Clinical samples

Tissue microarray (TMA) comprising 117 samples from OV cases was purchased from Wuhan Servicebio Technology Co., Ltd., and 47 OV samples were collected from patients who underwent cisplatin and paclitaxel chemotherapy at Shanghai Sixth People’s Hospital. All patients provided informed consent before sampling. This study was approved by the Research Ethics Committee of Shanghai Sixth People’s Hospital, affiliated with Shanghai Jiao Tong University School of Medicine.

Cell culture

Cisplatin-resistant A2780/DDP cells, human OV cell lines A2780, and OVCAR8 were preserved in the State Key Laboratory of Oncogenes and related genes, Shanghai Cancer Institute. The cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and 100 µg/mL penicillin/streptomycin (P/S) at 37 °C in a 5% CO2 atmosphere. A 10 mM Cisplatin (HY-17,394, MCE) was dissolved in sterile ddH2O and added to the culture medium when required.

Cell transfection

Short hairpin RNAs targeting HOXB2 and a randomized sequence were constructed into a GV112 vector (Genechem). Cells were infected with 1 × 106 recombinant lentivirus-transducing units and 5 µg/mL polybrene. After 72 h, 5 µg/mL puromycin was applied to exclude uninfected cells. The sequences were as follows:

shNC: TTCTCCGAACGTGTCACGT.

shHOXB2-1: GCTCATGATCTGGACGTGAAA.

shHOXB2-2: CCACGTCAAGATTTCTGATTT.

Small interfering RNAs (siRNAs) were also transduced into cancer cells by jetPRIME transfection reagent (Polyplus, France) to knock down HOXB2. The sequences were as follows:

siNC: sense 5’-UUCUCCGAACGUGUCACGUTT-3’.

antisense 5’-ACGUGACACGUUCGGAGATT-3’.

siHOXB2-1: sense 5’-GGCAGGUCAAAGUCUGGUUTT-3’.

antisense 5’-AACCAGACUUUGACCUGCCTT- 3’.

siHOXB2-2: sense 5’-GCCUUUAGCCGUUCGCUUATT-3’.

antisense 5’-UAAGCGAACGGCUAAAGGCTT-3’.

For DANCR overexpression, full-length human DANCR was cloned into a GV712 vector (Genechem), which was then transfected into cells using a jetPRIME reagent.

Half maximal inhibitory concentration (IC50) examination

A2780/DDP and OV8 cells were seeded and grown for 24 h in 96-well plates (5 × 103 cells/well). The original culture medium was replaced with different concentrations of the cisplatin solution. After 48 h of incubation, cell viability was determined using CCK-8, and the drug IC50 was calculated using GraphPad Prism 8.0.

Cell proliferation assay and plate colony formation assay

Different groups of cells were transferred to 96-well plates at a density of 1000 cells/well. Relative cell viability was detected at OD450nm after incubation with cell counting kit-8 (CCK-8, B34304, Bimake) for 1 h for the next five days.

For the colony formation assay, thousand cells from each cell group were seeded in 6-well plates and grown with cisplatin (10 µM for A2780/DDP and 5 µM for OV8) or equal PBS for two weeks. The clones were then fixed with 4% paraformaldehyde and stained with crystal violet.

Immunofluorescence and EdU assay

Cells from different groups were seeded in 15 µ slide 8-well plates (80,826, Ibidi). After treatment, cells were fixed with 4% paraformaldehyde, perforated with 0.1% Triton-X for 10 min, and blocked with 5% BSA for 30 min. Next, we incubated cell samples with anti-γH2A.X (1:100, GB111841, Servicebio) at 4 °C overnight and in the corresponding FITC-conjugated secondary antibody at room temperature for 1 h. Nuclei were then stained with DAPI for 10 min. A Confocal microscopy system was used for the image acquisition.

The EdU incorporation assay (KTA2030, Abbkine) was conducted following manual instructions to detect cell viability. Briefly, cells were co-cultured with 50 µM EdU for 2 h before being fixed and permeabilized. After the click reaction, cells were counterstained with DAPI and imaged by Confocal microscopy system. Likewise, cells were co-cultured with 50 µM EdU for 2 h and then be suspended, fixed, and peameabilized. After following click reaction, we used flow cytometry to quantify EdU-positive cells.

Cell apoptosis assay

Cell apoptosis rates were quantified by flow cytometry using the Annexin V-FITC/PI apoptosis kit (E-CK-A211, Elabscience) according to the protocol after 24–48 h of cisplatin treatment (20 µM for A2780/DDP, and 10 µM for OV8).

