Tissue specimens and ethical considerations

Ninety paraffin-embedded OS specimens and paired adjacent normal tissue samples were obtained from patients who underwent tumor resection at the Department of Orthopedics, Xiangya Hospital, Central South University. The patients’ demographic data, including sex, age, tumor histology, location, diameter, local recurrence, Enneking stage, metastasis, and prognosis, were collected from the hospital’s medical record management information system and used for statistical analysis. This study was approved by the Ethics Committee of Xiangya Hospital, Central South University with ethics approval number 2,021,101,013, and informed consent was obtained from the patients or their legal guardians.

TCGA data acquisition and processing

The sequencing data and clinical data of 84 OS patients in TARGET-OS cohort were downloaded from The Cancer Genome Atlas Program (TCGA) website (https://www.cancer.gov/ccg/research/genome-sequencing/tcga). Patients were separated into high- and low-risk group based on the median mRNA expression and survival curves were generated using the Kaplan–Meier method.

Cell lines, reagents and transfection

The human osteoblastic cell line hFOB1.19 and OS cell lines MG63 and U2OS were purchased from Pricella Company (Wu Han, China). Under the circumstance of 5% CO2 and 37 °C, MG63 and U2OS cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Biological Industries, Israel) while hFOB1.19 cells were maintained in DMEM/F12 medium (Pricella, Wu Han, China). Antibiotics and 10% fetal bovine serum (Gibco, USA) were added in the medium.

These antibodies used in the experiments were as follows: anti-CYFIP1 (ab108220), anti-RNMT (ab254842), anti-E-Cadherin (ab40772), anti-N-Cadherin (ab76011), anti-Caspase3 (ab32351), anti-FDX1 (ab108257), anti-Ki67 (ab15580), anti-CCND1 (ab16663), anti-DYKDDDDK (66008-4-ig, proteintech, China), anti-AURKAIP1 (821937, zenbio, China). Copper chelator, Cuprizone, (370-81-0, MCE) was purchased from MedChemExpress company.

CYFIP1-overexpressed lentivirus and negative control were purchased from Genechem Company (Shanghai, China). siRNAs and shRNAs were synthesized in General Biosystems Company (Nanjing, China). CYFIP1 stably expressed OS cells were infected with the lentivirus and selected with puromycin (1.2 mg/ml) for 4 weeks. Lipofectamine3000 (Invitrogen) was used to transfecte RNAs into tumor cells. The sequences are listed in Supplementary Table S1.

Immunohistochemistry (IHC) and immunocytochemistry (ICC)

Tissue slides and cell slides were incubated with the corresponding primary and secondary antibodies, details of which were implemented as described in our previous studies(Yuan et al. 2022).

RNA extraction and quantitative polymerase chain reaction (qPCR)

The total RNA was extracted using the AG RNAex Pro RNA extraction kit (AG, Changsha, China) and utilized to synthesize cDNA with the Reverse Transcription kit (AG, Changsha, China). qPCR analysis was carried out on the ABI7500 system using the TBGreen Premix Pro Taq HS Qpcr Kit (AG, Changsha, China). Lastly, we utilized the ΔΔCq method to calculate the relative expression levels of each sample and the results were expressed as 2−ΔΔCq. The primer sequences used for all qPCR experiments are listed in Supplementary Table S2.

Western blotting

Total proteins were extracted from different kinds of cell lysates, separated by 10% SDS polyacrylamide gel electrophoresis, and then transferred onto the polyvinylidene membrane. Incubating in 5% defatted milk, the membranes were incubated overnight with primary antibodies at 4℃ and were then incubated with secondary antibodies for 1 h. Finally, signals of the blots were visualized using an enhanced ECL kit (BL520A, Biosharp, China).

Cell proliferation and colony formation

CCK-8 kit (Beyotime, China) and EdU kit (RiboBio, China) were employed to assess cell proliferation. Around 5000 cells/well and 1.2 × 104 cells/well were seeded in 96-well plates, respectively. The kits’ working solutions were added as per the manufacturer’s instructions. Cell proliferation was measured using a multifunctional enzyme labeling instrument and fluorescence microscope. For colony formation assay, 1000 cells were seeded in a well of 6-well plates and cultured for 2 weeks.

