Cells and treatment
CRC cell lines (HCT116, HT29, WIDR and T87), lung cancer cell lines (A549, H460, H1299 and H1975) and a human bronchial epithelial cell line (BEAS-2B), human glioma cell lines (U251, U373 and U87), human hepatoma cell lines (HuH7, 7721, HepG2) and human embryonic kidney 293 cells (HEK 293T) were all purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The culture conditions used in this study are shown in Supplementary Table S1. High glucose Dulbecco’s modified Eagle’s medium (DMEM) and RPMI-1640 medium were supplied by HyClone (Thermo Fisher, USA). All culture media were supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher), 100 U/mL penicillin and 100 µg/mL phytomycin (Gibco, Thermo Fisher). All cultures were maintained at 37 °C and 5% CO2. To induce DNA damage, cells were treated with camptothecin (CPT, 1 µM, Selleck) for 1 h or etoposide (ETO, 100 μg/mL, Selleck) for 4 h, or γ-irradiation. An ATM inhibitor (ATMi, KU-55933), ATR inhibitor (ATRi, VE-821) and DNA-PK inhibitor (DNA-PKi, NU7441) were purchased from Selleck and used at a working concentration of 10 μM. During DNA damage induction by IR, CPT or ETO, the corresponding inhibitors were maintained in complete medium continuously unless indicated otherwise.
Plasmids, shRNA and sgRNA constructs
Plasmids containing full-length scaRNA2 (NR_003023) and truncated fragments were directly synthesized by OBIO Technology (Shanghai, China). The scaRNA2 overexpression plasmid was cloned into the lentiviral expression vector (GL132 pSlenti-EF1-EGFP-F2A-Puro-WPRE2-CMV-MCS). CRISPR-mediated knockout plasmids containing short guide RNAs targeting scaRNA2 were generated and cloned into the lentiviral vector H5070 (pCLenti-U6-spgRNA v2.0-CMV-Puro-3xFlag-spCas9-WPRE). shRNA hairpins targeting scaRNA2 were synthesized and cloned into the pLenR-GPH vector. The sequences of scaRNA2 shRNA and guide RNAs are listed in Supplementary Table S2. The DRR (pLCN DSB Repair Peporter, Addgene,98895) and HR mCherry (pCAGGS DRR mCherry Donor EF1a BFP, Addgene,98896) plasmids used in the DDR reporter assay were kind gifts from Jan Karlseder (Addgene plasmids) (23).
RNA isolation and quantitative RT‑PCR (RT–qPCR) assay
Total RNA was extracted from cultured cells using a MagaBio plus RNA Purified Reagent Kit (Bioer Technology, Hangzhou, China) and a GenePure Pro Automatic Nucleic Acid Extraction and Purification Instrument according to the manufacturer’s instructions. The quality of the total RNA was evaluated using a NanoDrop One spectrophotometer (Thermo Fisher, USA). cDNA was synthesized according to the manufacturer’s instructions using the PrimeScript RT Master Mix Kit (Takara, Japan). qRT–PCR was conducted using a TB Green Premix Ex TaqTM PCR kit (Takara, Japan) on a Quant Studio 1 (Applied Biosystems, Thermo Fisher, USA) Real-Time PCR system. Relative gene expression levels were determined using the 2-△△CT method. GAPDH was used as an internal control to calculate the relative expression of lncRNAs. Each PCR amplification was performed in triplicate, three independent trials. All the primer sequences are listed in Supplementary Table S2.
Lentiviral transfection and stable cell line construction
The packaged plasmids PSPAX2 and PMD2G and the corresponding target plasmids were cotransfected into 293T cells with 1 μg/μL PEI reagent. The virus supernatants were collected at 48 h and 72 h after transfection. To obtain stably transfected cell lines with overexpression or knockdown of scaRNA2, HCT116 and HT29 cells were transduced with the harvested viral supernatants in the presence of 5 μg/mL polybrene (Yeasen Biotech, Shanghai, China) and selected with 1.5 μg/mL puromycin (Beyotime Biotech, Shanghai, China) for two weeks. The cells were then seeded at a density of 0.5 cells/well into 96-well plates to obtain stably expressed cell clones. The overexpression and knockdown efficiency of scaRNA2 in these puromycin-selected cells were examined using RT–qPCR.
