Cell lines and cell culture
The human breast cancer cell lines MCF-7, ZR-75-1, BT474, SK-BR-3, MDA-MB-453, HCC-1806, MDA-MB-231, BT549 and breast epithelial cell line MCF-10A were purchased from American Type Culture Collection (ATCC, USA). Among them, MCF-7 and ZR-75-1 are considered luminal subtype. BT474 and SK-BR-3 represent the HER2-positive subtype, MDA-MB-453, HCC-1806, MDA-MB-231 and BT549 belong to the TNBC subtypes.
The cell lines were cultured in DMEM (Wisent, China), in addition, HCC-1806 were cultured in RPMI-1640 (Wisent, China), which were all supplemented with 10% FBS and penicillin-streptomycin (100 μg/ml). The cells were incubated at 37°C in a humidified atmosphere with 5% CO2.
Lentivirus and siRNA transfection
Lentivirus were generated to knockdown and overexpress IGF2BP3. The MDA-MB-231 and HCC-1806 cells were infected with IGF2BP3 knockdown (termed as shIGF2BP3-1 and shIGF2BP3-2) lentivirus and matched negative control vectors (termed as shRNA-NC), IGF2BP3 overexpress (termed as IGF2BP3) lentivirus and matched negative control vectors (termed as Vector), following the manufacturer's protocol (Obio Technology, China). We finally selected the stable cell lines with 3 μg/ml puromycin. Mechanistic studies used shIGF2BP3-1 structure with the higher efficiency named shIGF2BP3
The MDA-MB-231 and HCC-1806 cells were transfected with TET1-siRNA, TET2-siRNA, TET3-siRNA, NF1-siRNA (GenePharma, Shanghai, China) and non-silencing control. The IGF2BP3 negative control and knockdown TNBC cells MDA-MB-231 and HCC-1806 were transfected with NF1 siRNA and negative control vectors (shRNA-NC+Vector, shIGF2BP3+Vector, shRNA-NC+siNF1, shIGF2BP3+ siNF1). (GenePharma, China). The sequences of lentivirus and siRNAs were listed in Table S1.
Total RNA was extracted from clinical specimens or breasr cancer cells using Trizol reagent (Takara, Japan). RNA was used to reverse transcription with PrimescriptTM reverse transcription reagent (Takara, RR036A, Japan) following the manufacturer's protocol. qRT-PCR was performed with the Roche LightCycler 480 RT-PCR system (Roche, Switzerland). The relative RNA expression level was calculated by 2-ΔΔCt method and with the level normalized to β-actin. The specific PCR primers were purchased from Invitrogen Trading (Shanghai, China) and listed in supplementary Table S2.
Western blot analysis
Western blot was performed as previously described. Briefly, cells were ruptured with RIPA (P0013C, Beyotime, China). Whole cell lysates were fractionated and transferred to a PVDF membrane (Millipore, USA). The primary antibodies were anti-rabbit IGF2BP3 (14642-1-AP, proteintech, China), caspase3 (19677-1-AP, proteintech, China), caspase9 (10380-1-AP, proteintech, China), NF1 (27249-1-AP, proteintech, China), anti-mouse β-actin (66009-1-Ig, proteintech, China). The dilutions of antibodies were according to the product usage information.
Cell viability was detected using the CCK8 kit (Vazyme, Chian) as previously described. Briefly, cells were plated in a 96-well plate at 2000 cells/well. The absorbance was measured by OD450 for 2 h after adding 10% CCK8 solution.
Colony formation assay
Colony-formation assay was conducted as previously described. Cells were maintained in 6-well plates at 5×103 cells/well. After two weeks of culture, cells were stained with 0.1% crystal violet(C0121, Beyotime, China). The number of colonies was counted.
Following the manufacturer's instructions, EdU assays were performed using an EdU Cell Proliferation Kit (C0075, Beyotime, China). Briefly, cells were maintained in a 96-well plate at 2×104 cells/well and incubated with 50 μM EdU per well at 37°C for 2 h. Then cells were fixed with 4% paraformaldehyde and stained with 1× Click Reaction Buffer and 1× Hoechst 33342 solution. In the end, the cells were observed under the Zeiss fluorescence photomicroscope.