Transwell migration and scratch wound-healing assay

For transwell assay, 3 × 104 cells in 200 µL serum-free medium were added to upper chambers (24-well insert, Corning, USA). Bottom chambers contained 800 µL complete medium. After 24 h incubation, cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. Migrating cells in the bottom chamber were counted in five random fields under a microscope after scraping off cells in the upper chamber. In the scratch wound-healing assay, 3 × 105 cells were seeded in 6-well plates. When cells reached 90% confluence, a scratch was made using a 1 ml tip head. After 24 h, images were captured using an inverted microscope.

Flow cytometry

For cell apoptosis assay, cells were digested with EDTA-free trypsin, washed twice with pre-cooled PBS, and stained using an apoptosis detection kit (#FXP023, 4 A BIOTECH, China). Subsequently, the stained samples were analyzed by flow cytometry after incubation at room temperature in the dark for 10 min.

For reactive oxygen species (ROS) analysis, cells were seeded in 6-well plates and were stained with DHE (US Everbright, Suzhou) diluted with Hank’s solution (ratio 1:1000) in an incubator at 37 °C for 20 min. Then, cells were digested with trypsin and washed twice with pre-cooled PBS. Finally, the stained samples were detected by flow cytometry.

For mitochondrial membrane potential (MMP), cells were seeded in 6-well plates and were stained with TMRM (US Everbright, Suzhou) diluted with Hank’s solution (ratio 1:2000) in an incubator at 37 °C for 20 min. Then, cells were digested with trypsin and washed twice with pre-cooled PBS. Finally, the stained samples were detected by flow cytometry.

RNA stability assay

OS cells were seeded in 6-well plates overnight, and then treated with 5 µg/mL actinomycin D (#S8964, Selleck, USA) at the 0, 3, 6 h. Total RNA was then extracted using TRIzol reagent (AG, Changsha, China) and analyzed by qPCR. Eventually, the linear regression analysis was utilized to estimate the mRNA half-lives time.

RNA-sequencing

The total RNA was extracted from CYFIP1 stably expressed U2OS cells and the corresponding control cells using TRIzol reagent (AG, Changsha, China) according to the manufacturer’s protocol. The RNA sample quantification, qualification, library preparation, transcriptome sequencing and subsequent analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). Differential expression analysis was performed using the DESeq2(Love et al. 2014). Q value < 0.05 and foldchange > 2 or foldchange < 0.5 was set as the threshold for significantly differential expression gene (DEGs).

Mass spectrum and CO-IP

Cell lysis was performed using Pierce™ IP Lysis Buffer (XG351119, Thermo scientific, USA) according to the manufacturer’s instructions. Next, the supernatant was extracted by high-speed centrifugation, incubated with 5 µg of specific antibodies against CYFIP1 and rotated with magnetic beads overnight at 4 °C. For mass spectrum, the protein-beads complexes derived from U2OS cell lysis were washed 3 times and proteins were eluted by 20ul glycine-HCL solution. The mass spectrum and subsequent data analysis were constructed by Novogene Corporation (Beijing, China). For CO-IP, protein-beads complexes mixed with loading buffer were incubated in 100℃ 5 min and the supernatant was extracted by high-speed centrifugation for western blotting.

RNA immunoprecipitation (RIP)-sequencing or qPCR

RIP was conducted with the RNA immunoprecipitation Kit (BersinBio, Guangzhou, China) according to the manufacturer’s instructions. In brief, 5 µg of specific antibodies against rabbit immunoglobulin G, CYFIP1 or RNMT were incubated with prepared CYFIP1 stably expressed U2OS cells and the corresponding control cells lysates overnight at 4 °C and rotated with magnetic beads 1 h at the second day. Subsequently, the RNA-protein-beads complexes were washed 5 times and eluted by elution fluid mixed with proteinase K. Finally, phenol-chloroform-isopentanol mixture was utilized for RNA extraction. The sequencing library and subsequent data analysis were constructed by Novogene Corporation (Beijing, China). The relative interaction between CYFIP1-RNMT complexes and their targeted transcripts were validated by qPCR and normalized to the input.

