Cell culture

In this study, four strains of BRCA cells and one strain of human normal breast cells MCF-10A were employed. Purchased two strains of SKBR3 and BT474 cells in the Chinese Academy of Sciences and MCF-10A, MCF-7, and HCC1806 cells were kindly donated by the Translational Research Center of Guizhou Medical University. All cells were grown in Proxel's medium (DMEM, 1640) containing 10% fetal bovine serum (FBS) and penicillin–streptomycin solution at 37 °C and 5% CO2.

Clinical specimens and immunohistochemistry

The clinical specimen tissues were from the Department of General Surgery, Dejiang County People's Hospital, and the tumor tissues were from two HER2 + and HER2- BRCA patients and adjacent tissues, respectively. The patient and histopathologist agree to use this clinicopathological tissue. The use of this sample was reviewed and approved by the Ethics Committee of Dejiang County People's Hospital, and the principles of the Declaration of Helsinki were also followed. Each patient enrolled in this study has provided informed consent by signing a patient consent form. The process adheres to a specific protocol, including steps such as deparaffinization, dehydration, inhibition of endogenous peroxidase and non-specific antigens, antigen retrieval, incubation with primary antibodies, staining, and blocking. The experimental tissue sections are then examined using an inverted microscope.

Transient transfection

Ten thousand cells per well were plated in a six-well plate, and after 12 h, the cells were adherent, siRNA and overexpression plasmid were introduced into the cells with liposome Lipo3000 to obtain a temporarily silenced TTK cell line, and 12 h after transfection with fresh complete medium for follow-up experiments.

Quantitative real-time PCR

Extract the total RNA of the cells with an RNA kit, then reverse transcribe and amplify the cDNA. A quantitative polymerase chain reaction was performed on cDNA using primers ordered by Guizhou Weiboxin Biological Company. Finally, the BioRad CFX96 System was adopted to detect genetic changes through computational data.

Western blot analysis and antibodies

Cell total protein extraction, pure protein ultrasonic distraction collection, centrifugation, supernatant extraction, the addition of reagent ratio: lysate: protease inhibitor: phosphatase inhibitor = 100:1:1, BCA protein analysis kit quantification. The protein was separated by SDS–polyacrylamide gel electrophoresis. It is subsequently moved to a 0.45 mm membrane and incubated with the desired primary antibody for 24 h at 4 °C. The Te antibodies used in this experiment included TTK, mTOR, AKT, E Cadherin, N Cadherin, Vimentin, β-Actin, p-mTOR, p-AKT, Cleaved-casp3, and Cleaved-casp7. Following the protein is fully bound to the primary antibody, it is imaged in a chemical DocXRS + molecular imager using a chemiluminescence kit to unpack the communication of the protein, and the ImageJ software is processed with gray values.

The viability of CCK-8 cells and the IC50 value of the drug were measured

Cell viability is measured with the CCK-8 kit and resistance to pyrotinib, and the inhibition rate and IC50 value of pyrotinib before and after TTK knockout were verified. Prepare a 96-well plate, take the experimental cell digestion and off-center suspension, count 5000 cells by cell tallying, dilute with 100 μL of culture medium, seed each well, and continue to incubate in the incubator for 24 h. After watching the merisis of the cells, ten μL of pyrotinib at distinct consistency was mixed in every well and put in the incubator for 24 h. Remove the 96-well plate and add ten μL of CCK-8 reagent to each well in the dark. Wrap the 96-well plate with aluminum foil and continue incubating for four hours. Remove the 96-well plank, open the cap, and determine the OD value with an enzyme marker at 450 nm. GraphPad Prism 9.5 processes the data.

Wound healing test

Six-well plates were plated first, cells were adherent for eight hours for transfection, and when the cells were cultured to 90% concentration, a straight line was drawn on the cell monolayer with a ten μl pipette tip. The wound width was measured at 0 h and 48 h, respectively. The rate of cell migration was determined by calculating wound closure, normalized against the NC group. Wound healing trials were performed three times, and GraphPad Prism 9.5 processed data.

Plate cloning

200 SKBR3 and BT474 were added to each well of the six-well plate, and after three weeks of continuous culture, they were washed twice with PBS. The cells in each well are fixed with 3 ml of methanol for 30 min and twice with PBS. Cells are stained with 1% crystal violet for 30 min, washed off with slow water, and subsequently air dry. The data were then observed and counted under the microscope and processed with GraphPad Prism 9.5.

Flow cytometry was used to detect cell cycle and apoptosis

Cell Cycle Assay Kits are used; take 1 ml of cell suspension (1 × 10^6/mL), fix it with 70% cold ethanol 500ul for two hours, then wash twice in PBS, add 500ul PI/RNase staining solution, protect from light for 60 min at room temperature, and detect on the machine, the wavelength is 488 nm; The trial was conducted three times. Six-well plates were plated with approximately 2 × 10^6 cells per well using the Annexin V-FITC Apoptosis Assay Kit, and cells were resuspended with 100 ul of 1XAnnexin V Binding Buffer. Then, add 2.5 μl of Annexin V/FITC Reagent and 2.5ul of PI Reagent to each EP tube, mix well, and incubate for 20 min at room temperature. Add 400 μL of 1X Annexin V Binding Buffer to resuspend the cells, and proceed with immediate testing. The experiment was repeated three times.

Animal modeling

A mouse model of xenografts for breast cell carcinoma was established; SKBR3 and SKBR3 cells were directly injected into the left and right lower abdomen of nude mice, respectively, and approximately 10^7 cells were inoculated into each nude mouse, and the merisis of tumors was watched, and record the size of the tumor with vernier calipers once every three days. Following 30 days, the tumors were dislodged, photographed, and weighed, and tumor information was gathered for statistical analysis. The Animal Center and the Use Committee of Guizhou Medical University approved all animal experimental protocols.

Bioinformatics analysis

The databases employed in the study include Cancer Genome Atlas (TCGA), Breast Cancer Integrated Platform Data BCIP (http://www.omicsnet.org/bcancer/), UALCAN (http://ualcan.path.uab.edu/); The KEGG and GO pathways of TTK were enriched by Sangerbox technology to obtain the pathways, cell cycle and apoptosis-related to their expression.

Statistical analysis

The collected experimental data were analyzed by GraphPad Prism 9.5 software. One-way ANOVA was used for comparison between multiple groups, and LSD t test was used for pairwise comparison between groups, and on the condition that p < 0.05, the difference was significant or extremely significant. All experiments were performed with n ≥ 3.