Abstract
BackgroundHepatocellular 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.MethodsThe 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.ResultsCentromere 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 γ-H2AX expression and reduced colony formation.ConclusionCENPN functions as an oncogene in HCC and may be a therapeutic target and promising prognostic marker for HCC.
Highlights
Liver cancer is among the most common malignant tumors
To much better understand the potential molecular mechanism of Centromere protein N (CENPN) in HepG2 and Huh7 cells, we focused on three signaling pathways according to the gene set enrichment analysis (GSEA) results— P27, P21 and Rb/E2F1, which play a vital role in Hepatocellular carcinoma (HCC) and various cancers (Gartel, 2009; Razavipour, Harikumar & Slingerland, 2020; Rubin, Sage & Skotheim, 2020)
Our work showed for the first time that CENPN plays a carcinogenic role in HCC tumorigenesis
Summary
Liver cancer is among the most common malignant tumors. Despite a declining mortality rate, liver cancer remains to be one of the leading 10 causes of cancer-related fatalities in many countries (Siegel, Miller & Jemal, 2020). Hepatocellular carcinoma (HCC) is the most common type of liver cancer, as well as its occurrence and development are closely related to genetic changes, genetic susceptibility and alterations in key signaling pathways (Marquardt & Thorgeirsson, 2014). The Cancer Genome Atlas (TCGA) database was used to explore the correlations between the hub genes and clinical indexes of HCC patients. 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. 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
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