Irradiation
For ionizing radiation (IR) treatment, γ-irradiation was performed using a 60Co irradiator in the Radiation Center (Faculty of Naval Medicine, Naval Medical University, China). The dose rate was 1 Gy/min at room temperature. For cellular experiments with irradiation, doses ranging from 2 to 8 Gy were used. The doses for animal models are 15 Gy for local irradiation in CDX and PDX tumors. The flow chart for the local irradiation field and shielding is shown in Figs. S14 and S15.
5’-RACE and 3’-RACE
5’-RACE and 3’-RACE experiments were performed using a GeneRacerTM Kit (Invitrogen, USA) with 1.0 μg of purified total RNA isolated from HCT116 cells. All RACE assays were carried out according to the instructions of the manufacturer. The primers used for 3’ RACE and 5’ RACE are listed in Supplementary Table S5.
Fluorescence in situ hybridization (FISH) with the TSA technique
The subcellular localization of scaRNA2 was determined in HCT116 cells using an In Situ Hybridization Detection Kit (TSA technique) (Servicebio, Wuhan, China) following the manufacturer’s instructions. FISH probes against scaRNA2 were designed and synthesized using digoxigenin-labeled antisense riboprobes (Servicebio, Wuhan, China) (Table S3). Briefly, HCT116 cells were seeded on 14×14 Φ cover slips, fixed with 4% paraformaldehyde in DEPC at room temperature for 15 min, digested with proteinase K solution at 37 °C for 30 min, and incubated with 3% H2O2 at room temperature for 30 min. After incubation with prehybridization mix at 37 °C for 1 h, cell sections were incubated with hybridization mix containing 1 μM scaRNA2 or U6 probes in a humidity chamber and hybridized overnight at 37 °C. The next day, the cell slides were immersed in 2×SSC at 37 °C for 10 min, followed by two washes at 37 °C for 5 min with 1× SSC. After 30 min of blocking with 0.1% BSA, the cell sections were labeled with digoxin working solution at 37 °C for 50 min, followed by four washes at 37 °C for 5 min with 1× PBS. Afterward, cell sections were incubated with FITC-TSA working solution for 5 min in the dark. Subsequently, cell nuclei were stained with DAPI for 10 min in the dark. Fluorescence images were obtained using a ZEISS LSM primary microscope.
Isolation of nuclear and cytoplasmic fractions
The nuclear and cytosolic fractions of cellular RNA were isolated using the Cytoplasmic & Nuclear RNA Purification Kit according to the manufacturer’s instructions (Norgen Biotek, Canada). Briefly, HCT116 cells (2 × 106) were lysed with 200 μL ice-cold lysis buffer J for 5 min on ice. The lysate was transferred to a microcentrifuge tube with subsequent pelleting by spinning at 3500 × g for 10 min. The supernatant containing cytoplasmic RNA was transferred to a new tube and vortexed with 200 μL of buffer SK for 10 s. The cell pellets containing the nuclear RNA were resuspended in 400 μL of buffer SK for 10 s. Then, 200 μL of 96% ethanol was added, and the tubes were vortexed for 10 s. The sample mixtures were transferred to spin-columns to separate nuclear RNA using centrifugation (3500 g, 1 min). Next, all the columns were washed twice with 400 μL of wash solution, followed by a 2 min centrifuge to thoroughly dry the column. The samples were eluted from the columns using 50 μL of elution buffer to obtain the fractionated RNA. The concentrations of cytoplasmic and nuclear RNA were measured using a NanoDrop One spectrophotometer (Thermo Fisher, USA). Relative expression levels of U6, GAPDH and scaRNA2 were analyzed using RT–qPCR.
