Tissues and cell lines

Tissue samples from 80 HER2-positive breast cancer patients receiving trastuzumab treatment, definition of trastuzumab resistance, and establishment of two trastuzumab-resistant cell lines BT474-Tr and SKBR3-Tr were shown in our previous studies (Wang et al. 2022; Wang et al. 2023b). Trastuzumab-resistant cells were cultured in DMEM medium with 15 μg/mL trastuzumab, and trastuzumab was removed for at least 1 week before the functional experiments.

Quantitative reverse-transcription PCR (qRT-PCR)

RNA extraction was conducted by using TRIzol reagent (Invitrogen, USA), and qRT-PCR was carried out using SYBR Premix Ex Taq Kit (Takara Bio) as per manufacturer’s instructions. The relative gene expression was determined by 2−ΔΔCt method. In addition, isolation of cytoplasmic and nuclear RNA was carried out using Ambion® PARIS™ Kit (Thermo Fisher Scientific, USA) as per manufacturer’s instructions. The primer sequences used in this study are listed below:

Circ-NPTN: Forward: 5′-CTTGGGAATTCTGGCTGAAA-3′, Reverse: 5′-TGAGAGCTGGAGGTGAGGTT-3′

Circ-MRAP2: Forward: 5′-TGATTGGATTTTGGGTTGGT-3′, Reverse: 5′-GATTGCTGGGAGGTTCTGTT-3′

Circ-HSPB11: Forward: 5′-ACAAAATGCTCTTGCATGATTT-3′, Reverse: 5′-CTGGGACTCCCGTTAGCTCT-3′

Circ-PPID: Forward: 5′-TGGTGTCCAGTCTTTTTAAACTTG-3′, Reverse: 5′-TTGAGCTCTGCGGTACAATG-3′

Circ-RFC3: Forward: 5′-AGCCTATTCACTTGCTGATGG-3′, Reverse: 5′-AGAAGCTCATACAGCCTTCCA-3′

Linear PPID: Forward: 5′-TGATCGGGAGGGTTTACTGA-3′, Reverse: 5′-TTACTTGGCCAAACACCACA-3′

HER2: Forward: 5′-AATTCCAGTGGCCATCAAAG-3′, Reverse: 5′-TCCCGGACATGGTCTAAGAG-3′

XPO4: Forward: 5′-CCATCTTCCCAGATGAAGGA-3′, Reverse: 5′-GTGGGAACACGGTTATCAGG-3′

U6: Forward: 5′-CTCGCTTCGGCAGCACA-3′, Reverse: 5′-AACGCTTCACGAATTTGCGT-3′

ACTB: Forward: 5′-TGACGTGGACATCCGCAAAG-3′, Reverse: 5′-CTGGAAGGTGGACAGCGAGG-3′

Lentiviral vector, oligonucleotides, and transfection

The full-length of circ-PPID was cloned into pLV-circ-Puro lentiviral vector, followed by infection into BT474-Tr and SKBR3-Tr cells. The stable clones were screened by puromycin. Overexpression of circ-PPID was verified by qRT-PCR. For circ-PPID knockdown, two antisense oligonucleotides (ASOs) targeting circ-PPID junction site were designed and transfected into cells. The knockdown efficiency was verified by qRT-PCR. Besides, the well-verified siRNAs against METTL3, METTL14, and NAT10 were obtained from Sigma-Aldrich. Cell transfection was performed using Lipofectamine 3000 (Invitrogen) according to manufacturer’s protocols. The sequences used in this study are as follows:

ASO-NC: 5′-GCGTATTATAGCCGATTAAC-3′

ASO-circ-PPID#1: 5′-GAGCTTAAGTTTGTAGCTCTT-3′

ASO-circ-PPID#2: 5′-GTTTGTAGCTCTTGAACTAGA-3′

si-NC: 5′-TTCTCCGAACGTGTCACGT-3′

si-METTL3: 5′-GCCTTAACATTGCCCACTGAT-3′

si-METTL14: 5′-CCATGTACTTACAAGCCGATA-3′

si-NAT10: 5′-GCAATTGTACACAGTGACTAT-3′

Establishment of XPO4-/- cell lines

CRISPR-Cas9-mediated gene editing was used to generate XPO4 knockout cell lines.

