Zhang Zhenxing, Zhu Haoran, Zhao Chifeng, Liu Dong, Luo Jun, Ying Yukang, Zhong Yuan
Department of Stomatology, Taizhou Central Hospital (Taizhou University Hospital), Taizhou, Zhejiang Province, China.
Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi Province, China.
Mol Carcinog. 2023 Mar;62(3):332-347. doi: 10.1002/mc.23489. Epub 2022 Dec 1.
This study investigated the cancer-promoting effect of ferroptosis regulator DNA damage-inducible transcript 4 (DDIT4) and its relevant mechanisms. Vital ferroptosis-related genes were identified using bioinformatic methods on the basis of data collected from TCGA and seven other online databases. Cell Counting Kit-8 (CCK8), colony formation, wound-healing and transwell assays, and western blot analysis were conducted for verifying the biological role of DDIT4 in vitro. The immune score and tumor purity were calculated using R package "estimate." The relationship was identified between DDIT4 expression and immune cell infiltration using ssGSEA and CIBERSORT algorithms. R package "Seurat" was used to perform unsupervised clustering of the single cells, and "SingleR" was utilized for annotation. R package "STUtility" was employed to plot the spatial expression of DDIT4. For trajectory analysis, monocle was used to predict cell differentiation and demonstrate the expression of DDIT4 at each state. Here, DDIT4 overexpression was observed in Head and Neck Squamous Cell Carcinoma (HNSCC) cohort, and DDIT4 upregulation showed a positive correlation with larger tumor size, lymph node metastasis, more advanced TNM stage and higher tumor mutational burden (TMB). Moreover, DDIT4 knockdown could markedly inhibit the proliferation, colony formation, invasion and migration of HNSCC cells, as well as suppress the expression of HIF-1a, VEGF and vimentin. In comparison, DDIT4 overexpression showed a negative correlation with immune score and infiltrations of several immune cells. DDIT4 played crucial roles in the differentiation of CAFs and T cells. Collectively, this study demonstrates that DDIT4 contributes a critical role in HNSCC progression. The positive feedback regulation between DDIT4 and HIF-1a may be a potential target for HNSCC treatment.
本研究调查了铁死亡调节因子DNA损伤诱导转录本4（DDIT4）的促癌作用及其相关机制。基于从TCGA和其他七个在线数据库收集的数据，采用生物信息学方法鉴定了重要的铁死亡相关基因。进行细胞计数试剂盒-8（CCK8）、集落形成、伤口愈合和Transwell实验以及蛋白质免疫印迹分析，以验证DDIT4在体外的生物学作用。使用R包“estimate”计算免疫评分和肿瘤纯度。使用单样本基因集富集分析（ssGSEA）和CIBERSORT算法确定DDIT4表达与免疫细胞浸润之间的关系。使用R包“Seurat”对单细胞进行无监督聚类，并使用“SingleR”进行注释。使用R包“STUtility”绘制DDIT4的空间表达。对于轨迹分析，使用monocle预测细胞分化并展示DDIT4在每个状态下的表达。在此，在头颈部鳞状细胞癌（HNSCC）队列中观察到DDIT4过表达，并且DDIT4上调与更大的肿瘤大小、淋巴结转移、更晚期的TNM分期和更高的肿瘤突变负荷（TMB）呈正相关。此外，敲低DDIT4可显著抑制HNSCC细胞的增殖、集落形成、侵袭和迁移，以及抑制缺氧诱导因子-1α（HIF-1α）、血管内皮生长因子（VEGF）和波形蛋白的表达。相比之下，DDIT4过表达与免疫评分和几种免疫细胞的浸润呈负相关。DDIT4在癌相关成纤维细胞（CAFs）和T细胞的分化中起关键作用。总体而言，本研究表明DDIT4在HNSCC进展中起关键作用。DDIT4与HIF-1α之间的正反馈调节可能是HNSCC治疗的潜在靶点。
