliated Cancer Hospital of Xiangya School of Medicineof Central South University. Among these samples, there were 56 also contained adjacent non-cancerous epithelial tissues. All tissue samples were pathologically examined and independently confirmed. Following diagnosis, these patients received standard radiotherapy and were closely monitored accordingly. Comprehensive clinical information is listed in Supplemental Table
2. This research was approved by the Central South University Ethics Committee, and all participants provided informed consent.
Fluorescence in situ hybridization and in situ hybridization
Fluorescence in situ hybridization (FISH) and in situ hybridization (ISH) analyses were conducted using the in situ hybridization kit (BOSTER, Wuhan, China) according to the manufacturer’s instructions. Dig-labeled probes (Sangon Biotech, Shanghai, China) were used to detect circCDYL2 expression in nasopharyngeal carcinoma cells or clinical tissue samples. FISH employed fluorescence secondary antibody (Invitrogen, CA, USA), while ISH used DAB staining reagents (Meixin Biology, Fujian, China). Cell nuclei were counterstained with DAPI (Invitrogen, CA, USA, for FISH) or hematoxylin (for ISH). FISH slides were observed and photographed using confocal laser scanning microscopy (Perkin Elmer, MA, USA). The ISH slides were independently evaluated by two pathologists and analyzed using a semi-quantitative integral method. The sequences of circCDYL2 probes used in this study are provided in Supplemental Table
3.
Plasmids, siRNA, and cell transfection
The plasmid for overexpressing circCDYL2 was generated by cloning the second exon of the CDYL2 gene into the circular RNA expression vector pCirc. The EIF3D pcDNA3.1-3xFlag-C overexpression plasmid (NM_003753) was purchased from YouBio Biotech (Guangzhou, China), while the RAD51 overexpression plasmid (NM_002875) was purchased from Weizhen Biotech Co., Ltd. (Shandong, China). ASO-circCDYL2 and siRAD51 were purchased from RIBOBIO (Guangzhou, China). SiRNAs according to EIF3D, BRCA1, and 53BP1 genes were purchased from Qiagen Biotech (Beijing, China). Relevant sequences are provided in Supplemental Table
3. The overexpression vector or empty plasmid was transfected into nasopharyngeal carcinoma cells using Neofect DNA transfection reagent (Neofect Biotech, Beijing, China). SiRNA transfection was carried out using Hiperfect (Qiagen, Hilden, Germany).
RNA extraction and RT-qPCR quantification
Total RNA was extracted using the Trizol reagent (Solarbio Life Sciences, Shanghai, China) according to the manufacturer’s instructions. The RNA was reverse-transcribed into cDNA using the Vazyme reverse transcription kit (Nanjing, China). Real-time quantitative PCR was performed using SYBR Green (Bimake, Shanghai, China), and GAPDH was used as the internal reference. The primer sequences are listed in Supplemental Table
3.
Clonogenic assays
Cells that had been transfected with different overexpression vectors or siRNAs were seeded uniformly in 6-well plates. After 24 hours, cells were irradiated with single X-ray doses of 0, 2, 4, 6, and 8 Gy. The cells were then maintained in a cell culture incubator for 8 to 12 days. Then, fixation with 4% paraformaldehyde at room temperature and crystal violet staining was performed. The number of cell colonies was counted for each group, and dose-survival curves were generated for analysis.
Western blotting
Cell lysis was performed using RIPA buffer (Beyotime Biotechnology, Shanghai, China). Following protein extraction, samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with 10% skim milk for 1 hour at room temperature and incubated overnight at 4 °C with corresponding primary antibodies. After washing, the membranes were incubated with secondary antibodies for 2 hours at room temperature. Protein bands were detected using ECL reagent (Millipore, Billerica, MA, USA). Quantitative analysis of protein bands was performed using Image J. A list of antibodies used is provided in Supplemental Table
4.
Cycloheximide and MG132 treatment
The HNE2 and CNE2 cells were treated with cycloheximide (CHX, Beyotime, Shanghai, China) (50 μg/ml) or MG132 (TargetMol, Boston, USA) (20 μM) for 0, 6, 12, or 24 hours, respectively. Then the proteins were extracted for western blotting.
