El eje ADAR1-circRAB5A-BIP regula la resistencia a la radioterapia en el cáncer colorrectal mediante la coordinación de la autofagia protectora y la apoptosis.

Colorectal cancer (CRC) is the third most common malignancy globally, with more than 1.5 million new cases annually.^[1] It plays a crucial role in locoregional disease management and has attracted significant attention in recent years.^[2]
,
^[3] Despite technological advancements, intrinsic or acquired radioresistance affects 30%–40% of patients,^[4] leading to recurrence and poor survival. While dysregulation of DNA repair pathways and oncogenic signaling has been implicated, emerging evidence highlights non-coding RNAs,^[5]
,
^[6] particularly circular RNAs (circRNAs), as pivotal yet underexplored modulators of radiation responses.^[7] CircRNAs, formed by back-splicing of pre-mRNA, exhibit tissue-specific expression and regulate cancer progression by sponging microRNAs (miRNAs), interacting with RNA-binding proteins, or even encoding peptides.^[8]
,
^[9] Dysregulation of certain circRNAs always modulates signaling pathways by affecting the function of its target miRNA or proteins, thereby exhibiting the function of regulatory RNAs.^[10] Accumulating evidence has revealed their roles in chemoresistance and immune evasion,^[11] their involvement in radioresistance remains largely uncharacterized.

Endoplasmic reticulum (ER) stress adaptation has emerged as a survival mechanism in radioresistant tumors.^[12] The chaperone BIP, a master regulator of the unfolded protein response (UPR), stabilizes proteostasis, which modulates both pro-survival autophagy and apoptosis under extracellular stress.^[13] The role of BIP in balancing autophagy and apoptosis is a double-edged sword,^[14] which in some situations, BIP enhances autophagy and recycling of the UPR to maintain cell survival and inhibit apoptosis, which contributes to radioresistance.^[15] However, over-activated autophagy also leads to uncontrolled apoptosis. Therefore, revealing the role of autophagy in radioresistance is key to uncovering the mechanisms of BIP-mediated autophagy-apoptosis balance. Specifically, in CRC, the function of BIP, its relationship with autophagy, and the involvement of regulatory circRNAs in radioresistance warrant further exploration.

Adenosine deaminase acts on RNA 1 (ADAR1), an RNA-editing enzyme that catalyzes adenosine-to-inosine (A-to-I) conversions.^[16] As reported, overexpression of ADAR1 in tumors promotes metastasis and therapy resistance by altering mRNA splicing or microRNA (miRNA) targeting.^[17] Notably, Alu elements, which are primate-specific repetitive sequences that facilitate circRNA biogenesis, are hotspots for ADAR1 editing. ADAR1 can regulate the biogenesis of circRNAs by modulating Alu sequences, thereby promoting differential expression.^[18] A study by Tian et al. also highlighted that ADAR1 was upregulated during radioresistance^[19]; however, its underlying molecular mechanism and its regulation of circRNAs are still unclear.

Herein, we address these gaps by investigating the role of a novel circRNA, circRAB5A, and its regulatory network involving ADAR1 and BIP in CRC radioresistance. Using the Gene Expression Omnibus (GEO) dataset and bioinformatics analysis, we hypothesized that ADAR1 suppresses circRAB5A biogenesis by binding to Alu Jo/Jr elements in radioresistant CRC cells. The deficiency circRAB5A modulates BIP stability through TRIM21-mediated ubiquitination, thereby coordinating the autophagy-apoptosis balance to drive radioresistance. Our findings provide new insights into the molecular basis of the ADAR1/circRAB5A/BIP axis in CRC radioresistance and highlight potential therapeutic targets to enhance the efficacy of radiotherapy.

