Identification of disulfidptosis-related subtypes, characterization of tumor microenvironment infiltration, and development of a prognosis model in breast cancer
Introduction: Breast cancer (BC) is now the most common type of cancer in women. Disulfidptosis is a new regulation of cell death (RCD). RCD dysregulation is causally linked to cancer. However, the comprehensive relationship between disulfidptosis and BC remains unknown. This study aimed to explore the predictive value of disulfidptosis-related genes (DRGs) in BC and their relationship with the TME. Methods: This study obtained 11 disulfidptosis genes (DGs) from previous research by Gan et al. RNA sequencing data of BC were downloaded from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus database (GEO) databases. First, we examined the effect of DG gene mutations and copy number changes on the overall survival of breast cancer samples. We then used the expression profile data of 11 DGs and survival data for consensus clustering, and BC patients were divided into two clusters. Survival analysis, gene set variation analysis (GSVA) and ss GSEA were used to compare the differences between them. Subsequently, DRGs were identified between the clusters used to perform Cox regression and least absolute shrinkage and selection operator regression (LASSO) analyses to construct a prognosis model. Finally, the immune cell infiltration pattern, immunotherapy response, and drug sensitivity of the two subtypes were analyzed. CCK-8 and a colony assay obtained by knocking down genes and gene sequencing were used to validate the model. Result: Two DG clusters were identified based on the expression of 11DGs. Then, 225 DRGs were identified between them. RS, composed of six genes, showed a significant relationship with survival, immune cell infiltration, clinical characteristics, immune checkpoints, immunotherapy response, and drug sensitivity. Low-RS shows a better prognosis and higher immunotherapy response than high-RS. A nomogram with perfect stability constructed using signature and clinical characteristics can predict the survival of each patient. CCK-8 and colony assay obtained by knocking down genes have demonstrated that the knockdown of high-risk genes in the RS model significantly inhibited cell proliferation.  Discussion: This study elucidates the potential relationship between disulfidptosis-related genes and breast cancer and provides new guidance for treating breast cancer. Overall design: The Tumor Cell Line Comprehensive Analysis Database (DepMap Portal) was utilized to screen for cell lines for further experimental validation (18). Breast cancer cell lines (MDA-MB-468) were obtained from Sichuan Huijixin Biotechnology Co., Ltd. The MDA-MB-468 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) culture medium, supplemented with 10% Fetal Bovine Serum (FBS) in a standard humidified incubator with 5% CO2 at 37°C. The TMEM45A and SHCBP1 specific short hairpin RNAs (shRNAs) were synthesized from Chengdu Youkangjianxing Biotechnology Co., Ltd.  The sequences of shRNAs are as the following: sh- TMEM45A -1: 5'- TGCTGTTGACAGTGAGCGCGGTTAAAGTATTTGAATTTAATAGTGAAGCCACAGATGTATTAAATTCAAATACTTTAACCATGCCTACTGCCTCGGA-3'; sh- TMEM45A -2: 5'- TGCTGTTGACAGTGAGCGCGGTGTACAAAGAGTATTCTGATAGTGAAGCCACAGATGTATCAGAATACTCTTTGTACACCATGCCTACTGCCTCGGA'. sh- SHCBP1 -1: 5'-TGCTGTTGACAGTGAGCGCCACATTGATTTTTCAATTGAATAGTGAAGCCACAGATGTATTCAATTGAAAAATCAATGTGATGCCTACTGCCTCGGA-3'; sh- SHCBP1 -2: 5'- TGCTGTTGACAGTGAGCGCCAGCCAAATGTTGATATTAAATAGTGAAGCCACAGATGTATTTAATATCAACATTTGGCTGATGCCTACTGCCTCGGA -3'. The knockdown efficiency was evaluated using real-time quantitative PCR (RT-qPCR) after 48 h transfection. The primer sequences used in the experiment are as follows. For the TMEM45A gene, the primers include qpcr-TMEM45A-F (forward primer): TTGGATGCCCACACTATGA and qpcr-TMEM45A-R (reverse primer): TCCATGGTCAAGGAGTTACA. For the SHCBP1 gene, the primers consist of qpcr-SHCBP1-F (forward primer): CTGGAGTTACAGAAGGATGGTG and qpcr-SHCBP1-R (reverse primer): CCATAGAAGCCTGTGGAATGT. After the knockdown of TMEM45A and SHCBP1 in cell line MDA-MB-468, total mRNA from cells was extracted with TRIzol reagent (TaKaRa, Japan). Then, concentration and purity were evaluated by Nanodrop 2000 (Thermo Fisher, USA). After the RNA was reversely transcribed into cDNA with PrimeScript RT kit (TaKaRa, Japan) according to the instructions, SYBR Premix Ex Taq TM kit (TaKaRa, Japan) was applied for RT-qPCR, with ß-actin as the endogenous control gene.