The verified sgRNA against XPO4 (5′-GCACTCCTCCTACGCTGGAT-3′) was synthesized and inserted into pSpCas9(BB)-GFP plasmid, followed by transfection into BT474-Tr and SKBR3-Tr cells using Lipofectamine 3000. After 24 h, the GFP-positive cells were isolated using fluorescence-activated cell sorting and cultured in the form of a single clone. Knockout of XPO4 was tested by western blot.

Western blot

Total protein was isolated and separated on SDS-PAGE gel, followed by transfer onto PVDF membrane and blocking using 5% defatted milk. After incubation with primary and secondary antibodies, the protein signaling on PVDF membrane was tested using SuperSignal West Atto reagent (Thermo Fisher Scientific). The antibodies used in this study are anti-HER2 (#2165, CST), anti-p-ERK1/2 (#9101, CST), anti-ERK (#11257-1-AP, proteintech), anti-p-Akt (#4060, CST), anti-AKT (#60203-2-Ig, proteintech), anti-GAPDH (#60004-1-Ig, proteintech), anti-NAT10 (#13365-1-AP, proteintech), and anti-XPO4 (#25373-1-AP, proteintech).

Detection of cell proliferation and death

Cell viability was tested by CCK-8 assay. Cells were treated with 10 μL CCK-8 (MedChemExpress, USA) reagent for 2 h at 37 °C, followed by detection of absorbance at 450 nm, with six replicates in each well. For colony formation, cells were seeded into six-well plates and cultured for 10 days, followed by staining with crystalline violet. Cell death was determined by propidium iodide staining and analyzed by flow cytometry.

Orthotopic transplantation tumor model

Establishment of orthotopic transplantation tumor model was described previously (Wang et al. 2023a). In brief, BT474-TR cells were injected into the abdominal mammary fat pad of NOD/SCID mice, followed by treatment with vehicle or trastuzumab (20 mg/kg, intraperitoneal administration) once a week when the tumor volume reached about 0.1~0.15 cm3. At the end of the experiment, tumor tissues were obtained and weighed. The animal study was approved by the Animal Care and Use Committee of Peihua University.

RNA immunoprecipitation (RIP) and acetylated RIP (acRIP)

RIP assay was carried out by using the RIP-kit (Bes5101, BersinBio) as per manufacturer’s protocols. Cell lysates were incubated with magnetic beads and anti-NAT10 antibody at 4 °C overnight, followed by digestion with proteinase K. Then, RNA was extracted for qRT-PCR. For acRIP assay, the Magna m6A kit (#17-10,499, Millipore) was used and the anti-m6A antibody was replaced with the anti-ac4C antibody (#ab252215, Abcam). Briefly, total RNA was isolated and randomly digested into 100~200bp, followed by incubated with magnetic beads and anti-ac4C antibody at 4 °C overnight. The anti-IgG antibody was used as the negative control for RIP and acRIP assays.

Luciferase reporter assay

The full-length of HER2 mRNA exon 25 was synthesized and inserted after F-Luc in pmirGLO vector, followed by transfection into BT474-Tr and SKBR3-Tr cells. After 48 h, the luciferase activity was tested by the dual-luciferase reporter system (Promega, USA).

Detection of co-localization between circ-PPID and NAT10

Cell lysates were collected and incubated with biotin-labeled circ-PPID probe and streptavidin magnetic beads at 4 °C overnight. The pull-down proteins were washed and eluted by Laemmli buffer, followed by western blot analysis for NAT10 expression. In addition, Cy3-conjugated circ-PPID probe was synthesized and hybridized with fixed cells at 37 °C overnight. Then, cells were immune-stained with anti-NAT10 antibody; DAPI was used to stain the nuclei. The co-localization was analyzed by ImageJ software.

Statistical analysis

Figures were generated by GraphPad Prism. Experiments were repeated at least three times. Data are presented as mean ± standard deviation. Statistical differences were analyzed by two-tailed Student’s t test or one-way ANOVA. P<0.05 was considered statistically significant.