Materials and methods

Patient samples

Forty pairs of CRC tumor tissues and adjacent normal colorectal mucosal tissues were collected from patients who underwent radical resection at the Department of Gastrointestinal Surgery, Southwest Medical University (Luzhou, Sichuan, China), between September 2022 and September 2023. All patients received preoperative radiotherapy (45–50 Gy in 25–28 fractions) as part of their treatment regimen, and none of them had received neoadjuvant chemotherapy or other anti-cancer therapies prior to surgery. The radiosensitive and radioresistant groups were classified as follows. Patients were stratified into radiosensitive (n = 20) and radioresistant (n = 20) groups based on clinical outcomes. Radioresistance was defined as local tumor recurrence within six months post-radiotherapy, confirmed by endoscopic or imaging (CT or MRI) evidence. Radiosensitive patients showed no recurrence and achieved a complete or partial response as assessed by the RECIST 1.1 criteria^[20] within 12 months post-radiotherapy. The study was approved by the Ethics Committee of Southwest Medical University (Approval No. KY2022025) and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all the participants.### Cell culture and reagents

The human colorectal cancer cell lines SW480 (ATCC CCL-228), SW620 (ATCC CCL-227), HCT8 (ATCC CCL-244), HCT116 (ATCC CCL-247), and Caco-2 (ATCC HTB-37), and the normal human colon epithelial cell line NCM460 (CELLCOOK, CCC-223) were purchased from Guangzhou CELLCOOK Biotech Co. (Guangzhou, China). The cell lines were authenticated by short tandem repeat profiling (Genetic Testing Biotechnology Co., Guangzhou, China). The cells were maintained in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37 °C in a humidified incubator with 5% CO₂. All the cells tested negative for mycoplasma contamination prior to harvest.

For cell autophagy analysis, the autophagy inhibitor, chloroquine (CQ) (CAS No. 54-05-7) was purchased from Selleck (USA) and used at a working concentration of 10 μM. The autophagy inducer, Rapamycin (Rapa) (CAS No. 53123-88-9) was purchased from Selleck and used at a work concentration of 25 nM. MG132 (Sigma-Aldrich) was used as a proteasome inhibitor at a concentration of 10 μM. The cultured cells were treated for 6 h to block proteasomal degradation.### Construction of radiation-resistant cells

Irradiation-resistant SW480 cells (SW480-IR) were established using a fractionated irradiation protocol to mimic clinical radiotherapy regimens. Parental SW480 cells were cultured to 60% confluency in 10-cm dishes (Corning, USA) and exposed to 2 Gy per fraction at a dose rate of 0.75 Gy/min. After each irradiation, the cells were returned to the incubator (37 °C, 5% CO₂) and allowed to recover to 80% confluency before the next fraction. This process was repeated for 10 fractions, delivering a total cumulative dose of 20 Gy over five weeks (two fractions per week). After the final irradiation, the cells were cultured for an additional 3 weeks to stabilize the radioresistant phenotype.### Clonogenic survival assay

Radiosensitivity was assessed using clonogenic survival assay. Briefly, SW480 and SW480-IR cells were seeded at a low density of 1000 cells per well in a 6-well plate and exposed to 0, 2, 4, 6, or 8 Gy radiation. After 14 d, colonies (>50 cells) were fixed with 4% paraformaldehyde (PFA), stained with 0.5% crystal violet, and counted using the ImageJ software. Dose‒response curves were generated using the multi-target single-hit model (y = (1 − e
−
D/D0)n) in GraphPad Prism 9.0, where D represents the radiation dose (Gy), D0 represents the mean lethal dose (Gy), and n represents the extrapolation number. All experiments were performed with at least three independent biological replicates.### Plasmid construction and cell transfection

The full-length linear sequence of circRAB5A was PCR-amplified from SW480 cDNA. The amplicon, supplemented with circular sequences, was cloned into the EcoRI/BamHI sites of the pLCDH-mir vector (GeneChem, Shanghai, China) using T4 DNA ligase (NEB). The recombinant plasmid (pLCDH-circRAB5A) was verified using Sanger sequencing (Tsingke Biotechnology Co., Beijing, China). The construction of ADAR1, BIP, and TRIM21 expression vectors, as well as BIP wild-type or truncated recombinant expression vectors, were conducted by Tsingke Biotechnology Co. by cloning the full-length or truncated sequence into the pcDNA3.1 vector (Addgene). The mock vector served as a negative control (NC).