Immunofluorescence
Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 minutes, and permeabilized with Triton X-100. After that, the cells were blocked with 5% BSA and incubated overnight at 4 °C using the corresponding primary antibodies. Following PBS washing, cells were incubated with appropriate secondary antibodies for 40 minutes at 37 °C. DAPI staining was performed for 10 minutes, then the slides were sealed using a fluorescence quenching attenuator. Images were captured using a confocal laser scanning microscope (Perkin Elmer, MA, USA). Antibodies used are listed in Supplemental Table
4.
Comet assay
Comet assay was performed using the DNA damage Detection Kit (Jiangsu, China) following the manufacturer’s instructions. In brief, agarose gel was placed on slides, Irradiated cells were digested and resuspended in PBS at a concentration of 1 × 106 /ml, which was mixed with low melting point agarose gel and added to the slides. After solidification, another layer of agarose gel was added. Following incubation in lysis buffer and alkaline buffer, electrophoresis was run at 25 V for 30 minutes in a horizontal electrophoresis apparatus. Propidium lodide (PI) staining was done in the dark for 10 minutes, and DNA migration length was analyzed using a fluorescence microscope.
HR and NHEJ report assays
The DR-GFP-U2OS and EJ5-GFP-U2OS cells (gifted by Professor Shi Lei, Tianjin Medical University) were used to assess HR and NHEJ repair efficiency, respectively [
25]. Cells were seeded in 12-well plates and transfected with the corresponding plasmids or siRNA when they reached 50% confluence. SiBRCA1 and si53BP1 were used as positive controls for HR and NHEJ, respectively. The HA-I-SceI overexpression plasmid was transfected 24 hours later. After an additional 48 hours of incubation, GFP-positive cells were quantified using flow cytometry (Accuri 6, BD Bioscience, New Jersey, USA). At least 20,000 cells were collected for each treatment, and data were analyzed using FlowJo software.
Polysome profiling assays
Transfected cells were cultured in a medium containing 100 μg/ml of CHX for 5 minutes. After collection, cells were resuspended in cell lysis buffer (5 mM Tris, pH 7.4, 2.5 mM MgCl2, 1.5 mM KCl, 1 × Protease inhibitors, 100 μg/ml CHX, 2 mM DTT, 200 U/mL RNase inhibitor, 0.5% Triton X-100, 0.5% Sodium deoxycholate), centrifuged at 16,000×g, 4 °C for 7 min. Then, 500 ml of supernatant was transferred onto a sucrose gradient (Freshly prepared a 5–50% (w/v) sucrose density gradient in SW41 ultracentrifuge tubes). Centrifugation was carried out at 32,000×g, 4 °C for 2 hours using an ultracentrifuge (Beckman, California, USA). RNA from each fraction was extracted using Trizol (Invitrogen) for RT-qPCR analysis.
RNA pulldown and liquid chromatography coupled to tandem mass spectrometry
Cells transfected for 48 hours were lysed, and the supernatant was incubated with a biotin-labeled circCDYL2 probe (Sangon Biotech, Shanghai, China; Supplemental Table
3). Streptavidin magnetic beads were added and incubated overnight at 4 °C. After purification, proteins were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an UltiMate 3000 RSLCnano system and LTQ Orbitrap Velos Pro mass spectrometer (Thermo Scientific).
RNA immunoprecipitation (RIP)
The Magna RIP kit (Millipore, Massachusetts, USA) was used for RIP experiments according to the manufacturer’s instructions. Cells were lysed in lysis buffer and incubated with magnetic beads conjugated to specific target antibodies or negative control IgG antibodies overnight at 4 °C on a rotator. Immunoprecipitated RNA was purified, enriched, and quantified using RT-qPCR. Antibodies used are listed in Supplemental Table
4.
Immunohistochemistry
Immunohistochemistry was performed using the Elivision™plus (Mouse/Rabbit) Immunohistochemistry Kit (Kit-9902, Maxim, Fujian, China) following the manufacturer’s instructions. Two pathologists independently assessed stained slides. Semi-quantitative integral methods were used for analysis. Antibodies used are listed in Supplemental Table
4.
