Aberrant m5C hypermethylation mediates intrinsic resistance to gefitinib through NSUN2/YBX1/QSOX1 axis in EGFR-mutant non-small-cell lung cancer

Cell lines and reagents. The human lung adenocarcinoma cell lines HCC827, HCC4006, HCC2935, H1975, HCC2279 and H1650 cells were obtained from American Type Culture Collection (ATCC). PC-9 was kindly gifted by Prof. Jian Ding (Shanghai Institute of Materia Medica, Shanghai, China). All the cell lines were cultured at 37 °C in a humidified 5% CO2 incubator in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco). Gefitinib and osimertinib were purchased from Selleck Chemicals and dissolved to 10 mmol/L with DMSO as stock solutions for in vitro studies.

Patient samples. The pathological biopsies of lung adenocarcinoma tissues were collected from NSCLC patients with drug sensitivity or intrinsic resistance to EGFR-TKIs in the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Patients that received surgery, chemoradiotherapy or immunotherapy within the past six months were excluded. All tumor samples had either an exon 19 deletion (19del) or an exon 21 point mutation (L858R) in EGFR, but lacked the EGFR T790M mutation. Intrinsic resistance to EGFR-TKIs was defined as stable disease (SD) < 3 months or progression on the first imaging evaluation after EGFR-TKIs treatment. Clinical specimens of only intrinsic-resistant patients were taken before and after EGFR-TKIs treatment, because re-biopsy is important for exploring resistance mechanisms to select new therapeutic drugs for intrinsic-resistant patients. This study was approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University (the approval number 2019-KY-31) and complied with the Declaration of Helsinki. All subjects provided the written informed consent according to the institutional guidelines.

Cell viability assay. Cells were seeded at a density of 5 × 103 cells/well in 96-well plates in RPMI-1640 medium containing 10% FBS. After 24 h, cells were exposed to the indicated concentrations of gefitinib for another 24 h. Then 10 µL of CCK-8 reagent (Vazyme, China) was added to each well for 1 h at 37 °C, and the absorbance (optical density, OD) was read at a wavelength of 450 nm using EnVision® multimode plate reader (PerkinElemer). The IC50 values were calculated by concentration-response curve fitting using the four-parameter method.

Colony formation assay. Cells were seeded into 6-well plates at a density of 500–1000 cells per well. The medium was exchanged every 3 days for 3 weeks. Colonies were treated with fixation solution (10% methanol + 10% acetic acid) at room temperature for 15 min and then stained with a solution of 1% crystal violet in methanol for 15 min.

Flow cytometry detection. Cells were seeded in 6-well plates at a density of 2.5 × 105 cells/well. After 24 h, the cells were transfected with indicated siRNA and treated with DMSO or gefitinib (1 µM) for 72 h. For the cell cycle analysis, cells were fixed with 75% ethanol and then stained with PI/RNase (Beyotine, China) for 15 min at 37 °C. For the cell apoptosis analysis, the cells were stained with annexin V-FITC and PI according to Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, China). Then, samples were analyzed using a FACS Calibur flow cytometer (BD Biosciences).

Quantitative real-time PCR (qRT-PCR). The total RNA was extracted using TRIzol reagent (Invitrogen) and reversely transcribed to complementary DNA (cDNA) using iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed using iTaq Univer SYBR Green Supermix (Bio-Rad) according to the manufacturer’s instructions. GAPDH or 18 S rRNA was used as a housekeeping gene for normalization. Results were represented as fold expression. The primer pairs used for qPCR analysis were listed below:

NSUN2 forward: 5’- CAAGCTGTTCGAGCACTACTAC-3’,

NSUN2 reverse: 5’- CTCCCTGAGAGCGTCCATGA-3’;

YBX1 forward: 5’- GCGGGGACAAGAAGGTCATC-3’;

YBX1 reverse: 5’- CGAAGGTACTTCCTGGGGTTA-3’;

QSOX1 forward: 5’- TGAGAAAGTTTGGTGTCACCG-3’;

QSOX1 reverse: 5’- GGACCTGGATTCCATGAGCAC-3’;

GAPDH forward: 5’- GGAGCGAGATCCCTCCAAAAT-3’;

GAPDH reverse: 5’- GGCTGTTGTCATACTTCTCATGG-3’;

18S rRNA forward: 5’- GTAACCCGTTGAACCCCATT-3’;

18S rRNA reverse: 5’- CCATCCAATCGGTAGTAGCG-3’.