For knocking down, small interfering RNAs (siRNAs) targeting circRAB5A (sicircRAB5A), ADAR1 (siADAR1), BIP (siBIP), and TRIM21 (siTRIM21) were designed and synthesized by GeneChem. Off-target effects were minimized by selecting sequences with fewer than two mismatches with other human transcripts. The siRNA sequences are provided in Supplementary Table S1.

For plasmid transfection, SW480 and SW620 cells were seeded in 6-well plates (Corning) at a density of 3 × 10⁵ cells/well and cultured to 60%–70% confluency. Transfection was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. For siRNA transfection, cells were seeded at 2 × 10⁵ cells/well in 6-well plates and transfected with 50 nM siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. The cells were harvested 48 h post-transfection for downstream assays.### Identification of circRNAs

To characterize the circular structure of circRAB5A, multiple assays were conducted, including divergent/convergent PCR, RNase R digestion, oligo (dT) reverse transcription assay, and actinomycin D (ACT-D) assay, according to the published literature by Zhou et al.^[21]### Subcellular fractionation

Cytoplasmic and nuclear fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) following the manufacturer's protocol. qRT-PCR was performed to detect circRAB5A expression, using cytoplasmic (GAPDH) and nuclear (U6) markers used as controls.### Cell apoptosis assay

Apoptosis was assessed using an Annexin V-FITC/PI Apoptosis Detection Kit (Solarbio, Beijing, China). The cells (3 × 10⁵/well in 6-well plates) were irradiated and harvested 24 h later. The cells were collected, washed, and resuspended in binding buffer. After being stained with 2.5 μL Annexin V-FITC and 2.5 μL PI, apoptotic cells were analyzed by flow cytometry (NovoCyte, Agilent, USA).### Western blot (WB)

Total protein was extracted from SW480 and SW620 cells using RIPA lysis buffer (Beyotime, Jiangsu, China) supplemented with a protease inhibitor cocktail (Beyotime) and phosphatase inhibitor cocktail (Beyotime). The protein concentration was quantified using the BCA protein assay kit (Beyotime). Equal amounts of proteins were loaded and separated by 7.5%–15% SDS–PAGE gel and then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk or 3% FBS for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C: anti-BIP (Proteintech, 1:1000), anti-LC3B (CST, 1:2000), anti-p62 (CST, 1:2000), anti-p-Akt (CST, 1:1000), anti-Beclin1 (CST, 1:1000), anti-Flag (Sigma-Aldrich, 1:5000) and anti-GAPDH (Abclonal, 1:5000). After that, the membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies (Abclonal, 1:5000) for 2 h at room temperature. Finally, the protein bands were visualized using ECL Plus reagent (Bio-Rad, USA) and imaged using a ChemiDoc Imaging System (Bio-Rad). Quantification was performed using the ImageJ software, with the target protein levels normalized to those of GAPDH.### qRT-PCR assay

Total RNA was extracted from SW480 and SW620 cells using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Briefly, 1 × 10⁶ cells were lysed in 1 mL of TRIzol, incubated for 5 min at room temperature, and mixed with 200 μL of chloroform. After centrifugation (12,000 × g for 15 min), the aqueous phase was collected, and the RNA was precipitated with isopropanol, washed with 75% ethanol, and resuspended in RNase-free water. The RNA purity (A260/A280 ≥ 1.8) and concentration were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Next, 1 μg of RNA was reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit with Genomic DNA (gDNA) Eraser (Takara). The reaction mixture included 2 μL 5× gDNA Eraser Buffer, 1 μL gDNA Eraser, and the RNA template and was incubated at 42 °C for 2 min to remove genomic DNA. Reverse transcription was performed with 1 μL of PrimeScript RT Enzyme Mix, 1 μL RT Primer Mix, and 4 μL 5 × RT Buffer, and incubated at 37 °C for 15 min and 85 °C for 5 s. qPCR was performed using TB Green Premix Ex Taq II (Takara) on a Bio-Rad CFX96 Real-Time System. Each reaction contained 10 μL TB Green Mix, 0.8 μL 10 μM forward/reverse primers, 2 μL cDNA, and 7.2 μL RNase-free water. The cycling conditions was set as follow: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Relative expression was calculated using the 2⁻ΔΔCt method and normalized to that of GAPDH. The primer sequences for specific genes and internal controls are listed in Supplementary Table S2.### Fluorescence imaging for autophagy detection

