Wang Qingqing, Yu Xiaoyan, Zheng Zhewen, Chen Fengxia, Yang Ningning, Zhou Yunfeng
Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan, China.
Department of Radiation Oncology and Medical Oncology, Zhongnan Hospital, Wuhan University, Wuhan, China.
PeerJ. 2021 May 3;9:e11342. doi: 10.7717/peerj.11342. eCollection 2021.
Hepatocellular carcinoma (HCC) is one of the deadliest tumors. The majority of HCC is detected in the late stage, and the clinical results for HCC patients are poor. There is an urgent need to discover early diagnostic biomarkers and potential therapeutic targets for HCC.
The GSE87630 and GSE112790 datasets from the Gene Expression Omnibus (GEO) database were downloaded to analyze the differentially expressed genes (DEGs) between HCC and normal tissues. R packages were used for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses of the DEGs. A Search Tool for Retrieval of Interacting Genes (STRING) database was used to develop a protein-protein interaction (PPI) network, and also cytoHubba, Molecular Complex Detection (MCODE), EMBL-EBI, CCLE, Gene Expression Profiling Interactive Analysis (GEPIA), and Oncomine analyses were performed to identify hub genes. Gene expression was verified with a third GEO dataset, GSE25097. The Cancer Genome Atlas (TCGA) database was used to explore the correlations between the hub genes and clinical indexes of HCC patients. The functions of the hub genes were enriched by gene set enrichment analysis (GSEA), and the biological significance of the hub genes was explored by real-time polymerase chain reaction (qRT-PCR), western blot, immunofluorescence, CCK-8, colony formation, Transwell and flow cytometry assays with loss-of-function experiments in vitro.
Centromere protein N (CENPN) was screened as a hub gene affecting HCC tumorigenesis. Evaluation by Cox regression showed that a high level of CENPN expression was an independent danger variable for poor prognosis of HCC. GSEA showed that high CENPN expression was linked to the following pathways: liver cancer subclass proliferation, cell cycle, p53 signaling pathway, Rb1 pathway, positive regulation of cell cycle G1/S phase transition, and DNA damage response signal transduction by p53 class moderators. Further cell experiments showed that knocking down CENPN expression decreased the proliferation and colony-forming abilities of HepG2 and Huh7 cells as well as Ki67 expression in these cell lines. The cell cycle was arrested in G1 phase, which is consistent with previous experiments on CENPN downregulation., but neither migration nor invasion were significantly affected. Western blot results revealed that the expression of p53, p27, p21, CDK4, cyclin D1, CDK2, cyclin E, pRb, E2F1 and c-myc decreased after CENPN knockdown, but there was no significant change in total Rb levels. In addition, CENPN-knockdown cells subjected to irradiation showed significantly enhanced of -H2AX expression and reduced colony formation.
CENPN functions as an oncogene in HCC and may be a therapeutic target and promising prognostic marker for HCC.
肝细胞癌（HCC）是最致命的肿瘤之一。大多数HCC在晚期被检测到，HCC患者的临床治疗效果较差。迫切需要发现HCC的早期诊断生物标志物和潜在治疗靶点。
从基因表达综合数据库（GEO）下载GSE87630和GSE112790数据集，以分析HCC与正常组织之间的差异表达基因（DEG）。使用R包对DEG进行京都基因与基因组百科全书（KEGG）和基因本体论（GO）富集分析。利用检索相互作用基因的搜索工具（STRING）数据库构建蛋白质-蛋白质相互作用（PPI）网络，并进行细胞枢纽蛋白分析、分子复合物检测（MCODE）、欧洲生物信息学研究所（EMBL-EBI）、癌症细胞系百科全书（CCLE）、基因表达谱交互式分析（GEPIA）和Oncomine分析，以鉴定枢纽基因。用第三个GEO数据集GSE25097验证基因表达。利用癌症基因组图谱（TCGA）数据库探讨枢纽基因与HCC患者临床指标之间的相关性。通过基因集富集分析（GSEA）富集枢纽基因的功能，并通过实时聚合酶链反应（qRT-PCR）、蛋白质免疫印迹、免疫荧光、CCK-8、集落形成、Transwell和流式细胞术检测在体外进行功能缺失实验，探讨枢纽基因的生物学意义。
着丝粒蛋白N（CENPN）被筛选为影响HCC肿瘤发生的枢纽基因。Cox回归评估显示，高水平的CENPN表达是HCC预后不良的独立危险因素。GSEA显示，高CENPN表达与以下途径相关：肝癌亚类增殖、细胞周期、p53信号通路、Rb1通路、细胞周期G1/S期转换的正调控以及p53类调节剂的DNA损伤反应信号转导。进一步的细胞实验表明，敲低CENPN表达可降低HepG2和Huh7细胞的增殖和集落形成能力以及这些细胞系中Ki67的表达。细胞周期停滞在G1期，这与之前关于CENPN下调的实验一致，但迁移和侵袭均未受到显著影响。蛋白质免疫印迹结果显示，敲低CENPN后，p53、p27、p21、CDK4、细胞周期蛋白D1、CDK2、细胞周期蛋白E、pRb、E2F1和c-myc的表达降低，但总Rb水平无显著变化。此外，接受照射的CENPN敲低细胞显示γ-H2AX表达显著增强，集落形成减少。
CENPN在HCC中起癌基因作用，可能是HCC的治疗靶点和有前景的预后标志物。