Western blotting analysis. Total protein was extracted using RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitors (Roche) and phosphatase inhibitor cocktail (Sigma) and protein concentration was determined using BCA Protein Assay Kit (Beyotime, China). Harvested lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes, probed with primary antibodies and then incubated with horseradish peroxidase-conjugated secondary antibodies. The immunoreactive bands were visualized using the chemiluminescence (Thermo Scientific). Primary antibodies used in this study were listed as follows: anti-NSUN2 (Sigma, HPA037896), anti-EGFR (Cell Signaling Technology, #4267), anti-p-EGFR (Cell Signaling Technology, #3777), anti-QSOX1 (Abcam, ab235444), anti-YBX1 (Abcam, ab76149), anti-Cleaved PARP (Cell Signaling Technology, #5625), anti-Cleaved Caspase 3 (Cell Signaling Technology, #9661), anti-β-actin (Cloud Clone, CAB340Mi01) and anti-GAPDH (Cloud Clone, CAB932Mi01).

Immunohistochemistry (IHC) staining. Briefly, paraffin sections were heated at 60 °C for 4 h and deparaffinized with BioDewax and Clear Solution (ServiceBio, China). Heat-induced antigen retrieval was carried out using citric acid (PH 6.0) antigen retrieval buffer (ServiceBio, China) for 23 min at 95 °C. Endogenous peroxidase activity of tissues was blocked with 3% hydrogen peroxide at room temperature in darkness for 25 min. Then slides were blocked with 3% BSA for 30 min at room temperature and incubated with primary antibody overnight at 4 °C. Afterward, slides were incubated with horseradish peroxidase conjugated secondary antibodies at room temperature for 50 min. Images were scanned using the 3DHISTECH PANNORAMIC VIEWER. The concentration of anti-p-EGFR (Cell Signaling Technology, #3777), anti-NSUN2 (Sigma, HPA037896), anti-YBX1 (Abcam, ab76149), anti-QSOX1 (Abcam, ab235444), and ki67 (Servicebio, GB111499) were used according to supplier’s instructions.

Immunofluorescence (IF) staining. Briefly, cell climbing slides were permeabilization with permeabilize working solution (ServiceBio, China) for 20 min. Then slides were blocked with 3% BSA for 30 min at room temperature and incubated with primary antibody (anti-Cleaved Caspase 3, Cell Signaling Technology, #9661, 1:300 dilution) at 4 °C overnight. Afterward, the slides were incubated with secondary antibody (Cy3 goat anti-rabbit IgG (H + L), Abclonal, AS007, 1:200 dilution) for 1 h at room temperature in 3% BSA/PBS. Nuclei were stained with DAPI (Servicebio, China), and the slides were observed and imaged under a fluorescent microscope (ZEISS).

RNA interference. Cells were seeded in 6-well or 96-well plates at 40% confluence. After 24 h, cells were transfected with the indicated siRNA oligonucleotides using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Then, the cells were cultured for 72 h and harvested either for cell viability assay or western blotting analysis. The target sequences of siRNA oligonucleotides were as follows:

siNSUN2#1: 5’- GAGAUCCUCUUCUAUGAUCTT-3’;

siNSUN2#2: 5’- CACGUGUUCACUAAACCCUAUTT-3’;

siYBX1#1: 5’- GGAUAUGGUUUCAUCAACATT-3’;

siYBX1#2: 5’- CGUAACCAUUAUAGACGCUTT-3’;

siQSOX1: 5’- CCGGACAATGAAGAAGCCTTT-3’.

Plasmids and transfection. pcDNA3.1-NSUN2-WT, pcDNA3.1-NSUN2-Mut (C271/321A), PCMV-YBX1-WT, PCMV-YBX1-Mut (W65A), PLKO.1-shNSUN2 and PLKO.1-shYBX1 plasmids were kindly provided by Prof. Yun-Gui Yang (Beijing Institute of Genomics, Beijing, China). The plasmids GV493-shQSOX1, 3FLAG-EGFP-NSUN2-WT, 3 FLAG-EGFP-NSUN2-DM (C271/321A), 3FLAG-EGFP-YBX1-WT, 3FLAG-EGFP-YBX1-Mut (W65A), 3FLAG-EGFP-QSOX1-WT and 3FLAG-EGFP-QSOX1-Mut (C439/1201A) were purchased from GENECHEM (Shanghai, China). Transient transduction was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Then, the cells were cultured for 48 h and harvested for western blotting analysis. For lentiviral transduction, a second-generation lentivirus packaging system consisting of psPAX2 (Addgene) and PMD2.G (Addgene) was used to create virus particles. In brief, the plasmids were transfected into HEK293T packaging cells at 60% confluence using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. After an additional 48 h incubation, the supernatant was collected, filtered using a 0.45-µm filter (Millipore), and used to infect host cells in the presence of 6 µg/mL polybrene (Solarbio, China). The resultant stable polyclonal populations of transduced cells were then selected with puromycin or hygromycin (Solarbio, China) for two weeks, followed by validation by western blotting.

