Patients and tissue specimens
For this study, frozen tissue specimens obtained from 48 patients with skull base chordoma who underwent surgical resection at Beijing Tiantan Hospital between June 2016 and May 2018 were subjected to RNA sequencing. Paraffin-embedded tissue specimens obtained from 187 patients with primary skull base chordoma who underwent surgical resection at Beijing Tiantan Hospital between January 2008 and September 2014 were subjected to tissue microarray (TMA). These patients were followed up by clinical and radiographic examinations in November of 2019. Tumor recurrence was confirmed by clinical and imaging findings or histologic analysis of specimens from the second surgery. This study was approved by the Ethics Committee of Beijing Tiantan Hospital, Capital Medical University. Informed consent was obtained from all enrolled subjects, and the study was conducted in compliance with the principles governed by the Declaration of Helsinki.
RNA sequencing
RNA-Seq was performed on 48 skull base chordomas, and a total of 3μg of RNA per sample was adopted as input material for RNA sample preparations. Ribosomal RNA was first removed with an Epicentre Ribo-zero rRNA Removal Kit (RZH1046, Epicentre, Wisconsin, USA), and sequencing libraries were generated with the rRNA-depleted RNA using a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420L, NEB, Massachusetts, USA) following the manufacturer’s protocol. First-strand cDNA was synthesized using random hexamer primers, and second-strand cDNA was synthesized by incorporating dUTP. Double-stranded DNA was repaired via exonuclease/polymerase activities and then added adenylation to the 3’ end. After adapter ligation and library amplification, the library fragments were purified with an AMPure XP system (Beckman Coulter, California, USA) to select the fragments that were preferentially 150–200 bp in length. The strand marked with dUTP was not amplified, allowing strand-specific sequencing. Finally, the products were purified (AMPure XP system) and library quality was assessed on an Agilent Bioanalyzer 2100 system. After cluster generation, the libraries were sequenced on an Illumina Hiseq X platform and 150-bp paired-end reads were generated.
TMA construction
Paraffin-embedded primary chordoma tissue specimens from all 187 patients were assayed by TMA using a Tissue Array MiniCore (ALPHELYS, Plaisir, France). Three pathologists viewed the hematoxylin-eosin-stained slides, and the two most representative two-millimeter cores from each tissue slide were selected and removed to a new slide to construct the TMA. Four-micrometer sections from the TMA were then cut using a Leica RM 2135 Rotary Microtome (Rankin, Wetzlar, Germany) for subsequent immunohistochemical staining.
Immunohistochemistry (IHC)
The slides were placed in a BOND-III instrument manufactured by Leica Biosystems. A default IHC protocol was then chosen, and 20 minutes for epitope retrieval was set as the heat-induced epitope retrieval (HIER) parameter. BondTM Polymer Refine Detection (DS9800, Leica Biosystems, Wetzlar, Germany) was used for the detection of the primary antibody (anti-EZH2 antibody, 5246, CST, USA; anti-H3K27me3 antibody, 9733, CST, USA; anti-SIDT1 antibody, 55352-1-AP, Proteintech, USA). The slides were scanned using Aperio AT2 (Leica Biosystems) and the digital pictures were viewed using the digital slide-viewing software in Aperio AT2. We stratified the staining intensity on a scale of 0–3+ (0 = no staining, 1+ = weak, 2+ = moderate and 3+ = strong staining). An H-score was obtained by multiplying the staining intensity with a constant to adjust the mean to the strongest staining (H-score = 1 × [percent of 1+ cell] + 2 × [percent of 2+ cell] + 3 × [percent of 3+ cell]) to provide a score ranging from 0 to 300. We chose the median of the H-score as the cut-off value for separating patients into two groups, a high EZH2/H3K27me3/SIDT1-expression group and a low EZH2/H3K27me3/SIDT1-expression group.
Cell culture
The human chordoma cell line UM-Chor1 was purchased from the American Type Culture Collection (ATCC), and another human chordoma cell line, MUG-Chor1, was kindly provided by the Chordoma Foundation. Cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM, 30-2005, ATCC, Virginia, USA) supplemented with 18% RPMI-1640 Medium (30-2001, ATCC) and 10% fetal bovine serum (FBS, 10099-141, Gibco, California, USA) in a humidified incubator at 37℃ in 5% CO2. The culture medium was replaced every other day.
