Data Sheet 1_Systematic pan-cancer analysis identifies PKNOX1 as a potential prognostic and immunological biomarker and its functional validation.docx

BackgroundPBX/knotted1 homeobox 1 (PKNOX1), also known as PREP1, is a homeodomain transcription factor that has been reported to be involved in the progression of gastric adenocarcinoma and non-small cell lung cancer. However, its expression, clinical significance, and biological function in various human cancers have not been studied in depth, and its role and specific molecular mechanism in the occurrence and development of cancer remain unclear. Its potential as a cancer prognostic marker and therapeutic target has not yet been explored.
MethodsThe TCGA and GTEx databases were used to evaluate the differential expression of PKNOX1 pan-cancer, and PKNOX1 gene mutation information was obtained from the cBioPortal and GSCALite platforms. Cox regression and Kaplan-Meier analyses were used to evaluate the value of PKNOX1 in pan-cancer prognosis. The correlations between PKNOX1 expression and the expression of DNA methylation-related genes, immune-related genes, tumor mutation burden (TMB), and microsatellite instability (MSI) were analyzed using Spearman correlation. The correlations between PKNOX1 expression and MDSC immune infiltration and immune cells were analyzed using the TIDE algorithm and the ESTIMATE algorithm. PKNOX1 -interacting proteins and expression-related genes were analysed via the STRING and TIMER 2.0 platforms, and the functions of PKNOX1 in tumors and the cell pathways involved were predicted via KEGG enrichment analysis. In addition, the differential expression and function of PKNOX1 in HCC and breast cancer were explored via Western blotting and proliferation and migration-related experiments.
ResultsPKNOX1 is significantly highly expressed in most tumor types and is significantly associated with poor patient prognosis and increased clinicopathological stage. Tumor gene mutations and DNA methylation may explain the abnormal expression of PKNOX1. In most tumors, PKNOX1 expression was significantly correlated with MDSC immune infiltration and immune cells, methylation-related genes, immune-related genes, the TMB and MSI. The enriched KEGG pathways indicated that PKNOX1 is involved in cancer-promoting processes such as cell-cell adhesion, the cell cycle and cell proliferation and deterioration signalling pathways. In vitro experiments revealed that PKNOX1 is highly expressed in HCC and breast cancer cells and HCC tissues and promotes the growth, proliferation, migration and invasion of HCC and breast cancer cells.
ConclusionPKNOX1 is a promising prognostic and immune biomarker in pan-cancer and may play an important role in HCC and breast cancer progression and metastasis.

背景：同源盒基因PBX/Knotted1 1（PBX/knotted1 homeobox 1, PKNOX1），又称PREP1，是一种同源域转录因子，已有研究表明其参与胃腺癌与非小细胞肺癌的疾病进展。然而，该基因在多种人类癌症中的表达情况、临床意义及生物学功能尚未得到深入研究，其在癌症发生发展中的作用与具体分子机制仍不明确，其作为癌症预后标志物与治疗靶点的潜力也尚未得到探索。
方法：本研究借助癌症基因组图谱（The Cancer Genome Atlas, TCGA）与基因型组织表达（Genotype-Tissue Expression, GTEx）数据库评估PKNOX1在泛癌中的差异表达水平，并从cBioPortal与GSCALite平台获取PKNOX1的基因突变信息。采用Cox比例风险回归与Kaplan-Meier分析评估PKNOX1在泛癌预后中的价值；通过Spearman相关分析探究PKNOX1表达与DNA甲基化相关基因、免疫相关基因、肿瘤突变负荷（Tumor Mutation Burden, TMB）及微卫星不稳定性（Microsatellite Instability, MSI）表达的相关性；利用TIDE算法与ESTIMATE算法分析PKNOX1表达与髓系来源抑制细胞（Myeloid-derived suppressor cells, MDSC）免疫浸润及其他免疫细胞的关联。通过STRING数据库与TIMER 2.0平台分析PKNOX1的互作蛋白与表达相关基因，并借助KEGG富集分析预测PKNOX1在肿瘤中的功能及其参与的细胞通路。此外，本研究通过蛋白质印迹法（Western blotting）、增殖与迁移相关实验，探究PKNOX1在肝细胞癌（Hepatocellular carcinoma, HCC）与乳腺癌中的差异表达与功能。
结果：PKNOX1在多数肿瘤类型中呈显著高表达，且与患者不良预后及更高的临床病理分期显著相关。肿瘤基因突变与DNA甲基化水平异常或可解释PKNOX1的表达异常。在多数肿瘤中，PKNOX1的表达与MDSC免疫浸润、其他免疫细胞、甲基化相关基因、免疫相关基因、TMB及MSI均存在显著相关性。富集得到的KEGG通路结果显示，PKNOX1参与细胞-细胞黏附、细胞周期、细胞增殖等促癌过程及恶化相关信号通路。体外实验结果表明，PKNOX1在HCC细胞、乳腺癌细胞及HCC组织中呈高表达，并可促进HCC与乳腺癌细胞的生长、增殖、迁移与侵袭。
结论：PKNOX1是一种具有应用前景的泛癌预后与免疫生物标志物，或在肝细胞癌与乳腺癌的进展及转移过程中发挥重要作用。