Flow cytometry analysis
The apoptosis rate of cells was probed by Annexin V-APC/7-AAD apoptosis kit (MultiSciences Biotech, Hangzhou, China). Cells in different treatment groups were cultured for 24 h, and obtained 3 × 105 cells (including culture supernatant cells). Then resuspending cells with 300 μl 1×Binding Buffer, each tube add 10 μl 7-AAD and 5 μl annexin V-APC. After vortexing gently and incubating in the dark for 15 minutes, samples were analyzed by FCM and the BDFACSCalibur™ system.
Balb/c nude mice (4-6 weeks) were randomly divided into 4 groups (8 mice per group). Stable shRNA-NC, shIGF2BP3, shRNA-NC+siNF1 and shIGF2BP3+siNF1 MDA-MB-231 cells (1 × 107 cells) were injected into nude mice. Tumor volumes were measured every 4 days [volume = 0.5×length×(width)2]. Animal experiments were carried out following the ethical standards of experimental animal institutions approved by the Animal Management Committee of Nanjing Medical University.
Primary tissue samples
Twenty-seven pairs of TNBC and matched adjacent regions were provided by the First Affiliated Hospital with Nanjing Medical University from 2015 to 2016, in according with the ethical guidelines of the Declaration of Helsinki and approved by the ethics and research committee of the First Affiliated Hospital of Nanjing Medical University.
The cells were extracted according to the instructions of QIAmp DNA Mini Kit (Qiagen). After diluting 1 μL of DNA sample by 50 times, the DNA was converted to unmethylated cytosine bisulfite according to the instructions of the Epi Tect Bisulfite Kit (Qiagen). After the bisulfite modification, the DNA was eluted and purified and MS-PCR was then performed. Methylation specific primer sequences were shown in Table 2.
mRNA high-throughput sequencing
According to the manual's protocol, Trizol reagent (Takara, Japan) was used to isolate total RNA from stably IGF2BP3 knockdown or control MDA-MB-231 cells. After the samples were qualified, 3 μg RNA was selected, and the Small RNA Sample Pre Kit was used to construct the library. The filtered sequences were then compared to the reference genome (hg38). The library establishment and next generation sequencing (NGS) were performed by Beijing Allwegene (Beijing, China). Each group was sequenced in 3 replicates.
The RIP assay was performed using RNA immunoprecipitation lysis buffer (Millipore, USA). According to the manufacturer's instructions, MDA-MB-231 cell lysate was incubated with 5 µg of anti-rabbit IGF2BP3 (14642-1-AP, proteintech, China) or IgG at 4°C overnight. The RNA-protein complexes were adsorbed by protein A/G magnetic beads, and then performed RNA purification.
According to the manufacturer's protocol, index-encoded samples were clustered on the cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumia). Then the library was sequenced using the PE 150 sequencing strategy on the Illumina Hiseq 4000 platform of Beijing Allwegene (Beijing, China).
Total RNAs were isolated from MDA-MB-231 cells. After fragmentation, cell lysate was incubated with m6A antibody (ab208577, Abcam, US) for immunoprecipitation according to the protocol of the MeRIP m6A kit (Millipore, Germany). The RNA fragments are first ligated at the 3' end before the chemical reaction. They are then reverse transcribed before the 3' adapters are ligated to the final cDNA. During reverse transcription, only fragments containing internal m6A sites are misincorporated, which can be further detected by high-throughput sequencing. The library establishment and NGS were performed by Beijing Allwegene (Beijing, China).
mRNA stability analysis
IGF2BP3 knockdown and overexpression MDA-MB-231 and HCC-1806 cell lines were plated into 6-well plates and then treated with actinomycin D (ActD) 5 μg/ml at 0 h, 2 h, 4 h and 6 h before collection. Total RNA was isolated to quantify the relative NF1 mRNA levels.
The MDA-MB-231 and HCC-1806 cells were transfected with negative pGL3 reporter and luciferase vector (NF1-A, NF1-B, NF1-C, NF1-D, NF1-E, NF1-B-mut, NF1-C-mut). Firefly and renilla luciferase activities were tested by the Dual-Luciferase reporter assay system (Promega, USA). The primers for vectors are listed in Table S3.
The data were analyzed using the SPSS 19.0 software. All experiments were performed in triplicate unless otherwise stated. The linear correlation analysis evaluated the correlation between IGF2BP3 and NF1. For all the continuous variables, Student t-test and two-way ANOVA were used for comparing differences between groups. *P<0.05 was considered as statistically significant.