Lentiviral vector and transfection
Lentiviral vectors for INI1 knockdown and overexpression, and respective controls (termed shNC, shINI1, Vector, and INI1, respectively) were acquired from Genechem (Shanghai, China). Lentiviral vectors for SIDT1 knockdown and overexpression, and respective controls (termed shNC, shSIDT1, Vector, and SIDT1, respectively) were acquired from Syngentech (Beijing, China). Both UM-Chor1 and MUG-Chor1 cells were adherent cells and detached from the bottom of the culture flask by using trypsin digestion, and this reaction was terminated by adding culture medium. The cell suspension was centrifuged for 5 minutes at room temperature to collect the cells, and the cell pellets were ultimately resuspended using culture medium. After counting, 1 × 105 of the UM-Chor1 cells and 3 × 105 of the MUG-Chor1 cells were seeded into six-well plates and transfected with lentiviral vector at a MOI of 20. To establish stably transfected cell lines, 2 μg/mL puromycin was applied.
RNA extraction and qRT-PCR
Total RNA was extracted using a RNeasy Mini Kit (74104, Qiagen, Dusseldorf, Germany), and reverse transcription was performed with a High Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher, Massachusetts, USA) according to the manufacturer’s protocol. Subsequently, we executed qRT-PCR with a Power SYBR Green PCR Master Mix (4367659, Thermo Fisher) in a total reaction volume of 20μL; GAPDH was employed as a reference gene, and the levels of mRNAs were determined on a QuantStudio 5 instrument (Applied Biosystems, Massachusetts, USA). Amplification was conducted as follows: 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and 60°C for 60 seconds. For the quantitative analysis, relative expression levels were calculated based on CT values (corrected for GAPDH expression) according to the equation: 2-ΔCT(ΔCT = CT [gene of interest] - CT [GAPDH]). All qRT-PCR analyses were performed in triplicate. The primer sequences are provided in Supplementary Table 1.
Protein extraction and Western Blot assay
Tissue samples or cells were lysed using RIPA lysis buffer (C1050, Applygen, Beijing, China) along with a protease-inhibitor cocktail (P1265, Applygen) and a phosphatase-inhibitor cocktail (P1260, Applygen). The total protein concentration was determined using a BCA Protein Assay Kit (SK258437, Thermo Fisher). Equal amounts of total proteins were separated by SDS-PAGE (10% gels) for the detection of EZH2, H3K27me3, SIDT1, and ARAF (GAPDH was used as the protein loading control). After SDS-PAGE, the proteins on the gels were transferred to BioTrace nitrocellulose membranes (66485, Pall, New York, USA), blocked with 5% skim milk in Tris-buffered saline (TBS, pH 7.4; 20mM Tris-HCl, 150mM NaCl), and then incubated with anti-EZH2 (1:1000, 5246, CST, USA), anti-H3K27me3 (1:1000, 9733, CST, USA), anti-SIDT1 (1:1500, 55352-1-AP, Proteintech, USA), and anti-ARAF (1:600, sc-166771, Santa Cruz, USA) antibodies overnight at 4°C. The membranes were then incubated the following day with IRDye-labeled goat anti-rabbit or goat anti-mouse IgG at room temperature for one hour. Finally, the protein bands were scanned using a Li-COR Odyssey system (Li-COR Biosciences, Nebraska, USA). At least three independent experiments were performed, and a representative result is shown.
Cell growth and colony formation assays
A quantity of transfected cells was seeded into 96-well plates with 2.5 × 103 UM-Chor1 cells or 6 × 103 MUG-Chor1 cells per well. 10μL of CCK-8 (CK04, Dojindo, Kumamoto, Japan) solution was added to the wells at 0, 24, 48, 72, and 96 hours. Upon addition of the CCK-8 solution, the plates were incubated at 37°C for two hours, and the absorbance was determined at 450nm using a multimodal microplate reader (Tecan, Männedorf, Switzerland). For the colony formation assay, 2 × 103 transfected UM-Chor1 or MUG-Chor1 cells were seeded into six-well plates and incubated at 37°C in 5% CO2 for 14 days. The cell colonies were then fixed in 4% paraformaldehyde and stained with crystal violet solution in situ.