m7G meRIP-sequencing or qPCR

MeRIP experiment and high through-put sequencing and data analysis were conducted by Seqhealth Technology Co., LTD (Wuhan, China). Total RNAs were extracted from CYFIP1 stably expressed U2OS cells and the corresponding control cells and then undergone DNA digestion, quality testing, quantification. 50 µg total RNAs were used for polyadenylated RNA enrichment by VAHTS mRNA Capture Beads (VAHTS, cat. NO. N401-01/02). 20mM ZnCl2 was added to mRNA and incubated at 95℃ for 5–10 min until the RNA fragments were mainly distributed in 100-200nt. Then 10% RNA fragments was saved as “Input” and the rest was used for m7G immunoprecipitation. The specific anti-m7G antibody (#RN017M, MBL) was applied for m7G immunoprecipitation. RNA samples of both input and IP were prepared using TRIzol reagent (Invitrogen, cat. NO 15596026). After the stranded RNA sequencing using Illumina® platform, raw sequencing data undergone quality control, mapping, peak annotation, and subsequent data analysis. The m7G-modified transcripts were validated by qPCR and normalized to the input.

Mitochondrial proteomics

Mitochondrion were extracted using the mitochondrial extraction kit (SM0020, Solarbio, Beijing). Subsequently, mitochondrial proteins were extracted from cell lysates, quantified using BCA method, and separated by 12% SDS polyacrylamide gel electrophoresis. Then, the obtained proteins were undergone enzymolysis, label, enrichment, and Mass spectrometry analysis. Spectronaut (Version 15.3.210906.50606) was used to search all of the raw data thoroughly against the sample protein database. Database search was performed with Trypsin digestion specificity. Alkylation on cysteine was considered as fixed modifications in the database searching. Protein, peptide and PSM’s false discovery rate (FDR) all set to 0.01. For DIA data, the quantification FDR also set to 0.05. Quantity MS-level was set at MS2. The mass spectrum and subsequent data analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).

Xenograft mouse models

The animal experiments in this study obtained approval from the Animal Care and Ethics Committee of Xiangya Hospital of Central South University (Changsha, China). The tumor in mice did not exceed 20 cubic mm and any dimension was less than 20 mm in diameter, which met the animal ethical standards of Xiangya Hospital. In brief, four- to six-week-old female nude mice were divided into two group containing six mice, of which one group was injected CYFIP1 stably expressed U2OS cells while other group was injected control U2OS cells. Subsequently, the tumor volume of mice was measured every 5 days, and tumors were harvested after 20 days, with their weights measured.

Statistical analysis

GraphPad Prism 8 were used for data processing and statistical analysis of all the experimental results. The quantitative data were expressed as the mean ± SD and tested by student’s t-test was while the qualitative data expressed in frequency were analyzed by Chi-square test or Fisher’s exact test. The indicated P value (* P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) was considered statistically significant.

Table 1 Correlation of CYFIP1 expression with clinicopathological features of OS patientsFig. 1

CYFIP1 expression in osteosarcoma and its relationship to patient survival. A, B. Low CYFIP1 expression was detected in OS tissues while high CYFIP1 expression in their adjacent tissues. C. IHC staining showed the representative images of low or high expression of CYFIP1. D. Kaplan-Meier survival curve exhibited that low CYFIP1 expression was associated with the worse survival statuses in Xiangya hospital (Log-rank p = 0.0011, HR = 0.377, n[high] = 42, n[low] = 48 ). E. Kaplan-Meier survival curve exhibited that low CYFIP1 expression was associated with the worse survival statuses in TARGET-OS cohort (Log-rank p = 0.0006, HR = 0.261, n = 42 per group). The data represent the mean ± S.D. *P < 0.05 or **P < 0.01 indicates a significant difference between the indicated groups

Fig. 2

Overexpression of CYFIP1 inhibited the proliferation and metastasis while promoted the apoptosis of osteosarcoma cells in vitro. A, B. WB and qPCR exhibited the low expression of CYFIP1 in OS cells. C-F. WB and qPCR exhibited the overexpression efficiency of lentivirus transfection. G. Overexpression of CYFIP1 facilitated osteosarcoma cells apoptosis. H-K. CCK8. EdU and colony formation assays exhibited overexpression of CYFIP1 inhibited osteosarcoma cells proliferation. L, M. Overexpression of CYFIP1 inhibited osteosarcoma cells migration. The data represent the mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicates a significant difference between the indicated groups