Autophagosome formation was visualized using mCherry-GFP-LC3 dual-fluorescence reporter (Beyotime). SW480 cells were transfected with the pCMV-mCherry-GFP-LC3 plasmid using Lipofectamine 3000 (Invitrogen) 24 h prior to radiation treatment (4 Gy). The cells were fixed with 4% paraformaldehyde 24 h post-irradiation and imaged using a Zeiss fluorescence microscope (Leica Microsystems) with a 63 oil immersion objective. Autolysosomes (red puncta, mCherry+GFP−) and autophagosomes per cell (yellow puncta, mCherry+GFP+) were counted manually from three random fields using the ImageJ software.### Fluorescence imaging and fluorescence in situ hybridization (FISH) of circRAB5A

CircRAB5A subcellular localization was detected using a FISH kit (Riobio, Guangzhou, China)^[22] with a Cy5-labeled DNA probe targeting the circRAB5A back-splice junction. The cells were seeded on cover-slips, fixed with 4% PFA, permeabilized with 0.5% Triton X-100 (Beyotime), and hybridized with a 50 nM probe in hybridization buffer at 37 °C overnight. After washing with 2 × SSC, the nuclei were stained with DAPI. Images were acquired using a fluorescence microscope (Zeiss).### Bioinformatics analysis

The prediction of RNA-binding proteins with Alu Jo/Jr sequences were performed using catRAPID online bioinformatics software^[23] according to the manufacturer's instructions. The prediction results were provided in Supplementary Tables S3 and S4.### RNA pull-down

RNA-protein interactions were detected using the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific) with biotinylated probes targeting circRAB5A (GenePharma, Guangzhou, China). In brief, 1 × 10⁷ SW480 or SW620 cells were lysed in 1 mL of RNA lysis buffer with protease inhibitor cocktail and 100 U/mL RNase inhibitor (Takara) for 15 min on ice. Biotinylated probes were then mixed with 500 μL of cell lysate and incubated at 37 °C for 1 h to allow RNA-protein binding. Next, streptavidin magnetic beads were added to the probe-lysate mixture. The samples were rotated at 4 °C for 2 h to capture the biotinylated complexes. The beads were then washed with wash buffer to remove non-specific binding. The bound proteins were eluted with 50 μL of elution buffer at 95 °C for 5 min and analyzed by WB. NC probe pull-down served as a negative control.### RNA immunoprecipitation (RIP) assay

RNA-protein interactions were validated using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) following the manufacturer's protocol. In brief, 1 × 10⁷ SW480 or SW620 cells were lysed in 1 mL RIP lysis buffer containing protease inhibitor cocktail and 100 U/mL RNase inhibitor for 30 min on ice. The magnetic beads were then washed with RIP wash buffer and incubated with 5 μg anti-BIP antibody (CST) or normal IgG (Millipore, negative control) at 4 °C for 2 h. Next, the lysates (500 μL) were added to the antibody-bound beads and rotated at 4 °C overnight to co-immunoprecipitate the RNA‒protein complexes. The next day, the beads were washed with RIP wash buffer, and bound RNA was extracted using TRIzol reagent (Invitrogen) and analyzed by qRT-PCR.### Ubiquitination assay

