Xu Lijun, Yang Lei, Zhang Dan, Wu Yunxi, Shan Jiali, Zhu Huixia, Lian Zhengyi, He Guying, Wang Chongyu, Wang Qingqing
Department of General Surgery, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong, China.
Department of Clinical Biobank & Institute of Oncology, Affiliated Hospital of Nantong University & Medical School of Nantong University, Nantong, China.
Comput Struct Biotechnol J. 2024 Mar 6;23:1201-1213. doi: 10.1016/j.csbj.2024.02.016. eCollection 2024 Dec.
Cuproptosis, i.e., copper-induced programmed cell death, has potential implications in cancer therapy. However, the impact of the cuproptosis-related gene (CRG) dihydrolipoyl dehydrogenase (DLD) on breast cancer (BC) prognosis remains underexplored.
We employed real-time quantitative PCR and multiplexed immunostaining techniques to quantify DLD expression in both BC and the adjacent non-cancerous tissues. Immunofluorescence analysis was employed to assess the influence of DLD on immune cells and immunological checkpoints in the BC microenvironment. DLD knockdown experiments were conducted in BC cell lines MDA-MB-468 and SK-BR-3, with knockdown efficiency validated via western blot. Subsequently, we performed the cell counting kit-8 (CCK-8) assay, clone formation assay, Transwell migration assay, and invasion assay. To construct a prognostic model, we employed a Lasso-Cox regression analysis of immune-related genes associated with DLD. Additionally, we established a competing endogenous RNA network based on CRGs to evaluate potential regulatory pathways.
Compared to the adjacent tissues, BC tissues exhibited markedly elevated DLD expression levels. In vitro experiments demonstrated that DLD knockdown effectively inhibited BC cell migration, invasion, and proliferation. DLD exhibited positive correlations with CD68 macrophages and PD-L1 in the tumor, as well as with macrophages and CD4 T cells in the stroma. Tumor regions with high DLD expression were enriched in PD-L1 and macrophages, while stromal regions with high DLD expression contained CD4 T cells and macrophages. The AUC values for 1-, 3-, and 5-year overall survival in TCGA-BRCA training set were 0.67, 0.66, and 0.66, respectively. A nomogram with a C-index of 0.715 indicated that risk score, tumor stage, and age could serve as independent prognostic factors for BC.
Our findings underscore the significant predictive significance of DLD in BC and its influence on the tumor microenvironment. DLD represents a promising diagnostic and prognostic marker for BC, offering novel avenues for the identification of therapeutic targets and the enhancement of immunotherapy in BC.
铜死亡，即铜诱导的程序性细胞死亡，在癌症治疗中具有潜在意义。然而，铜死亡相关基因（CRG）二氢硫辛酰胺脱氢酶（DLD）对乳腺癌（BC）预后的影响仍未得到充分研究。
我们采用实时定量PCR和多重免疫染色技术来量化BC组织和相邻非癌组织中DLD的表达。采用免疫荧光分析来评估DLD对BC微环境中免疫细胞和免疫检查点的影响。在BC细胞系MDA-MB-468和SK-BR-3中进行DLD敲低实验，并通过蛋白质免疫印迹法验证敲低效率。随后，我们进行了细胞计数试剂盒-8（CCK-8）检测、克隆形成检测、Transwell迁移检测和侵袭检测。为构建预后模型，我们对与DLD相关的免疫相关基因进行了Lasso-Cox回归分析。此外，我们基于CRG建立了竞争性内源性RNA网络，以评估潜在的调控途径。
与相邻组织相比，BC组织中DLD表达水平显著升高。体外实验表明，DLD敲低有效抑制了BC细胞的迁移、侵袭和增殖。DLD在肿瘤中与CD68巨噬细胞和PD-L1呈正相关，在基质中与巨噬细胞和CD4 T细胞呈正相关。DLD高表达的肿瘤区域富含PD-L1和巨噬细胞，而DLD高表达的基质区域含有CD4 T细胞和巨噬细胞。在TCGA-BRCA训练集中，1年、3年和5年总生存率的AUC值分别为0.67、0.66和0.66。C指数为0.715的列线图表明，风险评分、肿瘤分期和年龄可作为BC的独立预后因素。
我们的研究结果强调了DLD在BC中的显著预测意义及其对肿瘤微环境的影响。DLD是一种有前景的BC诊断和预后标志物，为识别治疗靶点和增强BC免疫治疗提供了新途径。
