Study on the clinical implications and biological functions of zinc finger and BTB domain containing 9 in breast cancer

ZHANG Haiyan; TANG Tong

doi:10.3969/j.issn.1009-0460.2026.01.003

Abstract：Objective To explore the clinical significance and biological functions of zinc finger and BTB domain-containing protein 9(ZBTB9)in breast cancer.Methods Multi-omics data of breast cancer were obtained from The Cancer Genome Atlas-Breast Cancer cohort and Gene Expression Omnibus database.After standardized processing,elastic network and support vector machine were used to construct a prognostic risk model.Kaplan-Meier curve,forest plot and Wilcoxon rank-sum test were combined to analyze the prognostic differences and ZBTB9 expression characteristics.Breast cancer tissues and adjacent non-tumor tissues were collected from the Second Affiliated Hospital of Anhui Medical University.The expression of ZBTB9 was detected by immunohistochemical H-SCORE system and grouped accordingly.Taking normal breast epithelial cell line MCF-10A and breast cancer cell lines as research objects,a ZBTB9-targeted small interfering RNA(siRNA)knockdown system was constructed.After verifying the knockdown efficiency by real-time fluorescence quantitative PCR and Western blot,methyl thiazolyl tetrazolium(MTT)assay and colony formation assay were used to evaluate cell proliferation ability.Wound healing assay,Transwell migration and invasion assays were performed to detect cell migration and invasion abilities,and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)staining was used to detect cell apoptosis.Results Multi-dimensional validation using public databases and clinical samples showed that the expression level of ZBTB9 in breast cancer tissues was significantly higher than that in adjacent normal tissues(P＜0.05).Clinical prognostic analysis revealed that the overall survival rate of patients in the ZBTB9 high-expression group was significantly lower than that in the low-expression group(HR=6.44,95％CI:1.38-30.05,P=0.007).Further Cox regression analysis incorporating multiple clinicopathological parameters such as age and clinical stage indicated that high ZBTB9 expression was an independent risk factor for the prognosis of breast cancer patients(HR=6.756,95％CI:1.209-37.761).Receiver operating characteristic curve analysis showed that the area under the curve for evaluating the prognosis of breast cancer patients was 0.68(95％CI:0.53-0.83),suggesting a moderate prognostic predictive efficacy.At the cellular experimental level,compared with the normal breast epithelial cell line MCF-10A,the mRNA and protein expression levels of ZBTB9 in breast cancer cell lines(such as MDA-MB-231 and BT-549)were significantly upregulated.After knocking down ZBTB9 expression via siRNA,the viability(MTT assay)and colony-forming ability of breast cancer cells were significantly decreased,the wound healing rate and the number of transmembrane cells in Transwell migration and invasion assays were significantly reduced,and TUNEL staining showed that the cell apoptosis rate was significantly increased.All the above differences were statistically significant(all P＜0.05).Conclusion ZBTB9 is highly expressed in breast cancer tissues and serves as an independent poor prognostic factor.Knockdown of ZBTB9 can inhibit the proliferation,migration and invasion of breast cancer cells and promote their apoptosis,suggesting that ZBTB9 may be a potential target for prognostic evaluation and targeted therapy of breast cancer.