Table2_Integrative multiomics evaluation reveals the importance of pseudouridine synthases in hepatocellular carcinoma.xlsx

Background: The pseudouridine synthases (PUSs) have been reported to be associated with cancers. However, their involvement in hepatocellular carcinoma (HCC) has not been well documented. Here, we assess the roles of PUSs in HCC.
Methods: RNA sequencing data of TCGA-LIHC and LIRI-JP were downloaded from the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), respectively. GSE36376 gene expression microarray was downloaded from the Gene Expression Omnibus (GEO). Proteomics data for an HBV-related HCC cohort was obtained from the CPTAC Data Portal. The RT-qPCR assay was performed to measure the relative mRNA expression of genes in clinical tissues and cell lines. Diagnostic efficiency was evaluated by the ROC curve. Prognostic value was assessed using the Kaplan-Meier curve, Cox regression model, and time-dependent ROC curve. Copy number variation (CNV) was analyzed using the GSCA database. Functional analysis was carried out with GSEA, GSVA, and clusterProfiler package. The tumor microenvironment (TME) related analysis was performed using ssGSEA and the ESTIMATE algorithm.
Results: We identified 7 PUSs that were significantly upregulated in HCC, and 5 of them (DKC1, PUS1, PUS7, PUSL1, and RPUSD3) were independent risk factors for patients’ OS. Meanwhile, the protein expression of DKC1, PUS1, and PUS7 was also upregulated and related to poor survival. Both mRNA and protein of these PUSs were highly diagnostic of HCC. Moreover, the CNV of PUS1, PUS7, PUS7L, and RPUSD2 was also associated with prognosis. Further functional analysis revealed that PUSs were mainly involved in pathways such as genetic information processing, substance metabolism, cell cycle, and immune regulation.
Conclusion: PUSs may play crucial roles in HCC and could be used as potential biomarkers for the diagnosis and prognosis of patients.

背景：假尿苷合酶（pseudouridine synthases, PUSs）已被证实与癌症存在关联，然而其在肝细胞癌（hepatocellular carcinoma, HCC）中的作用尚未得到充分阐释。本研究旨在评估PUSs在HCC中的功能角色。
方法：分别从癌症基因组图谱（Cancer Genome Atlas, TCGA）及国际癌症基因组联盟（International Cancer Genome Consortium, ICGC）下载TCGA-LIHC队列与LIRI-JP队列的RNA测序数据；从基因表达综合数据库（Gene Expression Omnibus, GEO）获取GSE36376基因表达芯片数据集；从CPTAC数据门户获取乙型肝炎病毒（hepatitis B virus, HBV）相关肝细胞癌队列的蛋白质组学数据。采用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction, RT-qPCR）检测临床组织与细胞系中基因的相对mRNA表达水平。通过受试者工作特征（receiver operating characteristic, ROC）曲线评估诊断效能；采用卡普兰-迈耶（Kaplan-Meier）曲线、Cox比例风险回归模型及时间依赖性ROC曲线分析预后价值。利用GSCA数据库分析拷贝数变异（copy number variation, CNV）；采用基因集富集分析（Gene Set Enrichment Analysis, GSEA）、基因集变异分析（Gene Set Variation Analysis, GSVA）及clusterProfiler分析包开展功能富集分析。采用单样本基因集富集分析（single-sample Gene Set Enrichment Analysis, ssGSEA）与ESTIMATE算法进行肿瘤微环境（tumor microenvironment, TME）相关分析。
结果：本研究筛选出7个在HCC中显著上调的PUSs，其中5个（DKC1、PUS1、PUS7、PUSL1及RPUSD3）为患者总生存期（overall survival, OS）的独立危险因素。同时，DKC1、PUS1及PUS7的蛋白表达水平同样上调，且与不良预后相关。上述PUSs的mRNA及蛋白表达均对HCC具有较高的诊断效能。此外，PUS1、PUS7、PUS7L及RPUSD2的CNV亦与预后显著相关。进一步功能富集分析显示，PUSs主要参与遗传信息加工、物质代谢、细胞周期及免疫调控等生物学通路。
结论：PUSs在HCC的发生发展中可能发挥关键作用，可作为患者诊断与预后评估的潜在生物标志物。