临床肿瘤学杂志

目的 探讨锌指和BTB结构域包含蛋白9(ZBTB9)在乳腺癌中的临床意义和生物学功能。方法 从癌症基因组图谱-乳腺癌队列及基因表达汇编数据库获取乳腺癌多组学数据，经标准化处理后，采用弹性网络、支持向量机构建预后风险模型，结合Kaplan-Meier曲线、森林图及Wilcoxon秩和检验分析预后差异与ZBTB9表达特征；收集安徽医科大学第二附属医院乳腺癌组织及癌旁组织样本，通过免疫组化H-SCORE评分系统检测ZBTB9表达并分组。以正常乳腺上皮细胞系MCF-10A及乳腺癌细胞系为研究对象，构建ZBTB9靶向小干扰RNA(siRNA)敲降体系，经实时荧光定量PCR和蛋白质印迹检验证敲降效率后，采用噻唑蓝（MTT）、克隆形成实验评估细胞增殖能力，通过划痕、Transwell迁移及侵袭实验检测细胞迁移侵袭能力，原位末端转移酶标记（TUNEL）染色检测细胞凋亡情况。结果 公共数据库及临床样本多维度验证表明，乳腺癌组织中ZBTB9表达量显著高于癌旁正常组织（P<0.05）。临床预后分析显示，ZBTB9高表达组患者总生存率显著低于低表达组（HR=6.44,95%CI:1.38～30.05,P=0.007）；进一步纳入年龄、临床分期等多项临床病理参数进行Cox回归分析，结果表明ZBTB9高表达是乳腺癌患者预后的独立危险因素（HR=6.756,95%CI:1.209～37.761）；受试者工作特征曲线分析显示其评估乳腺癌患者预后的曲线下面积为0.68(95%CI:0.53～0.83)，提示具有中等预后预测效能。细胞实验层面，与正常乳腺上皮细胞MCF-10A相比，乳腺癌细胞系（MDA-MB-231、BT-549等）中ZBTB9的mRNA及蛋白表达水平均显著上调；通过siRNA敲降ZBTB9表达后，乳腺癌细胞的活力（MTT实验）、集落形成能力显著下降，划痕愈合率及Transwell迁移、侵袭穿膜细胞数明显减少，同时TUNEL染色检测显示细胞凋亡率显著升高，上述差异均具有统计学意义（均P<0.05）。结论 ZBTB9在乳腺癌组织中高表达且为独立不良预后因素，敲降ZBTB9可抑制乳腺癌细胞增殖、迁移和侵袭并促进其凋亡，提示ZBTB9可能成为乳腺癌预后评估及靶向治疗的潜在靶点。

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.