RNA immunoprecipitation sequencing (RIP-seq.) and PCR (RIP-qPCR)
RIP was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore, USA) according to the manufacturer’s instructions. In brief, 2×106 HCT116 cells were harvested and lysed with RIP lysis buffer containing protease inhibitor cocktail. The cell supernatants were incubated with magnetic beads conjugated with ATR antibody (CST, 13934s) or IgG (Millipore, AP101) for 2 h at 4 °C. RNase-free DNase I and Proteinase K were consecutively used to remove DNA and protein from the RIP complex. The resulting RNA was subjected to qRT–PCR to detect the enrichment of scaRNA2.
Colony formation assay
Cells were harvested and seeded in 6-well plates for 24 h, followed by treatment with different doses of IR, camptothecin, etoposide or olaparib. Cell densities and drug treatment concentrations are listed in Supplementary Table S6. After these treatments, the cells were incubated with drug-containing medium for 10-14 days. Cells were dyed with crystal violet solution (Beyotime, Shanghai, China) for 15 min after fixation with 4% paraformaldehyde for 30 min at room temperature. The images were taken with a HiCC-IB Automatic Colony Counting Analysis System (Wanshen, Hangzhou, China). Cell colonies were defined as 0.5-1 mm in diameter cell assemblies. Experiments were repeated in triplicate. Data from experimental groups were normalized to their respective control groups.
Flow cytometric cell apoptosis and cell cycle analysis
Cells were seeded in six-well plates at a density of 1×106. On the second day, cells were irradiated with 8 Gy or treated with 1 μM CPT for 1 h or 100 μg/mL ETO for 4 h. Apoptosis of cells was assessed using an Annexin V FITC Apoptosis Detection Kit (Dojindo, Japan) according to the manufacturer’s instructions. For the cell cycle assay, cells were collected and fixed with 70% ethanol at 0, 4, 8, 12 and 24 h after different treatments overnight. The cells were treated with 100 μg/mL RNase (Thermo Fisher, USA) and 40 μg/mL propidium iodide (Beyotime, Shanghai, China) for 30 min at 37 °C. The percentages of cells distributed in G0/G1-, S-, and G2/M-phase were determined using flow cytometry (Beckman, USA).
Neutral comet assay
DNA DSBs were evaluated using a neutral single-cell gel electrophoresis assay kit (Trevigen, USA) according to the manufacturer’s instructions. In brief, NC and scaRNA2 knockdown cells were trypsinized and resuspended at a concentration of 1×105 cells/mL. The cell suspension was then mixed with low melting agarose (LMA) (37 °C) at a ratio of 1:10. Then, 50 μL of cell suspension was immediately dribbled onto the slide. The slides were immersed in precooled lysis buffer at 4 °C overnight and then immersed in prechilled neutralizing buffer for 30 min. After electrophoresis, the slides were immersed in DNA precipitation solution and 70% ethanol successively at room temperature for 30 min each. The slides were air dried at room temperature and stained with 100 μL of diluted SYBR® Green Gold at 4 °C for 5 min. Comet images were visualized using a fluorescence inverted microscope (LSM Primovert, ZEISS). Quantification of images was achieved by analyzing at least 5 randomly selected comets per slide with CaspLab software.
