Table_5_ASF1B: A Possible Prognostic Marker, Therapeutic Target, and Predictor of Immunotherapy in Male Thyroid Carcinoma.xlsx

BackgroundThyroid carcinoma (TC) is the most common malignant endocrine tumor worldwide. Several studies have documented that male patients with TC have a higher rate of metastasis and disease recurrence than female patients. However, the mechanism underlying this observation is not completely clear. The goal of our research was to investigate the potential key candidate genes and pathways related to TC progression in male patients at the molecular level.
MethodsA total of 320 samples were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Hub genes were screened out using weighted gene coexpression network analysis (WGCNA) and a protein–protein interaction (PPI) network analysis. Survival analysis was used to identify hub genes associated with disease-free survival (DFS) rates. Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression (ESTIMATE) data were used to assess the relationship between hub genes and immune cell infiltration. The molecular mechanism and biological functions of hub genes were explored using RT-qPCR, Western blot, Cell Counting Kit-8 Assay, flow cytometry, Transwell assays, and scratch assays.
ResultsForty-seven hub genes were identified, and the survival analysis demonstrated that anti-silencing function 1B (ASF1B) was the sole independent risk factor for poor DFS in male TC patients. Possible associations between the results from the ESTIMATE analysis showed that the ASF1B expression level was related to the ESTIMATE score, immune score, and T-cell regulatory (Treg) infiltration level. Through in vitro cell function experiments, we verified that knockdown of ASF1B inhibited KTC-1 cell proliferation, promoted cell apoptosis, and blocked cell cycle. The silencing of ASF1B reduced protein kinase B (AKT), phospho-AKT (p-AKT), and forkhead box p3 (FOXP3) in KTC-1 cells. Moreover, FOXP3 overexpression markedly restored the cell migration, invasion, and proliferation abilities repressed by ASF1B knockdown.
ConclusionsOur results indicate that ASF1B can be considered a prognostic marker, therapeutic target, and predictor of immunotherapy response in male thyroid cancer patients. However, further in-depth studies are required to validate this finding.

背景 甲状腺癌（Thyroid carcinoma, TC）是全球范围内最常见的内分泌恶性肿瘤。多项研究证实，男性甲状腺癌患者的转移率与疾病复发率均高于女性患者，但该现象的分子机制尚未完全阐明。本研究旨在从分子层面探究与男性甲状腺癌患者疾病进展相关的潜在关键候选基因及通路。
方法 本研究从癌症基因组图谱（The Cancer Genome Atlas, TCGA）及基因型-组织表达（Genotype-Tissue Expression, GTEx）数据库中获取了共计320份样本。通过加权基因共表达网络分析（WGCNA）与蛋白质-蛋白质相互作用（PPI）网络分析筛选枢纽基因。采用生存分析鉴定与无病生存率（DFS）相关的枢纽基因。利用ESTIMATE算法（Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression）评估枢纽基因与免疫细胞浸润的关联。通过实时定量聚合酶链反应（RT-qPCR）、蛋白质印迹（Western blot）、细胞计数试剂盒-8（Cell Counting Kit-8，CCK-8）实验、流式细胞术、Transwell实验及划痕实验，探究枢纽基因的分子机制与生物学功能。
结果 本研究共鉴定出47个枢纽基因，生存分析结果显示，抗沉默功能蛋白1B（ASF1B）是男性甲状腺癌患者不良无病生存率的唯一独立危险因素。ESTIMATE分析结果的关联分析表明，ASF1B的表达水平与ESTIMATE评分、免疫评分及调节性T细胞（Treg）浸润水平相关。通过体外细胞功能实验，本研究验证了敲低ASF1B可抑制KTC-1细胞增殖、促进细胞凋亡并阻滞细胞周期。在KTC-1细胞中，沉默ASF1B可降低蛋白激酶B（AKT）、磷酸化AKT（p-AKT）及叉头框蛋白P3（FOXP3）的表达水平。此外，过表达FOXP3可显著恢复ASF1B敲低所抑制的细胞迁移、侵袭与增殖能力。
结论 本研究结果表明，ASF1B可作为男性甲状腺癌患者的预后标志物、治疗靶点及免疫治疗反应预测因子。但仍需开展进一步的深入研究以验证本研究发现。