Patient samples and cell lines
Breast cancer and para-cancerous tissues of 48 patients were collected from the Department of Breast and Thyroid Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from June 2019 to December 2020. After careful examination of the tissues by three pathologists, the patients were diagnosed with breast cancer. Patients who received any treatment (including surgery, chemotherapy, immunotherapy, etc.) before the surgery were excluded from the study. Informed consent was signed by all patients. The study was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. The study was in accordance with the Declaration of Helsinki.
The cell lines used in the study included human normal breast cell line MCF-10A, human breast cancer cell line MCF-7, MDA-MB-231, SKBR3, T47D and BT-549. MCF-10A, MCF-7, MD-MB-231, T47D and BT-549 were purchased from American Type Culture Collection (ATCC). SKBR3 was purchased from China Center for Type Culture Collection (CCTCC). Cells except for MCF-10A were cultured in DMEM medium supplemented with 10% FBS (Gibco, US) and antibiotics (Penicillin 100 U/ml, Streptomycin 100 mg/ml) (Gibco, US). For MCF-10A, the culture conditions were: mixture of DMEM and F12 medium (1:1) (Gibco, US), antibiotics (Pen/Strep), 5% horse serum (Ausbian, AUS), insulin (10 µg/ml) (Thermo Fisher Scientific, US), epidermal growth factor (20 ng/ml) (Gibco, US), choleramycin (100 ng/ml) (Sigma, US) and hydrocortisone (0.5 µg/ml) (Tocris, US). Cells were cultured at 37 ℃ with 5% CO2. Cells were cultured for up to 20 passages.
Cell proliferation assay
Cell proliferation was measured by CCK-8 (Cell Counting Kit-8, Dojindo, Japan) and EdU assays. For the CCK-8 assay, 1 * 103 cells were seeded and cultured in 96-well plates for 24 h, 48 h, 72 h, 96 h and 120 h; 10 µl CCK-8 solution was added to each well. After adding the solution, the culture was continued for 4 h, and the absorbance was measured every 30 min using a microplate reader at 450 nm.
EdU proliferation assay was carried out using Click-iT Plus EdU Alexa Fluor 594 flow cytometry assay kit (Thermo Fisher Scientific, Catalog No. C10646) according to the manufacturer's instructions. Images were captured by the confocal microscope (Olympus, Japan) at ten randomized fields.
Cell cycle assay
2 * 105 cells were seeded in a 12-well plate for 48 h, and then cell cycle assays were performed. The procedures are briefly described: cells were collected by centrifugation at 8500 rpm for 5 min, and 70% cold ethanol was added to the cells dropwise. Cells were then fixed at 4 °C for 30 min. Next, wash the cells with 1× Phosphate Buffer Saline (PBS) twice. After staining the cells with propidium iodide (PI) antibody for 30 min, the cell cycle was measured by flow cytometry.
Cell invasion assay
Breast cancer cell invasive ability was measured by transwell insert chambers (Corning, NY, USA) pre-coated with Matrigel (Corning Inc.). In brief, 2 * 104 cells were seeded into the upper chamber containing 200 µl serum-free medium, and 500 μl culture medium with 20% FBS was added into the bottom chamber. After 24 h, invading cells were fixed with 4% paraformal-dehyde and stained with 0.1% crystal violet. Cells were counted using a microscope for 5 fields randomly at 100× magnification.
Immunohistochemistry and HE staining
Hematoxylin and eosin (HE) staining was conducted following the standard protocol. For immunohistochemistry staining, tissue slides (6 μm) were deparaffinized and hydrated. Antigen retrieval was performed using citrate solution (PH 6), and the tissues were then blocked with 2% BSA for 2 h at room temperature. Next, tissues were incubated overnight with primary antibody (anti-YTHDF1: Abcam, catalog No. 230330) at 4 °C. Subsequently, tissues were incubated with HRP-conjugated antibody for at least 1 h in the darkroom at room temperature. Images were captured with the microscope (Leitz, Italy), and the results were analyzed using ImageJ software (version 1.8.0, NIH, US). The IRS (immunoreactive score) scoring system to determine immunohistochemical positivity and the protein expression levels. IRS = SI (Staining Index) * PP (Proportion of Positive tumor cells). SI ranges from 0 to 3 and PP from 0 to 4. IRS score ranges from 0 to 12. For protein expression, IRS 0–1: no expression, 2–3: low expression, 4–8: moderate expression, 9–12: high expression [22]. The mean integrated optical density (IOD) values of YTHDF1 and FOXM1 in tumor tissues were calculated by Image Pro-Plus 6.0 software (MEDIA CYBERNETICS, US).