Western blot analysis
Total protein was extracted from cells using M-PER Protein Extraction Reagent (Thermo Scientific, USA) containing a proteinase inhibitors cocktail (MCE, USA), phosphatase Inhibitor Cocktail I (MCE, USA) and phosphatase Inhibitor Cocktail II (MCE, USA). The cell lysate was smashed by sonication for 15 cycles (Power 6%, ultrasound for 1.5 s, interval of 1 s) in an ultrasonic cell crusher (Qiqian Electronic Technology, Shanghai, China) and centrifuged at 14,000 × g for 15 min at 4 °C. The protein concentrations were determined using a BCA protein assay kit (Beyotime, Shanghai, China). For WB analysis, 15 μg protein lysates were separated using a 10% or 12.5% SDS PAGE gel preparation kit (Epizyme Biomedical Technology, Shanghai, China). The proteins were transferred to 0.2 μm PVDF membranes (Millipore, USA) using an eBlotTM L1 Wet Protein Film Transfer Instrument (GeneScript, China). The PVDF membranes were incubated overnight at 4 °C with the selected primary antibodies against the target proteins (Supplementary Table S4) and then incubated with secondary antibodies for 2 h at room temperature. The western blot signals were visualized with chemiluminescence using an ECL kit (Thermo Fisher, USA) and a GelView detection system (Biolight, Guangzhou, China). Relative protein expression was quantified using ImageJ software. GAPDH was used as the internal control, and the relative content of each target protein gray value was normalized to GAPDH. Contrast gray value of phosphorylated protein = gray value of phosphorylated protein/gray value of nonphosphorylated protein.
Immunoprecipitation (IP)
Immunoprecipitation was performed using a Pierce™ Crosslink IP Kit (Pierce, 26147) according to the standard protocol. Briefly, NC and scaRNA2 knockdown cells were lysed with 500 μL IP lysis buffer with protease inhibitor cocktail plus phosphatase inhibitor cocktail (MCE) and centrifuged at 14,000 × g at 4 °C for 15 min. Total protein lysates (1 mg) were used per immunoprecipitation. Nonspecific binding of protein lysates was excluded by incubating with 80 μL control agarose resin for 1 h and rotating at 4 °C. Antibodies against ATR, Mre11 and Exo1 or control IgG (5 μg) (Supplementary Table S4) were cross-linked with 20 μL Protein A/G Plus Agarose for 1 h and rotated at room temperature. Then, protein lysates were loaded onto the selected antibody cross-linked columns overnight at 4 °C. The following day, the immunoprecipitation complex was washed five times with IP lysis buffer, eluted with 40 μL elution buffer, and analyzed using western blotting along with 5% input controls as described above.
RNA pulldown assay and mass spectrum analysis
The sense and antisense strands of scaRNA2 or truncated scaRNA2 were obtained with in vitro transcription from the MB1985 pSPT18 vector (OBIO BioTech., Shanghai, China) and purified using a QIAquick Nucleotide Removal Kit (Qiagen, Germany) according to the manufacturer’s protocol. Sense and antisense RNAs were biotinylated with the Biotin RNA Labeling Mix (Roche, Switzerland), T7 RNA polymerase (Roche, Switzerland) and SP6 RNA polymerase (Roche, Switzerland), respectively. The RCR products were purified using a RNeasy Mini Kit (Qiagen, Germany). The quality was verified using a NanoDrop One (Thermo Fisher, USA). Then, 3 µg of each kind of purified biotinylated transcript was heated at 90 °C for 2 min, left on ice for 2 min and incubated with 5× Annealing Buffer (Beyotime, Shanghai, China) for 20 min at room temperature. Thereafter, the biotinylated transcripts were incubated with protein lysates from HCT116 cells (1 mg) at 4 °C for 1 h to form RNA–protein complexes. Then, the complex was incubated with Dynabeads MyOne™ (Thermo Fisher, USA) beads for 1 h at room temperature in the dark to isolate the labeled complexes from other components. Dynabeads MyOne™ beads were then washed three times with PBS and elution buffer. The proteins pulled down by sense, antisense scaRNA2 or truncated scaRNA2 were analyzed using western blotting and mass spectrometry (Thermo Fisher, USA). Mass spectra results were analyzed using MaxQuant (version 1.5.5.1) software.