The shRNA sequences are:

shNSUN2#1: 5’- GCTGGCACAGGAGGGAATATA-3’;

shNSUN2#2: 5’- CACGTGTTCACTAAACCCTAT-3’;

shYBX1: 5’- GGTTCCCACCTTACTACAT-3’;

shQSOX1: 5’- CCGGACAATGAAGAAGCCTTT-3’.

RNA stability assay. Cells were seeded in 6-well plates, and then treated with Actinomycin D (5 µg/mL, MedChemExpress) for 0, 2, 4, 6, and 8 h. The same number of H1650 cells were collected and total RNA was extracted by TRIzol reagent (Invitrogen). An equal-volume RNA was reverse-transcribed into cDNA, and then qRT-PCR was used to detect the mRNA level of QSOX1. The relative abundance of mRNA at each time point relative to t = 0 time point was calculated.

Protein translation assay. Protein translation levels were determined in vitro utilizing the puromycin (P8230, Solarbio, China). Cells were treated with 200 ng/mL puromycin for indicated time points. The cells were lysed, and the protein expression was assayed by western blotting with GAPDH as the reference.

Dual-luciferase reporter assay. Luciferase reporter plasmids containing CDS-QSOX1-wild type or CDS-QSOX1-mutant (C439/1201A/C74T) were synthesized by Genechem (Shanghai, China) and cloned into GV272 vector. NSUN2 knockdown cells were plated in a 96-well plate and transfected with 200 ng luciferase reporter plasmids and 10 ng of Renilla luciferase control reporter vectors using Lipofectamine 3000. Then cells were lysed after 48 h and subjected to the luciferase activity analysis using a Dual Luciferase Reporter Assay Kit (E1910, Promega). The firefly-luciferase activity was measured using the EnVision® multimode plate reader (PerkinElemer) and normalized by the renilla-luciferase activity.

RNA immunoprecipitation (RIP). H1650 cells were harvested and washed twice with cold PBS, and the cell pellet was incubated with RIP lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, Protease Inhibitor, RNase Inhibitor) on ice for 30 min. One tenth portion of the cell lysate was used as input. The rest of the cell lysate was incubated with either Rabbit IgG-coated beads or anti-YBX1 (Abcam, ab76149)-coated beads for 4 h at room temperature. Afterward, the beads-antibody-protein-RNA complex was washed five times with ice-cold washing buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, RNase inhibitor). Then, immunoprecipitated sample was digested with proteinase K and the RNA was precipitated with glycogen (Thermo Scientific, AM9516).

. Total RNA was extracted by TRIzol reagent followed by quantitative RT-PCR.

Animal studies. The nu/nu athymic BALB/c female mice (6–8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and maintained under specific pathogen-free conditions. For xenograft implantation, PC-9-Mock, PC-9-NSUN2-WT, PC-9-NSUN2-DM, H1650-shCtrl, H1650-shNSUN2, H1650-shYBX1, H1975-shCtrl, H1975-shQSOX1 cells (6.0 × 106 cells/100 µL) were suspended in PBS and subcutaneously injected into the right flank of all mice. About two weeks after injection with PC-9-Mock, PC-9-NSUN2-WT, PC-9-NSUN2-DM cells, mice were administrated with 25 mg/kg gefitinib (MB1112, meilunbio, China) or 0.5% CMC-Na via gavage once daily for consecutive 10 days. Tumor volume was measured every 3 days and calculated by caliper measurements of the width (W) and length (L) of each tumor using the following formula: V=(L×W2)/2. Mice were sacrificed and tumors were collected for further analysis. Ethical approvals for the animal experiments were obtained from the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China).

RNA isolation and purification. Total RNA was extracted from cells using Trizol reagent (Invitrogen) and mRNA enrichment was performed using Dynabeads mRNA Purification Kit (Invitrogen, 61,006) according to the manufacturer’s instructions with certain modifications. Briefly, RNA rebound and the DNase treatment of RNA were performed for the removal of contaminated rRNA and DNA, in the mRNA specimens, respectively. Finally, the concentration of the purified mRNA was determined using Qubit fluorometer (Invitrogen).