RNA immunoprecipitation (RIP)
RIP was carried out to detect mRNAs that bind to the YTHDF1 protein. The RIP was conducted using a Megna RNA-binding protein kit (Millipore, US). 10 µl YTHDF1 or IgG antibody (Cell signaling Technology, US) was incubated with protein A/G magnetic beads for at least 4 h at 4 °C. After lysing the cells with RIP buffer, the cells were incubated with the washed magnetic beads at 4 °C overnight. Then RNA was extracted by Trizol reagent (Invitrogen, US), and the expression of the target genes was detected by RT-qPCR.
Cross-linking immunoprecipitation (CLIP)
The CLIP assay was conducted according to the protocol as previously described [23]. Briefly, 2 * 107 MCF-7 cells were irradiated with the UV crosslinker (Thermo Fisher Scientific, US) (wavelength 260 nm). The cells were then lysed with the RIPA buffer and co-incubated with protein A/G magnetic beads at 4 °C for at least 4 h. The immune complexes were then eluted at 60 °C for 20 min with a 50 mM Tris–HCl solution (pH 7.8). To isolate the RNA from the immune complexes, we use chloroform to extract RNA. Finally, RT-qPCR was used to detect and analyze the expression level of the target genes.
m6A immunoprecipitation (m6A-IP)
1.5 µg of IgG or m6A antibody was conjugated to protein A/G beads at 4 °C overnight. Next, incubate 300 µg fragmented RNA with the IgG or m6A antibody in the immunoprecipitation buffer containing RNase inhibitor at 4 °C overnight. RNA was eluted from the beads using elution buffer and was extracted for RT-qPCR by phenol and ethanol.
Polysome profiling
Polysome profiling was conducted according to the protocols described previously [24]. After treated with 0.1 mg/ml cycloheximide for 10 min, cells were washed with cold PBS and lysed with polysome lysis buffer (10 mM Tris–HCl, pH 7.4; 10 mM MgCl2; 0.3 mM NaCl; 1% Triton X-100; RNase inhibitor and 100 µg/ml cycloheximide). Cell lysates were centrifuged at 2000 rpm for 5 min and 12,000 rpm for 15 min. RNA concentration was detected by Nanodrop (Thermo Fisher Scientific, US). 500 µg RNA was then added in freshly prepared sucrose gradient solutions (10–50%) and centrifuged at 30,000 rpm for 4 h at 4 °C. 1 ml fractions were collected. Total RNA was extracted by RNeasy Mini Kit (QIAGEN, US) for qRT-PCR analysis.
Lentivirus production and cell transduction
HEK-293 T cells were used to produce lentivirus particles. Briefly, pLKO.1 puro-sh-YTHDF1#1/sh-YTHDF1#2 or pLKO.1 puro control vector, helper vector pxPAX2 and envelope vector pMD2.G were transfected into HEK-293 T cells using Lipofectamine 2000 (Thermo Fisher Scientific, US). Supernatants containing lentivirus particles were collected and filtered after 48 h transfection. 1 * 104 cells were incubated with 1 ml supernatants for 24 h and were selected using 4 μg/ml puromycin. 1 μg/μl puromycin was used as a selective pressure to get stably transfected cell lines. sh-YTHDF1#1 and sh-YTHDF1#2 were synthesized by GenePharma (CN). Oligo sequence of sh-YTHDF1#1: 5′-CGGTGGGACAAATGTGAACAT-3′; sh-YTHDF1#2: 5′-CCCGAAAGAGTTTGAGTGGAA-3′; sh-YTHDF1-3: 5′-GTTCGTTACATCAGAAGGATA-3′. sh-FOXM1#1: 5′-GCTGGGATCAAGATTATTA-3′; sh-FOXM1#2: 5′-GGCTGCACTATCAACAATA-3′. Scramble shRNA was purchased from Addgene (#1864, US).