NHEJ and HR reporter assay
The NHEJ and HR reporter assays were performed according to our previous study (19). Briefly, the pLCN DSB Repair Reporter (DRR) plasmid was transfected into HeLa cells, and stable DDR expression clones were selected using 400 μg/mL neomycin. Then, the cells were transduced with scaRNA2 knockdown or vector lentiviruses and selected using 0.5 μg/mL puromycin. To induce DSB formation, the I-SceI-expressing plasmid and HR donor (pCAGGS DRR mCherry Donor EF1a BFP) plasmid were cotransfected into the above selected cells. After 48 h, transfected cells were harvested and examined for the expression of GFP and mCherry by CytExpert flow cytometry (Beckman, USA).
Chromatin fractionation
For preparation of the chromatin fraction, a chromatin extraction kit (Abcam, UK) was utilized according to the manufacturer’s recommendations. Briefly, after different treatments, 2 x 106 NC and scaRNA2 knockdown cells were lysed with 200 μL lysis buffer containing protease inhibitor cocktail. The cell suspension was vortexed vigorously for 10 s and centrifuged at 5000 rpm for 5 min. The supernatant was collected and incubated with 50 µL working extraction buffer on ice for 10 min and sonicated 2 x 20 s to increase chromatin extraction. Thereafter, the samples were centrifuged at 12,000 rpm at 4 °C for 10 min, and the supernatant was transferred to a new tube and mixed with chromatin buffer at a 1:1 ratio. The chromatin-binding proteins were analyzed using western blotting. Quantification of protein bands was analyzed using ImageJ software. Histone 3 was used as the internal control.
Immunofluorescence (IF) staining and imaging analysis
Immunofluorescence staining assays were performed according to our previous study (19). Briefly, NC and scaRNA2 knockdown cells were seeded on circular coverslips with a diameter of 14 mm in 12-well plates at a concentration of 1 x 105 cells per well. For immunostaining, cells were fixed in 4% paraformaldehyde for 15 min at different time points (30 min-24 h) after 6 Gy irradiation. Then, the coverslips were permeabilized in 0.5% Triton X-100 on ice for 10 min, followed by blockage with 5% goat serum in PBS for 1 h at room temperature. Subsequently, the coverslips were incubated at 4 °C with the appropriate primary antibodies overnight, followed by incubation with the appropriate fluorophore-conjugated secondary antibodies for 2 h in the dark at room temperature. All antibodies utilized in our study are listed in Supplementary Table S4. The nuclei were stained with 0.1 μg/mL DAPI (Beyotime, Shanghai, China) for 15 min and mounted with mounting media (Beyotime, Shanghai, China).
For the detection of RPA2, RAD51, CtIP, Mre11, DNA2, and Exo1 foci induced by IR, cell coverslips were incubated with preextraction buffer (25 mM HEPES pH 7.5, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 300 mM sucrose, 0.5% Triton X-100) for 5 min on ice before fixation. For the detection of ssDNA upon BrdU labeling, cells were labeled with 10 µM BrdU (Sigma, USA) for 24 h prior to exposure to IR. Before fixation, the cells were treated with fresh preextraction buffer as described above.
The images were photographed with a Zeiss LSM 880 confocal microscope (Zeiss, Germany) under 63× oil objective lens magnification, and raw data were processed with Zen 2.3 software. For the quantification of foci number approximately ten fields of view were randomly captured from distinct images.
