Primary tumor (T) and adjacent non-tumorous (N) tissue samples were obtained from 77 patients at Jiangxi Cancer Hospital. These patients had not receive any adjuvant treatments, such as radiotherapy or chemotherapy, prior to surgery. All patients have written informed consent and all experiments were approved by the Medical Ethics Committee of Jiangxi Cancer Hospital with the ethical code 2023ky182.
USP42 expression data, measured by HTSeq-FPKM, was obtained from TCGA cohort pan-cancer database (https://portal.gdc.cancer.gov/) and Genotype-Tissue Expression (GETx) project database (https://www.gtexportal.org). The Wilcoxon rank-sum test was used to analyze disparities between tumor and non-tumorous specimens. Survival curve for the USP42-high and -low groups were generated using the Gene Expression Profiling Interactive Analysis (GEPIA) database(http://gepia.cancer-pku.cn/), with quartile USP42 expression levels as the basis for the analysis.
The human breast cancer cell lines MCF-7, MDA-MB-231, and BT549 were obtained from the Cell Resource Center of Beijing Xiehe, while the mammary gland epithelial cell line MCF10A was obtained from the American Type Culture Collection (ATCC). MCF-7 and BT549 cells were cultured in RPMI-1640(Gibco-BRL, Australia). MDA-MB-231 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL, Australia). MCF10A cells were cultured in Mammary Epithelial Basal Medium (MEBM) with 100 ng/ml cholera toxin, 1% solution of Pen/Strep, and 20ng/ml EGF. All media were supplemented with 10% fetal bovine serum (FBS, Gibco-BRL, Australia). Cells were cultured under humidified conditions at 37 °C and 5% CO.
The human USP42 shRNA plasmid and negative control shRNA (shNC) were acquired from Invitrogen (Thermo Fisher Scientific, Inc). The targeted sequences were as follows: Human USP42-sh1 (5'-CGAGTTCATCTGTACCTGATA-3'), Human USP42-sh2(5'-CGTCTCTTGTCTTCACTGATA-3'), HumanUSP42-sh3(5'-GCGTCTCTTGTCTTCACTGAT-3') and shNC (5'-CCGGCAACAAGATGAAGAGCACAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT-3'). Lentiviral vectors carrying USP42 shRNA or shNC were constructed and added to the culture media of MCF-7 and MDA-MB-231 cells. After 48 h later, the stable cell lines were selected using puromycin. In specific experiments, cells were treated with the JNK inhibitor SP600125 or the p38-MAPK inhibitor SB203580(MCE, USA), according to the manufacturer's instructions.
Proteins were extracted from cells or tissues using RIPA lysis buffer. The extracted protein samples were subsequently loaded onto SDS-PAGE gels, where proteins were separated into distinct bands based on their molecular weights via electrophoresis. Following separation, the proteins were transferred from the gel onto a PVDF membrane. Specific antibodies targeting the protein of interest were added to the membrane to allow the formation of antigen-antibody complexes. To minimize background signals, unbound antibodies were removed using multiple washing steps. Protein detection was performed using a chemiluminescent reagent. The resulting protein bands were visualized and analyzed using a Bio-Rad ChemiDoc XRS system to determine the presence and relative abundance of the target protein. The experimental procedure aligned with methodologies described in the literature. The protein marker used in western blot was the GoldBand Plus 3-color Regular Range Protein Marker (8-180 kDa, Yeasen Biotechnology [Shanghai] Co., Ltd.). Specific information for the antibodies used in the experiment is as follows: USP42 (1:500, sc-390604, Santa Cruz Biotechnology, Inc., USA), JNK (9252, Cell Signaling Technology, 1:1000), p-JNK (9251, Cell Signaling Technology, 1:1000), p38 (9212, Cell Signaling Technology, 1:1000), p-p38 (9211, Cell Signaling Technology, 1:1000), β-actin (sc-47778, Santa Cruz Biotechnology, 1:1000).
Total RNA was extracted from samples using TRIzol reagent and subsequently transcribed into complementary DNA (cDNA). The resulting cDNA was combined with PCR primers, SYBR Green fluorescent dye, and PCR reaction components. PCR amplification was performed under specific thermal cycling conditions involving repeated replication of the DNA sequence through controlled temperature fluctuations. During PCR amplification, fluorescence signals were detected, and the signal intensity of each PCR cycle was recorded. Based on the obtained cycle threshold (Ct) values, the relative quantity of target RNA in the samples was analyzed using the relative quantification method with a standard curve. The corresponding primer sequences were as follows: USP42,5'- AATCTTCAGACCCATCAGCCT-3'(forward) and 5'- AGAACCTGCATCCATGTCTCC-3(reverse); GADPH: 5'-GGTGAAGGTCGGAGTCAACG-3'(forward) and 5'-CAAAGTTGTCATGGATGHACC - 3'(reverse). ALL experiments were conducted in triplicates to ensure reproducibility and statistical significance.