BIP ubiquitination was assessed using co-immunoprecipitation (co-IP) to detect ubiquitin conjugation. Briefly, SW480 cells were transfected with a circRAB5A overexpression plasmid, sicircRAB5A, or respective controls for 48 h. To block proteasomal degradation, the cells were treated with 10 μM MG132 (Sigma-Aldrich) for 6 h prior to lysis. The cells were then lysed in 500 μL IP lysis buffer (Beyotime) supplemented with a protease inhibitor cocktail, phosphatase inhibitor cocktail, and 10 mM N-ethylmaleimide (Sigma‒Aldrich). The lysates were incubated on ice for 30 min and then centrifuged at 14,000 × g for 15 min at 4 °C, after which the supernatants were collected. Next, 500 μg of total protein was pre-cleared with 20 μL protein A/G agarose beads (Invitrogen) for 1 h at 4 °C. The supernatants were incubated with 2 μg of anti-BIP antibody (CST) or normal IgG (Millipore, negative control) overnight at 4 °C. The next day, 20 μL of protein A/G beads were added and rotated for 2 h at 4 °C to capture the antibody‒protein complexes. The beads were washed with IP lysis buffer, and the bound proteins were eluted and analyzed by WB.### Animal study

Animal experiments were approved by the Ethics Committee of Southwest Medical University (Approval No. swmu20230056) and conducted in accordance with the ARRIVE guidelines.^[24] A total of 30 four-week-old female BALB/c nude mice (SPF grade) were purchased from Hunan SJA Laboratory Animal Co. (Hunan, China). The mice were housed under controlled conditions (22 °C ± 2 °C, 12 h light/dark cycle) with free access to food and water. Stable knockdown of circRAB5A and BIP was achieved by lentivirus expressing shRNA targeting circRAB5A (sh-circRAB5A) and BIP (sh-BIP) and verified by qRT-PCR. CRC xenografts were established by subcutaneous injection of 5 × 10⁶ SW480 cells into the right axilla of the mice. When the tumor volume reached ~75–100 mm³ (7 d post-injection), mice were randomly assigned to five groups (n = 6 per group) using a random number table method. Prior to tumor irradiation, all the mice were anesthetized using a 5% isoflurane and oxygen mixture for rapid induction of anesthesia and maintained at 1.5%–2% isoflurane concentration during the irradiation procedure to induce a stable plane of anesthesia while minimizing respiratory and cardiovascular stress. Then, the mice received local tumor irradiation using an X-RAD 320 Biological Irradiator (Precision X-ray, USA) at a dose rate of 0.5 Gy/min. The mice in the sh-NC + radiation group received 3 Gy in 2 fractions (consecutive days), and those in the sh-BIP group received a single dose of 2 Gy. Tumor growth was monitored twice weekly using calipers. The tumor volume was calculated using the following formula: volume (mm³) = 0.5 × width² × length. Four weeks after tumor implantation, the mice were euthanized using an intraperitoneal injection of tribromoethanol (20 μL/g, AbMole, USA) following the cervical dislocation method. Last, tumors were harvested, weighed, and processed for subsequent analysis.### Immunohistochemistry and TUNEL staining

Xenograft tumor tissues were fixed in 4% PFA, paraffin-embedded, and sectioned into 5-μm slides. The sections were then deparaffinized, rehydrated, and rinsed with PBS. After incubation with 3% H₂O₂ to block endogenous peroxidase activity, the slides were treated with a 5% BSA solution (Beyotime) for 30 min at room temperature. The primary antibody, rabbit anti-Ki67 (Abclonal), was diluted 1:200 with PBS, and the slides were incubated overnight at 4 °C. The next day, the secondary goat anti-rabbit HRP (Abclonal) was diluted 1:400 with PBS and incubated with the slides for 2 h at room temperature. A DAB staining kit (Abcam, USA) was used to develop a brown color. TUNEL staining was performed according to the manufacturer's protocol using a TUNEL staining kit (Beyotime Biotechnology). Finally, the nuclei were stained with hematoxylin, and images were acquired using an Olympus microscope (Japan).### GEO analysis

Differentially expressed circRNAs in radioresistant CRC were identified using publicly available transcriptomic data from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The raw data of the GSE186940 dataset by Shao. Y. et al.^[25] were downloaded, preprocessed, and visualized using R 4.3.1. The thresholds of the differentially expressed circRNAs were set as: |log₂(fold change)| > 1, FDR