Cell transfection
Transfection of plasmids was performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. YTHDF1-WT (Addgene #70087), YTHDF1-MUT, FOXM1-WT (Addgene #68811, FOXM1 isoform 3) and FOXM1-MUT, CCNB1 (Addgene #39871) overexpression plasmids were used for transfection. Detailed information on the mutations was shown in Additional file 2: Fig. S2. The sequences of siRNAs targeting YTHDF1 (si-YTHDF1) were as follows: siYTHDF1-1: 5′-GAACAAAAGGACAAGAUAAUA-3′, si-YTHDF1-2: 5′-CAAAAGGACAAGAUAAUAAAG-3′.
RT-qPCR
Total RNA was extracted from cells using TRIzol Reagent (Life Technologies) according to the manufacturer's instructions. RNA was transcribed into cDNA using the Reverse Transcription Kit (Takara, JP). RT-qPCR was conducted by SYBR Green Master Mix (#4309155, Applied Biosystems, US). The procedure was set as follows: Hold 95 °C for 10 min; Denature 95 °C 15 s and Anneal/Extend 60 °C 1 min for 40 cycles. Primers were listed in Additional file 5: Table S1.
Western blot and antibodies
Cells were washed with cold Phosphate Buffered Saline (PBS, Thermo Fisher Scientific, US) twice before protein extraction. Total proteins were extracted from cells using RIPA buffer (Sigma Aldrich, US) supplemented with the protease inhibitor cocktail (Roche, Shanghai, CN) according to the manufacturer's instructions. 25–30 μg proteins were loaded onto the 4–12% SDS-PAGE (BeyoGel, CN). Electrophoresis was performed at 120 V for 1 h. Proteins were then transferred to the PVDF membranes at 250 mAh for 2 h. After blocking with 5% non-fat milk for 30 min, membranes were incubated with primary antibodies overnight at 4 °C. Western blot images were captured by a myECL imager (Thermo Fisher Scientific, US). Primary antibodies used in the present study were listed as follows: FOXM1 (A2493, ABclonal), YTHDF1 (17479-1-AP, Proteintech), ZEB1 (21544-1-AP, Proteintech), N-cadherin (22018-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), Vimentin (10366-1-AP, Proteintech), Snail (ab216347, Abcam), β-Actin (ab8226, Abcam), anti-Flag tag (SAB4301135, Sigma-Aldrich), anti-HA-tag (05-904, Sigma-Aldrich).
In vivo assays
Animal Experiments were conducted under the approval of the ethics committee of Tongji Medical College, Huazhong University of Science and Technology (Protocol #TJU 2018-0025). NOD/SCID immune-deficient mice were purchased from Shanghai Experimental Animal Center. 2 * 106 MCF-7 cells transduced with sh-NC or sh-YTHDF1-were subcutaneously injected into the mice (5/group). Tumor width and length were measured every 7 days. Tumor volume = (length * width2)/2. After 7 weeks, mice were sacrificed, and the weight of tumors was detected. Xenografts were collected for HE staining, immunohistochemistry staining and western blot analysis.
For spontaneous lung metastasis assay, 4 * 106 sh-NC or sh-YTHDF1#2 transduced MCF-7 cells were injected into the mammary fat pads of the NOD/SCID mice (5/group). The primary tumor was removed when its volume reached 150 mm3. The mice were sacrificed, and lung metastasis nodules were counted 12 weeks after the removal.
Statistical analysis
Data were expressed as mean ± standard derivation (SD). A T-test was used for comparison between two groups. A one-way ANOVA test followed by an SNK test was used for comparison between three groups. Kaplan–Meier curve was used for survival analysis. All data were analyzed using GraphPad Prism 9.0 (GraphPad Software, US). Statistical significance is considered as *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were three independent repetitions.