Transwell invasion assay
3 × 104 transfected UM-Chor1 cells or 1 × 105 transfected MUG-Chor1 cells were seeded into the Matrigel-coated upper chambers of transwell plates in 100μL of serum-free medium, while the following wells were filled with 600μL of complete culture medium containing 10% FBS as a function of chemoattractant which can induce the cells in the upper chamber to the lower one. After incubation for 48 hours, non-invading cells were scraped away carefully with a cotton swab, while cells that invaded to the lower chamber were fixed in 4% paraformaldehyde and stained with crystal violet solution in situ. We observed the invading cells in at least five separate fields of vision using a microscope.
Chromatin immunoprecipitation (ChIP) sequencing
UM-Chor1 cells of the shNC and shINI1 groups underwent ChIP sequencing as follows. After cross-linking by formaldehyde, the nuclear extracts of the cells were collected, and the chromatin was immunoprecipitated with anti-H3K27me3 antibody (10μg, 9733, CST, USA) or anti-IgG antibody (8μg, Millipore, USA). High-throughput DNA sequencing libraries were prepared by the GeneCreate Biological Engineering Co., Ltd (Wuhan, China) using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Catalog No. ND607, Vazyme, Nanjing, China). The library products that corresponded to 200-500 bp were enriched, quantified and then sequenced on a Novaseq 6000 sequencer (Illumina, California, USA). The data were analyzed and mapped to the hg19 genome using STAR software with default parameters. MACS2 and Bedtools software were used for peak calling and identification of different binding peaks. Function annotation of peak-related genes was then conducted.
ChIP-qPCR
UM-Chor1 cells from the shNC and shINI1 groups were applied to ChIP-qPCR as follows. Cells were crosslinked with 1% formaldehyde for 10 minutes by inverting the flasks at room temperature and quenched with 0.125M glycine for 5 minutes. The cells pellets were washed repeatedly in PBS and then lysed in lysis buffer (50mM HEPES, 150mM NaCl, 1mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and complemented with protease-inhibitor cocktail) for 10 minutes. After centrifugation, the supernatant was discarded, and the pellet was lysed in lysis buffer and subjected to sonication. Sheared chromatin was incubated with anti-H3K27me3 antibody (10μg, 9733, CST, USA) or anti-IgG antibody (8μg, Millipore, USA) bound to PierceTM Protein A/G Agarose Beads (Thermo Fisher) overnight, followed by elution and reverse cross-linking at 65°C overnight. TE buffer (10mM Tris-HCl, 1mM EDTA) was added to the DNA elution buffer followed by RNase treatment (0.5 mg/mL) at 37°C for 30 minutes and by proteinase K treatment (0.3 mg/mL) at 51°C for one hour, and the DNA was isolated and purified subsequently. ChIP-qPCR was applied to confirm the ChIP sequencing results. The primers of ChIP-qPCR are listed in Supplementary Table 1.
Xenograft mouse model
To establish the chordoma model, 6 × 106 transfected UM-Chor1 cells were suspended in a mixture of 100μL of PBS and 100μL of Matrigel and then injected subcutaneously into the left axilla of female BALB/c nude mice (4 weeks of age, Beijing Vital River Laboratory Animal Technology, China). After 7 weeks, the tumor xenografts were extracted, weighed and used for protein extraction. Tumor volume was determined with the following formula: volume = (length × width2)/2.
Pan-cancer analysis
The TIMER 2.0 was used to analyze the difference in SIDT1 expression between normal tissue and tumor tissue in 33 types of cancer. The Kaplan-Meier analysis was used to explore the impact of SIDT1 on the prognosis in patients with head and neck squamous cell carcinoma (HNSC), lower-grade glioma (LGG) and rectum adenocarcinoma (READ) using the GEPIA website. The LIMMA R package was adopted to screen the genes correlated with SIDT1. The clusterProfiler R package and enrichplot R package were applied to perform the KEGG pathway enrichment analysis.
Statistical analysis
Data are presented as the mean ± SEM. Statistical analyses were performed using SPSS v24.0 software (IBM Corporation, New York, USA) and GraphPad Prism 8.0. Variables were analyzed using the Chi-squared test, Fisher’s exact test, unpaired Student’s t test, and Mann-Whitney U test for comparisons between two groups. Kaplan-Meier curves and the log-rank test were applied for univariate survival analysis. Statistically significant variables were further analyzed by multivariate Cox regression analysis. For all statistical analyses, a p value less than 0.05 was considered to be statistically significant.