Subcutaneous tumor xenografts
Four-week-old female BALB/c nude mice were housed in the SPF center of Hunan Cancer Hospital. Animal experiments (including care, injections, euthanasia, dissection, and sample collection) were conducted following an approved animal experiment protocol. Mice were randomly divided into four groups (n = 5), with each mouse being subcutaneously injected with 3 × 106 CNE2 cells transfected with vector, circCDYL2 plasmid, negative control, or ASO-circCDYL2 subcutaneously. Tumor volume and weight were monitored every 2 days. After 18 days, 6 Gy of ionizing radiation was applied to the transplanted tumor site. Mice were observed for an additional 10 days and then euthanized. Tumor tissues were dissected, measured, fixed, paraffin-embedded, and used for subsequent experiments.
Statistical analysis
Statistical analysis was carried out by using GraphPad Prism 8.0.2. Two-tailed t-tests were used to analyze differences between any two groups, and one-way analysis of variance (ANOVA) was used to analyze differences among multiple groups. All data are presented as mean ± standard deviation (SD), and p-values less than 0.05 were considered statistically significant.
Discussion
Radiotherapy is currently one of the most commonly used treatments for tumors, alongside surgery and chemotherapy. Its fundamental mechanism involves the induction of irreparable DNA DSBs within cancer cells using ionizing radiation, ultimately leading to cell death [
3,
26]. Tumor cells’ resistance to radiation represents a primary cause of treatment failure. The DNA damage repair system promptly activates a DNA damage response following a DNA double-strand break, recruiting numerous DNA damage repair proteins to the damaged sites and initiating a cascade reaction that facilitates DNA repair [
33]. The main pathways for repairing DSBs include HR and NHEJ [
34]. HR is predominantly mediated by RAD51. Upon DSB occurrence, the MRN (MRE11-RAD50-NBS1) sensor complex recognizes and binds to the structure, subsequently recruiting and activating ATM, leading to phosphorylation of H2AX (γH2AX) and amplifying the initial signal. Subsequently, numerous DNA damage repair proteins are recruited to the sites of DNA damage [
35,
36]. CtIP, EXO1, and DNA2 exonuclease facilitate end resection to generate single-stranded DNA (ssDNA), which is then coated by RPA [
37,
38]. The BRCA1-PALB2-BRCA2 complex recruits RAD51 to the damage site, displacing RPA and enabling RAD51 to utilize the undamaged homologous sister chromatid as a template for precise repair [
25,
39]. In contrast, the NHEJ pathway is mediated by 53BP1, which inhibits DNA end resection, followed by direct ligation of the DNA double-strand break ends by the Ku80-Ku70 heterodimer and DNA-PKcs [
40,
41]. In our study, we observed that circCDYL2 promotes HR repair rather than NHEJ, as evidenced by the DR-GFP and EJ5-GFP reporter systems. Examination of key molecules in the HR pathway unveiled that circCDYL2 enhances RAD51 expression. Further investigation revealed that circCDYL2 recruits EIF3D to promote RAD51 translation initiation, thereby fostering homologous recombination repair and contributing to radiation resistance in nasopharyngeal carcinoma.
Tumor cells evade radiation-induced cell death by proficiently repairing DNA double-strand breaks through the homologous recombination repair pathway, a primary contributor to radiotherapy resistance. Consequently, targeting the homologous recombination repair pathway emerges as a pivotal strategy for sensitizing tumor cells to radiotherapy. A comprehensive understanding of the regulatory mechanisms governing HR repair pathways and the identification of potential targets and molecular markers for radiotherapy sensitization hold significant clinical importance. These insights can lead to improved therapeutic outcomes, reduced tumor recurrence rates, and the ability to identify radiosensitive individuals, thereby mitigating the unnecessary side effects associated with excessive ionizing radiation.