Tissue samples were fixed on slides in formalin, embedded in paraffin, and sectioned. Tissue sections were treated for antigen retrieval, including protein dissociation, antigen retrieval, and antigen exposure, to enhance antibody binding efficiency. Non-specific binding was blocked using non-specific proteins, such as bovine serum albumin (BSA), to prevent antibody binding to non-target areas. Tissue samples were incubated with primary antibodies, which allowed them to bind specifically to the target protein. The samples were washed multiple times to remove unbound primary USP42 antibodies (sc-390604, Santa Cruz Biotechnology, Inc.USA), and reduce background signals. Secondary antibodies labeled with fluorophores or enzymes were added, which specifically bound to the primary antibodies to enhance the signal. The samples were washed multiple times to remove unbound secondary antibodies, and to reduce the background signals. The corresponding substrate was added to enzyme-labeled secondary antibodies, and the reaction products were observed. A microscope was used to examine the tissue sections and determine the expression of the target protein based on staining intensity and location. USP42 expression scores in tumor cells were determined by combining the proportion of positively stained tumor cells and the intensity of staining, as previously described.
For Cell Counting Kit 8 (CCK-8) assays, breast cancer cells were seeded into 96-well plates at a density of 3,000 cells per well. The CCK-8 reagent (K1080, APExBIO Technology LLC, USA) was added to the wells at 0, 24, 48, 72 h and 96 h. The plates were incubated at 37 °C for 2 h, after which absorbance was measured at 450 nm using a microplate reader. For the colony formation assays, 1500 cells were seeded in each well of 6-well plate containing 3 mL of medium per well. After approximately 14 days of incubation, the resulting cell colonies were fixed with a 4% paraformaldehyde solution for 15 min. Subsequently, the colonies were stained with a 0.2% crystal violet solution (Solarbio, China) and quantified using Image J software. All experiments were performed in triplicates to ensure reliability and consistency.
The treated cell samples were collected and processed appropriately, including washing and centrifuging, to obtain single-cell suspension. The cell samples were then stained with apoptosis markers, such as Annexin V and Propidium Iodide (PI). The stained cell samples were injected into a flow cytometer (FACSVantage SE; BD, Franklin Lakes, NJ, USA), which utilized lasers to irradiate cells and detect the fluorescence signal intensity within them. Based on the staining characteristics of Annexin-V/PI (FA101-01, TransGen Biotech Co.,Ltd, Beijing, China), the flow cytometer identified and differentiated cells in various states, including live, apoptotic, and necrotic cells, and necrotic cells. The proportion of apoptotic cells and other relevant parameters in the samples were analyzed based on the data obtained from the flow cytometer.
Animal experiments were approved by the Animal Ethics Committee of Kangtai Medical Laboratory Service Hebei Co. on March 14, 2022 (approval number: MDL2022-03-14-02) and conducted with the formal approval of the animal care committees. Nude mice were maintained in sterile conditions, including sterilized food and water, and regular cleaning and disinfection of the cages and the equipment. Each cage contained up to six nude mice. The laboratories maintained strict control over temperature (22-24 °C) and relative humidity (40-60%). The animals were housed in isolation cages with filtered air vents to prevent the entry of pathogen entry. Due to their susceptibility to infections, their health, including activity levels, appetite, and any signs of illness, were regularly monitored. Nude mice were randomly assigned to either the sh-USP42 or control vector group (n = 4 per group). Tumor growth was initiated by subcutaneously injecting MDA-MB-231 cells into 3-week-old female nude mice at a concentration of 1 × 10^6 cells in 200 µl of a 1:1 PBS -Matrigel mixture. Tumor dimensions were evaluated every three days using caliper, and tumor volume was calculated using the formula ((L×W)/2). At the end of the animal experiments, mice were euthanized by cervical dislocation. Xenografted tumors were surgically excised, and protein levels within the tumors were determined using western blot analysis. All experiments were conducted in accordance with relevant guidelines and regulations. All studies involving live animals is reported in accordance with the ARRIVE guidelines.
Statistical analysis was performed using SPSS software (version: 21.0, Chicago, USA). Continuous data were presented as means ± standard deviation (SD), whereas categorical data were expressed as frequency counts and percentages. Student's t-test was used for comparisons between two groups. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was conducted, followed by the Student-Newman-Keuls test for post-hoc multiple comparisons. The association between USP42 expression and clinicopathologic characteristics was evaluated using the χ2 test or Fisher's exact test as appropriate. Differences in survival curves were assessed using the log rank test. A p < 0.05 was considered statistically significant.