Protein extraction and western blotting

Cell lysates were extracted using RIPA lysis buffer (G2033, Servicebio). Total cell protein was normalized using the BCA method (23,227, Thermo Fisher) and separated using 8–12% SDS-PAGE gel electrophoresis before being transferred onto a nitrocellulose membrane. After blocking with 5% milk for 1 h, the membrane was incubated with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature. Antibodies used were as follows: anti-HOXB2 (1:1000, AY2582, Abways), anti-Beta Actin (1:5000, AB0035, Abways), anti-Beta tubulin (1:5000, 10086-1-AP, Proteintech), anti-γH2A.X (1:1000, GB111841, Servicebio), anti-Chk2 (1:10000, CY5633, Abways), anti-phospho Chk2 Thr68 (CY8878, Abways), anti-Chk1 (1:1000, 2360, Cell Signaling Technology), anti-phospho Chk1 Ser345 ((1:1000, 2348, Cell Signaling Technology), anti-ABCA1 (1:1000, ab66217, Abcam), anti-ABCG1 (1:1000, 13578-1-AP, Proteintech), anti-phospho Erk1/2 Thr202/Tyr204 (1:2000, 4370, Cell Signaling Technology), and anti-Erk1/2 (1:1000, 4695, Cell Signaling Technology). Enhanced chemiluminescence was conducted using an HRP substrate (WBKLS0500, Merck) and visualized by a Bio-Rad imaging system.

RNA extraction and quantitative real-time PCR (qPCR)

The total RNA of cells was extracted and reverse transcribed using RNA TRIzol reagent (9108, Takara) and PrimeScript RT reagent kit (RR037A, Takara) following the instructions. qPCR was conducted with SYBR Master Mix (B21703, Bimake) on a 7500 Real-time PCR system (Applied Biosystems) at the commonly recommended thermal cycling settings. The 2(−ΔΔCt) method was used to calculate the relative mRNA expression level using the expression of 18 s as the reference gene. Primer sequences were as follows:

18s-F ATCACCATTATGCAGAATCCACG,

18s-R GACCTGGCTGTATTTTCCATCC;

HOXB2-F CGCCAGGATTCACCTTTCCTT.

HOXB2-R CCCTGTAGGCTAGGGGAGAG;

ABCA1-F CATCTGGTTCTATGCCCGCT,

ABCA1-R TCTGCATTCCACCTGACAGC;

ABCA7-F TCCTGACCTCTCTGTCCCG,

ABCA7-R GGAGCTGGACCGGCTGT;

ABCB4-F GAAAGGCCAGACACTAGCCC,

ABCB4-R ACCATCGAGAAGCACTGTCC;

ABCC3-F GCCAAGAGGAACTTGACCCC,

ABCC3-R GACCTGGATGTCTAGGCTGTG;

ABCC5-F GCAGGGGCGCAGGAAT,

ABCC5-R GCTGGTTCTCTCCCTCACAC;

ABCD1-F CATGTTCTACCACAGGCCCA,

ABCD1-R GTGATGGAGAGCAGGGCAAT.

ABCG1-F CGTGGGCCCAGTGACAG;

ABCG1-R CCCTTCGAACCCATACCTGAC,

DANCR-F CAGTGCCACAGGAGCTAGAG;

DANCR-R GCAGCCTGTCCCTAACAGAA;

Mouse xenograft models

Subcutaneous xenograft models were constructed by subcutaneously implanting 2 × 106 shNC or shHOXB2 A2780/DDP cells into female BALB/c nude mice (6–8 weeks old). Once visible xenografts were grown, the mice with different cells were randomly divided into PBS and CDDP (cisplatin treated) groups. Equal volumes of PBS and 5 mg/kg CDDP solution were injected intraperitoneally every three days. Three weeks later, the mice were euthanized with CO2, and the xenografts were measured and weighed before being fixed with 4% paraformaldehyde. All mouse experiments were approved by the Shanghai Sixth People’s Hospital Research Ethics Committee affiliated with the Shanghai Jiao Tong University School of Medicine.

Immunohistochemistry and TUNEL assay

Immunohistochemistry (IHC) was routinely performed on paraffin sections following the instructions and observed under an optical microscope. The sections were dewaxed, rehydrated, and heated in a citrate-based solution for antigen retrieval. After blocking in 10% BSA, the sections were stained with the following primary antibodies: anti-HOXB2 (1:100, AY2582, Abways) and anti-Ki67 (GB111141, Servicebio), followed by incubation in HRP conjugated secondary antibodies. The sections were then reacted with DAB substrate and counterstained with hematoxylin before being dehydrated, mounted, and covered.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed to detect apoptotic cells in paraffin sections of mouse xenografts using a TUNEL kit (GB1507-50T, Servicebio) following the manual instructions.

Chromogenic in situ hybridization (CISH)

CISH was routinely performed on paraffin sections following the protocols. The sections were dewaxed, dehydrated, repaired, and digested using protease K. After pre-hybridization, the sections were incubated in hybridization solution containing probes. The sections were then blocked by rabbit serum, color developed, and counterstained the nucleus before sealing and imaging. In this article, we used five probes targeting DANCR. Probe sequences are as follows:

TGCGACGGGCGCACAAACCAGAGA;

GCACTTCCGCAGACGTAAGAGACG;

CTGCACGGACACGTGGTTGCTACA;

GGGTCAGCTGCATTGAGTTAGCGG;

TTCTCCACCAGTCGGAGGTGGCAG.

Statistical analyses

Data are expressed as mean ± standard deviation. The survival time was analyzed using the Kaplan-Meier method, and the P-value was calculated using the log-rank test. The results of the correlation analyses between HOXB2 and the target genes were described using Pearson’s correlation coefficient. The statistical significances between groups were calculated using the chi-square test, Student’s t-test, or ANOVA, as appropriate.