Integrated single-cell and bulk RNA sequencing analysis reveals paraptosis-related biomarkers and immune microenvironment regulation in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited targeted therapeutic options and poor prognosis. Paraptosis, a non-apoptotic programmed cell death modality characterized by endoplasmic reticulum dilation and mitochondrial swelling, plays a critical role in tumor suppression and chemosensitivity. However, the molecular mechanism of paraptosis-related genes (PRGs) in TNBC progression and immune microenvironment remodeling remains poorly defined. As a pivotal calcium release channel on ER, ITPR3 is speculated to participate in endoplasmic reticulum stress and paraptosis execution, but its diagnostic value and regulatory role in TNBC have not been systematically elucidated. Two TNBC datasets were downloaded from the GEO database. PRGs were intersected with TNBC differentially expressed genes, and six hub genes were screened using random forest, SVM-RFE and multivariate logistic regression. ROC curve, nomogram, calibration curve and decision curve analysis were applied to evaluate the diagnostic value of hub genes. Furthermore, external validation in the independent TCGA-TNBC cohort was performed to further verify the robustness and diagnostic efficacy of this six-gene diagnostic model. Consensus clustering was used to classify TNBC into subtypes, followed by immune infiltration and inflammatory cytokine correlation analysis. Weighted gene co-expression network analysis (WGCNA) was performed to identify key modules and perform Gene Ontology (GO) / Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Single-cell transcriptomics was used to explore the cell-type-specific expression patterns of ITPR3 . We used the TIMER and HPA databases to verify the mRNA and protein expression levels of ITPR3 across pan-cancers and breast tissues. Finally, qRT-PCR, CCK-8 and EdU assays were conducted to verify the expression and biological function of ITPR3 in TNBC cells. A total of 13 differentially expressed PRGs were identified, and six hub genes including ITPR3 , CASP3 , CCR4 , CDKN3 , LPAR1 and MYLK were finally screened. The six hub genes and the combined diagnostic model exhibited satisfactory diagnostic performance. TNBC was divided into two distinct subtypes with significant differences in clinical features, immune cell infiltration and inflammatory cytokine levels. WGCNA identified two core modules closely correlated with subtypes, which were enriched in cell cycle, DNA replication, p53 signaling pathway and platinum drug resistance. Single-cell analysis confirmed that ITPR3 was highly expressed in tumor epithelial cells. In vitro experiments confirmed that ITPR3 was highly expressed in TNBC cell lines, and knockdown of ITPR3 markedly inhibited cell proliferation. This study reveals the expression characteristics and immune regulatory roles of hub PRGs in TNBC. The constructed diagnostic model has good predictive value, and TNBC subtypes present obvious heterogeneity in clinical and immune landscapes. ITPR3 is upregulated in TNBC and can promote tumor cell proliferation, which may serve as a candidate diagnostic biomarker and potential therapeutic target for TNBC.