Patient-derived organoids and treatment
Patient-derived organoids (PDOs) were constructed to preclinically determine the effects of scaRNA2 knockdown on tumor growth. Tumor tissues from rectal cancer patients in the Department of Colorectal Surgery, Shanghai Changhai Hospital (Shanghai, China) were used for the establishment of PDO models. Briefly, fresh resected tumor tissue was minced, rinsed with iodophor solution 3 times, cut into ~0.5 mm3 pieces and centrifuged at 200 × g for 3 min to remove the supernatant. The fragmented tissue was incubated in 4 mL digestion buffer (Advanced DMEM F12 (Gibco, USA) containing 500 U collagenase (Gibco, USA) on an orbital shaker at 37 °C for 2 h. After digestion, the suspension was pipetted 20 times with a 1 mL pipette tip, allowed to stand for 3 min, transferred to the supernatant and centrifuged for 1 min at 2500 rpm. Red blood cell lysis buffer (the volume of precipitation: red blood cell lysis buffer =1:3) was added to lyse the precipitate, shaken at room temperature for 4 min, and then mixed with DPBS followed by centrifugation at 2500 rpm for 1 min. The pellet was resuspended with Matrigel (Corning, USA) on ice and 30 µL of Matrigel resuspension was added to each well of a 48-well plate at 37 °C for 15 min. Upon complete gelation, 500 μL of Colorectal Cancer Organoid medium (Biogenous, Hangzhou, China) was added to each well and the plates were transferred to humidified incubators. Culture media was changed every 4-5 days, and organoids were passaged every 2-4 weeks.
Organoid Treatment: Cells from organoids were plated into 24-well plates after 3 days of culture and then infected with scaRNA2 knockdown or vector lentiviruses. After culturing for 48 h, organoids were irradiated with 8 Gy γ-rays. Then, images were taken with a light microscope every day for up to 8 days. The average number of organoids per field was quantified.
Tumor xenograft models and treatments
Cell-derived xenograft (CDX) model
In vivo subcutaneous tumor growth assays were performed in male BALB/c nude mice (4-5 weeks old) purchased from the Experimental Animal Center of Naval Medical University. HCT116 cells were harvested and resuspended in culture medium and the same volume of Matrigel (Corning, USA) at a concentration of 2.0 × 107 cells/ml. Each mouse was injected subcutaneously with 100 μL cell suspension on the left and right thigh. When tumor sizes reached 100 mm3, the mice were randomly divided into four groups: vector group, scaRNA2 knockdown group, vector + irradiation group, and scaRNA2 knockdown + irradiation group (n = 5). Then, 100 μL (108 IU/mL) of lentiviruses carrying scaRNA2 sgRNA or vector were intratumorally injected at multiple points. The injections were delivered twice every two days. Thereafter, tumor-bearing mice received local irradiation with a 60Co irradiator at a dose of 15 Gy, as depicted in Fig. S14A. For the analysis of tumor growth, tumor size was measured every 3 days for a month with a digital caliper using the formula: volume = length × width2 × 1/2. At the end of treatment, the mice were euthanized and the excised tumors were fixed and serially sliced for histological and immunohistochemical analyses.
Patient-derived xenograft (PDX) model
For the patient-derived xenograft (PDX) model, tumor tissues from rectal cancer patients were implanted into nude mice and established by LIDE Biotech Co., Shanghai, China. Tumor tissues from mice bearing the passage 3 PDX model (COPF161282) were injected subcutaneously into the dorsal flank of male BALB/c nude mice. When the tumors reached 100 mm3, lentiviruses carrying scaRNA2 sgRNA or vector were intratumorally injected. The lentiviruses were injected every two days three times, and then the mice were randomly divided into four groups as in the CDX models (n = 5). Tumors in the flank of PDXs were locally irradiated with a 60Co irradiator at a dose of 15 Gy, and the abdomen of mice was covered with custom lead jigs (Fig. S15B). The tumor size was monitored every 3 days for a month. Tumor volume was calculated in accordance with the formula: volume = length × width2 × 1/2. At the endpoint, the mice were euthanized and the tumors were excised, measured, weighed and fixed for analysis with histology and immunohistochemistry.
Histology and Immunohistochemistry
Tumor tissues from CDX and PDX were collected and fixed with 4% paraformaldehyde overnight and sectioned into slides at a thickness of 5 μm. For histological analysis, slides were stained with hematoxylin and eosin (H&E). For immunohistochemistry, slides were deparaffinized, rehydrated, and treated with 3% H2O2 for 25 min in the dark. After extensive washing, the slides were blocked with 3% BSA in PBS for 30 min and incubated overnight at 4 °C with the indicated primary antibodies, followed by incubation with the appropriate fluorophore-conjugated secondary antibodies. Antibody information is shown in Supplemental Table S4.
