Patients and tissue samples

The fresh tumor tissues and para-cancer tissues of 16 BC patients were obtained from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All patients did not receive chemotherapy or radiotherapy prior to surgery and signed written informed consent. The study was reviewed and approved by the Hospital Ethics Committee of Huazhong University of Science and Technology, and all subjects signed the informed consent form of Helkisian.

Cell lines

BC cell lines MDA-MB-231 and MCF7 were purchased from the Wuhan typical culture collection center (CCTCC), and normal breast epithelial cells MCF10A were purchased from Wuhan Procell Life Science&Technology Co.,Ltd. (Wuhan, China). DMEM cell medium containing fetal bovine serum (FBS) and MCF10A special medium were used and cultured in cell incubators under conditions of 37℃, 5% CO2.

RT-qPCR

The mRNA expression levels of CD24 and other genes in BC tissues and cells were detected using RT-qPCR according to the manufacturer's instructions. β-actin was used as a control. Total RNA was extracted using the ultrapure RNA extraction Kit (Yeasen, Shanghai, China), and cDNA was synthesized using the ABScript II cDNA First Strand Synthesis Kit for the QPCR Kit (Abclone, Wuhan, China).

For miRNA quantification, RiboBio Co., Ltd. (Guangzhou, China) designed a convex-ring TM miRNA RT-qPCR primer set specifically for miR-125a-5p. CDNA was synthesized using miRNA first strand cDNA synthesis kit (Vazyme, Nanjing, China).

RT-qPCR was performed with the cDNA obtained in the above steps as a template, and three replicate wells were set in each group. PCR was performed for 40 cycles of 5 min at 95 ℃, 12 s at 95 ℃, 20 s at 58 ℃ and 20 s at 72 ℃.

The primers were listed in supplementary Table 1.

Western blot (WB)

Cells were lysed with protease inhibitor RIPA lysis buffer (Meilun, Wuhan, China) ice. Protein extracts (30 μg) were electrophoretically separated on SDS–polyacrylamide gel and then transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% milk for 1 h at room temperature, and then incubated with antibodies overnight. Secondary antibodies (1:5000, AS014, ABclone, Wuhan, China) were added, incubated for 1 h at room temperature and imaged on a chemiluminescent imaging system. The following antibodies were used: anti-β-actin (1:5000, ABclone, AB clone, Wuhan, China), anti-CD24 (1:1000, AB290730, ABCAM, Cambridge, UK), anti-Ki67(1:1000, AC002, ABclone, Wuhan, China, anti-Vimentin (1:1500,10,366–1-AP, proteintech, Wuhan, China), anti-E-cadherin (1:1000, A20798, ABclone, Wuhan, China). Anti-ZNF460 (1:1000, A17621, ABclone, Wuhan, China).

Cell Counting Kit-8 (CCK-8) assay

The proliferation ability of BC cells was evaluated by the CCK-8 method (Meilun, Wuhan, China). 5 × 103 cell suspensions were inoculated into 96-well culture plates containing 100 μL medium. 10 μL CCK-8 reagent was added to each well after 1, 2, 3, 4 and 5 days, respectively. After incubation at 37℃ for 1 h, absorbance was determined at 450 nm.

Colony formation assay

Colony formation assays were performed to test the clonal ability of BC cells. The 1 × 103 cells were cultured in 6-well plates for 14 days, and the medium was changed every 5 days. The colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 15 min. Colony Formation is determined by counting the number of stained colonies.

Cell migration and invasion

Wound healing assays were used to test cell migration. A total of 2 × 105 cells were seeded in 6-well plates, scraped with sterile plastic tips after 12 h of culture, washed with medium, and then cultured for 24 h and 48 h. At different time points, images of the plates were obtained using an inverted microscope.

For the invasion assay, 1 × 105 cells in serum-free medium were placed in the upper chamber coated with matrigel. The medium containing 10% FBS was added to the lower chamber. After incubation for 24 h, the residual cells on the upper membrane were removed with absorbent cotton. Cells that had migrated or invaded the membrane were fixed with paraformaldehyde and stained with 0.1% crystal violet. Take an image using an inverted microscope.

Luciferase reporter assays

Binding motifs to ZNF460 in the CD24 promoter region were identified by JASPAR (http://JASPAR.genereg.net/). The complete and mutated CD24 promoter sequences were synthesized and then inserted into the pGL3-basic vector and cotransfected into 293 T cells with the ZNF460 plasmid. MiR-125a-5p mimics were cotransfected into 293 T cells with wild-type and mutant LINC00525 plasmids. All vectors were validated by sequencing and luciferase activity was assessed using a dual luciferase detection kit (Yeasen, Shanghai, China) according to the manufacturer's instructions.

Biotin-labeled miRNA pull-down assay

Biotin-labeled miR-125a-5p and miRNA control were transfected into BC cells and lysis were harvested 48 h later. The Streptavidin Dynabeads (Merck, Darmstadt, Germany) were washed and resuspended in buffer. Equal volumes of biotin-labeled miR-125a-5p and miRNA control were then added to the buffer. After incubation at room temperature for 10 min, the coated beads were separated with a magnet and washed 3 times. The isolated RNA was then analyzed by RT-qPCR.

RNA immunoprecipitation (RIP)

An EZMagna RNA immunoprecipitation (Millipore, Bedford, MA, USA) was used according to the manufacturer's protocol. MCF-7 and MDA-MB-231 cells were lysed in RIP lysis buffer (containing protease inhibitor and phosphatase inhibitors) for 30 min, the cell extracts were incubated with magnetic beads conjugated with specific antibody or control IgG at 4 ℃ overnight. The magnetic beads were washed and incubated with protease K to remove the protein. Finally, the purified RNA was analyzed by RT-qPCR.

Establishment of xenografts and in vivo studies

4-week-old female BALB/C nude mice were bred under specific pathogen-free conditions and operated according to protocols approved by the Ethics Committee for Wuhan University of Science and Technology animal experiments. MCF7 cells stably transfected with 1 × 107 control shRNA or sh-CD24 were injected subcutaneously into one side of the nude mice. Tumor volume was measured with a caliper and recorded every 5 days. Euthanasia was performed after 25 days of growth, and tumor tissue was excised for subsequent experiments.

Statistical analysis

GraphPad Prism 8 software was used to plot and analyze the original data. The statistical results were the results of three independent repeated experiments unless otherwise stated test for differences in two different sets of data using unpaired Student's-T test, and analysis of differences between multiple sets of data using one-way ANOVA. In vitro experimental data were expressed as mean ± standard deviation. A P value of less than 0.05 was considered statistically significant.