Detection of m5C level. The level of 5-methylcytosine (m5C) in mRNA was detected using MethylFlash 5-mC RNA Methylation ELISA Easy Kit (EpiGentek). 200 ng purified mRNA was bound to each well and incubated at 37 °C for 90 min. Then the wells were washed three times and incubated with 50 µL of the 5-mC Detection Complex Solution at room temperature for 50 min. Next, the wells were washed five times and incubated with 50 µL of Fluorescence Development Solution at room temperature for 4 min in darkness. The fluorescence was measured by EnVision microplate reader (PerkinElmer) within 10 min at 530ex/590em nm.

Methylated RNA immunoprecipitation (MeRIP). Purified mRNA was fragmented into around 100-nucleotide-long fragments using RNA Fragmentation Reagents (Invitrogen, AM8740). About 400 ng of fragmented mRNAs were mixed with 2.5 µg of anti-m5C antibody (Abcam, ab10805) in immunoprecipitation buffer and incubated by rotating at 25 °C for 1 h. The mixture was then immunoprecipitated by incubation with prewashed Protein A Magnetic Beads (Thermo Scientific, 10002D) at 4 °C for 5 h. After extensive washing, the bound RNA fragments were eluted from the beads by proteinase K digestion at 55 °C for 60 min. Finally, RNAs were isolated from the eluate by phenol-chloroform extraction and ethanol plus glycogen (Thermo Scientific, AM9516) precipitation for qRT-PCR analysis.

RNA-seq. RNA-seq libraries of the cell samples (20–30 ng mRNA) were constructed with the KAPA Stranded mRNA-Seq Kit (Illumina platform) following the manufacture’s protocol. Libraries sequencing were executed using paired-end mode on the Illumina HiSeq-PE150 instrument.

RNA-BisSeq. The process of RNA fragmentation and bisulfite conversion were performed as previously described with minor modifications [19]. Briefly, purified mRNA was mixed with Luciferase at a ratio of 300:1 and the Luciferase functioned as methylation conversion control. The libraries of the bisulfite-treatment samples were prepared using the KAPA Stranded mRNA-Seq Kit according to the manufacturer’s instructions (Illumina platform). Libraries sequencing were performed using paired-end mode by the Illumina HiSeq-PE150 system.

RNA-Seq bioinformatics analyses. Quality control of RNA-Seq data was processed by FastQC. Adaptors were trimmed using cutadapt and low-quality bases were removed with Trimmomatic. The clean raw reads were mapped to the hg38 genome with the Ensembl 78 genome annotation with default parameters by hisat2 and Htseq-count was selected for counting the number of reads mapped to each Ensembl gene with the following parameters: -m union -s no. DESeq2 was chosen for analyzing the genes expression by calculating the TPM of the gene in each sample. Differentially expressed genes were assigned with cut-off |FoldChange| ≥ 1.2 and adjusted p-value < 0.05.

RNA-BisSeq bioinformatics analyses. RNA-BisSeq reads for each sample were extensively filtered and processed by FastQC which was similar with “RNA-Seq bioinformatics analyses” section described. Clean reads of Bis-treated libraries were mapped to human genome (hg38) using meRanGs, a splice-aware RNA-BSseq alignment tool available with meRanTK. The processed reads with lengths greater than 35 nt were defined as clean reads. The sample with C to T conversion rates > 99% were filtered for further analysis. The m5C sites within the genome were extracted by meRanCall with the following criteria: -mBQ 20 -mr 0.1. In order to identify the credible m5C sites, only candidate m5C sites with coverage depth (methylated C number + nonmethylated C number) ≥ 20, methylated cytosine depth ≥ 3 and m5C methylation level ≥ 0.1 in at least half replicates in any one condition were kept and used for differential methylation analysis after calling. The m5C sites were annotated using BEDTools intersectBed. The top hits with |Methylation level A – Methylation level B| > 0.05 were considered as differentially meythylated m5C sites. Cluster profiler was used to analyze the significantly differential genes for GO and KEGG pathway enrichment. The GO terms and KEGG pathways with adjusted p-value < 0.05 were considered as the significantly differential pathways.

Statistical analysis

Data were presented as the means ± standard deviation (SD), unless stated otherwise. Two-tailed Student’s t test was used to compare means between groups as indicated. The OS and RFS were assessed using the Kaplan-Meier analysis and the p values were calculated using the two-sided log-rank test. *p < 0.05 was considered significant.

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