TUNEL staining was conducted to evaluate apoptosis in resected tissue according to the manufacturer’s instructions (Servicebio, Wuhan, China). Briefly, after deparaffinization and antigen retrieval, sections were incubated with 1% Triton X-100 in PBS for 20 min at room temperature. Then, the sections were incubated with reaction solution containing 1 µL TdT, 5 µL dUTP and 50 µL equilibration buffer at 37 °C for 2 h in a humidified box. Following PBS washing, sections were stained with DAPI for 10 min at room temperature. The slides were scanned using a panoramic slice scanner (3DHISTECH, Hungary). ImageJ software was used for quantitation of TUNEL-positive cells.
Patient sample collection and tissue microarray analysis
Eighty locally advanced rectal cancer patients who underwent neoadjuvant chemoradiotherapy from May 2016 to October 2019 at the Department of Colorectal Surgery, Shanghai Changhai Hospital, Naval Medical University (Shanghai, China) were enrolled in the study. For inclusion, the patients were required to have only one primary lesion, have completed standard neoadjuvant chemoradiotherapy and undergone surgical resection and have survived more than 1 month after surgery. Tumor tissues and tissues adjacent to carcinoma were obtained after surgery and then paraffin-embedded into a microarray. Neoadjuvant chemoradiotherapy was performed by the 3D conformal technique, which is conventionally fractional. The total dose was 50 Gy (2 Gy per fraction). The interval between preoperative radiotherapy and surgery was generally 6-8 weeks. Tumor responsiveness to neoadjuvant chemoradiotherapy was represented using a Tumor Regression Grade (TRG), TRG 0 (complete regression): no visible tumor cells under microscope; TRG 1 (close to complete regression): microscopy showed only single or small tumor cells. TRG 2 (partial regression): significant regression with more residual tumors than single or small tumor cells; TRG 3 (poor or no regression): extensive residual tumor without significant regression.
scaRNA2 and ATR expression in the microarray was detected with FISH immunofluorescence. The procedures of scaRNA2 probe hybridization were consistent with the above FISH method. After FITC-TSA incubation, the slides were incubated at 4 °C overnight with ATR primary antibody (Abcam, ab289363, 1:50) in a humidified chamber, followed by the corresponding goat anti-rabbit Cy3 secondary antibody for 1 h at 37 °C in the dark. Image information on the tissue slice was scanned using a panoramic slice scanner (3DHISTECH, Hungary). CaseViewer 2.4 software was employed at 1-400x arbitrary magnification for observation. The TMA plug-in in Halo V3.0.311.314 software (Indica labs, USA) was used to analyze the data, and the Indica Labs-Highplex FLv3.1.0 module was used to quantify the number of positive cells and total cells in the target area of each slice. The area of positive cells (cells/mm2) = the number of positive cells (cells/analysis area in mm2).
Clinical analysis for tissue microarray
The clinical analysis of scaRNA2 levels was independently and semi-quantitatively assessed by two pathologists using the histochemistry score (H-score, ranging from 0 to 300). We evaluated scaRNA2 using the ratio of the H-score in cancer tissues to that in adjacent tissues. The cutoff value for the scaRNA2 level was deduced according to a receiver operating characteristic (ROC) curve, and patients were categorized into two groups based on their scaRNA2 level (high or low).
Statistical analysis
Graphs and all statistical analyses were generated using GraphPad Prism 8.0 software. Quantitative data are presented as the means ± standard deviation (SD). For experiments including only two groups, means were evaluated using a two-tailed unpaired Student’s t test. For multiple group comparisons, data were analyzed using one-way or two-way ANOVA followed by Tukey’s multiple-comparisons test or Dunnett’s test. p < 0.05 was considered statistically significant. All experiments were performed at least three times independently.