circRNAs exert substantial influence over gene expression at multiple levels and have emerged as a cutting-edge and high-interest area in biomedical research. CircRNAs are widely distributed across various tissues and cells, exerting regulatory control over gene expression through diverse mechanisms. These mechanisms primarily include acting as miRNA sponges [
21,
42] or binding to proteins [
18,
43], thereby modulating downstream gene transcription or splicing processes [
44,
45]. Additionally, circRNAs participate in epigenetic regulation [
46], with certain instances of direct encoding of small peptides with biological functions [
47,
48]. However, the exploration of circRNAs in the context of radiation resistance, particularly concerning DNA damage repair in nasopharyngeal carcinoma, remains limited. In this study, we have identified an elevated expression of circCDYL2 in nasopharyngeal carcinoma, and this upregulation is associated with an unfavorable prognosis for patients. By utilizing the DR-GFP and EJ5-GFP reporter systems, we have elucidated that both the overexpression and knockdown of circCDYL2 exert a significant impact on HR repair rather than NHEJ. Immunofluorescence assays have demonstrated that circCDYL2 promotes the formation of RAD51 foci, a crucial protein within the HR pathway, in nasopharyngeal carcinoma cells following radiation treatment. Our western blotting analysis has further validated that circCDYL2 elevates RAD51 expression. Subsequent investigations have unveiled that circCDYL2 actively enhances RAD51’s translation, thus promoting HR repair and contributing to radiotherapy resistance in nasopharyngeal carcinoma. It is worth noting that RAD51, being a pivotal component in HR repair, exhibits high expression in various solid tumors, and therefore, holds potential as a novel target for cancer treatment [
49‐
51].
The role of circCDYL2 in malignant tumors and its underlying mechanisms remains to be fully elucidated. Previous research has confirmed that circCDYL2 promotes migration in colorectal cancer by binding to Ezrin and activating the AKT pathway [
52]. Additionally, in breast cancer, circCDYL2 forms a complex with GRB7 and FAK, sustaining downstream signaling pathways of HER2 and contributing to trastuzumab resistance [
53]. In our study, we provide the first evidence of circCDYL2’s involvement in promoting radiotherapy resistance in nasopharyngeal carcinoma. This unique mechanism centers around circCDYL2 acting as a scaffold molecule. Specifically, it binds to a portion of the 5’UTR of RAD51 mRNA through complementary base-pairing. Simultaneously, it recruits translation initiation factor EIF3D, to the 5’UTR of RAD51 mRNA, thus facilitating translation of RAD51 and enhancing RAD51 protein expression.
After mRNA maturation, the process of protein translation becomes a critical regulatory step. Protein translation encompasses three fundamental phases: translation initiation, elongation, and termination, all of which depend on the coordinated efforts of various translation-related factors [
54,
55]. The translation initiation complex assumes a pivotal role in numerous tumor types [
56‐
58]. Nonetheless, research on translation initiation in the context of radiation resistance is scarce. In our study, we employed RNA pull-down and mass spectrometry analysis to identify interactions between circCDYL2 and translation initiation complex-related proteins, including EIF3D. The EIF3 protein family, which constitutes the largest group of translation initiation factors in eukaryotes, comprising 13 family members, plays a central role in eukaryotic translation initiation [
59]. Among these members, EIF3D holds particular significance. It has been previously reported that EIF3D directly interacts with RBMS1, with both factors binding to the 5’UTR and 3’UTR of SLC7A11, respectively. This interaction leads to the regulation of SLC7A11 expression by RBMS1 through EIF3D, consequently inhibiting ferroptosis and promoting the progression of lung cancer [
60].
In summary, circCDYL2 has been a relatively unexplored molecule, and its function in tumors has remained elusive. Our study marks the first discovery of circCDYL2’s pronounced expression in nasopharyngeal carcinoma, where it correlates with a poor prognosis. In both in vivo and in vitro settings, we demonstrated that circCDYL2 actively promotes radiotherapy resistance in nasopharyngeal carcinoma cells. The mechanistic basis for this effect is rooted in circCDYL2’s role as a scaffold molecule. It facilitates the binding of EIF3D to RAD51 mRNA, thereby accelerating translation initiation and enhancing HR repair. This mechanism ultimately contributes to radiotherapy resistance in nasopharyngeal carcinoma (Fig.
7F). These findings underscore the critical importance of the circCDYL2/EIF3D/RAD51 axis in mediating radiotherapy resistance in nasopharyngeal carcinoma. Moreover, circCDYL2 has the potential to serve as a prognostic marker for adverse outcomes in nasopharyngeal carcinoma. Targeting circCDYL2 and RAD51 could represent promising therapeutic strategies for sensitizing nasopharyngeal carcinoma to radiotherapy.
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