Cai Changzhou, Wang Weilin, Tu Zhenhua
Key Laboratory of Precision Diagnosis and Treatment for Hepatobiliary and Pancreatic Tumor of Zhejiang Province, First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310003, China.
Ward of Liver transplant, Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery. First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310003, China.
J Cancer. 2019 Jan 1;10(2):355-366. doi: 10.7150/jca.27832. eCollection 2019.
Methylation plays a significant role in the etiology and pathogenesis of hepatocellular carcinoma (HCC). The aim of the present study is to identify aberrantly methylated-diferentially expressed genes (DEGs) and dysregulated pathways associated with the development of HCC through integrated analysis of gene expression and methylation microarray. Aberrantly methylated-DEGs were identified from gene expression microarrays (GSE62232, GSE74656) and gene methylation microarrays (GSE44909, GSE57958). Functional enrichment and pathway enrichment analyses were performed through the database of DAVID. Protein-protein interaction (PPI) network was established by STRING and visualized in Cytoscape. Subsequently, overall survival (OS) analysis of hub genes was performed by OncoLnc. Finally, we validated the expression level of CDCA5 by quantitative real-time PCR (qRT-PCR) and western blotting, and performed Immunohistochemical experiments utilizing a tissue microarray. Cell growth assay and flow cytometry were behaved to explore the function of CDCA5. Aberrantly methylated-DEGs were enriched in biological process, molecular function, cellular component and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Among them, cell cycle was enriched most frequently, and some terms associated with cancer were enriched, such as p53 signaling pathway, pathways in cancers, PI3K-Akt signaling pathway and AMPK signaling pathway. After survival analysis and validation in TCGA database including methylation and gene expression status, 12 hub genes were identified. Furthermore, the expression level of new gene CDCA5 was validated in HCC cell lines and hepatic normal cell lines through qRT-PCR and western blotting. In additional, immunohistochemistry experiments revealed higher CDCA5 protein expression from HCC tumor tissues compared with paracancer tissues by tissue microarray. Finally, through loss of function, we demonstrated that CDCA5 promoted proliferation by regulating the cell cycle. In summary, the present study implied possible aberrantly methylated-differentially expressed genes and dysregulated pathways in HCC by bioinformatics analysis and experiments, which could be helpful in understanding the molecular mechanisms underlying the development and progression of HCC. Hub genes including CDC20, AURKB, BIRC5, RRM2, MCM2, PTTG1, CDKN2A, NEK2, CENPF, RACGAP1, GNA14 and especially the new gene CDCA5 may serve as biomarkers for diagnosis, treatment and prognosis of HCC.
甲基化在肝细胞癌（HCC）的病因学和发病机制中起着重要作用。本研究的目的是通过对基因表达和甲基化微阵列的综合分析，鉴定与HCC发生发展相关的异常甲基化差异表达基因（DEGs）和失调通路。从基因表达微阵列（GSE62232、GSE74656）和基因甲基化微阵列（GSE44909、GSE57958）中鉴定出异常甲基化的DEGs。通过DAVID数据库进行功能富集和通路富集分析。利用STRING建立蛋白质-蛋白质相互作用（PPI）网络，并在Cytoscape中进行可视化。随后，通过OncoLnc对枢纽基因进行总生存（OS）分析。最后，我们通过定量实时PCR（qRT-PCR）和蛋白质印迹法验证了CDCA5的表达水平，并利用组织芯片进行了免疫组织化学实验。进行细胞生长测定和流式细胞术以探究CDCA5的功能。异常甲基化的DEGs在生物学过程、分子功能、细胞成分和京都基因与基因组百科全书（KEGG）通路中富集。其中，细胞周期富集最为频繁，并且还富集了一些与癌症相关的术语，如p53信号通路、癌症中的通路、PI3K-Akt信号通路和AMPK信号通路。在包括甲基化和基因表达状态的TCGA数据库中进行生存分析和验证后，鉴定出12个枢纽基因。此外，通过qRT-PCR和蛋白质印迹法在HCC细胞系和肝正常细胞系中验证了新基因CDCA5的表达水平。另外，免疫组织化学实验通过组织芯片显示，与癌旁组织相比，HCC肿瘤组织中CDCA5蛋白表达更高。最后，通过功能缺失实验，我们证明CDCA5通过调节细胞周期促进增殖。总之，本研究通过生物信息学分析和实验揭示了HCC中可能存在的异常甲基化差异表达基因和失调通路，这有助于理解HCC发生发展的分子机制。包括CDC20、AURKB、BIRC5、RRM2、MCM2、PTTG1、CDKN2A、NEK2、CENPF、RACGAP1、GNA14在内的枢纽基因，尤其是新基因CDCA5，可能作为HCC诊断、治疗和预后的生物标志物。
