In our studies of this paper, WGCNA was employed to further explore gene expression alternation between HBV associated HCC tissues and adjacent normal tissues. There are many advantages of WGCNA over other traditional methods for differential expression analysis, because it focuses on co-expression patterns and the biofunctional relevant modules that consist of related genes will be discovered. Key/hub genes in the modules related to some specific traits may serve as clinical detective biomarkers or therapeutic targets 24–26.
HCC is a common consequence of HBV chronic infection. HBV chronic infection can change the expression of genes of hepatic cells in different ways. HBV-DNA integrations randomly distributed among chromosomes into host chromosomes HBV-infected liver cells, and the integrations of HBV will alternate the expression of cellular genes near the integrating sites 27. Noncoding RNAs, such as miRNAs, lncRNAs and circRNAs, also take parts in the pathogenesis of HBV-associated HCC 28–30. Viral protein HBx plays important roles in hepatocarcinogenesis by interfering with telomerase activity 31, affecting hepatocellular apoptosis 32, 33, and upregulating the transcriptional activation of human telomerase transcriptase34. Interesting, our GO cellular component enrichment results of genes of module turquoise, which is the co-expression module most positively related to the tumor trait, showed that some genes of this module were enriched in telomeric region.
In our study, modules changed significantly between HBV associated HCC tissues and normal adjacent normal tissues included the midnight blue, magenta, turquoise, royal blue modules which were up-regulated in HCC, whereas the blue, tan, yellow modules which were down-regulated in HCC. These up-regulated modules and down-regulated modules mentioned above were classified into two main groups through eigengene adjacency clustering as shown in Fig. 2 (A). Among these modules, the turquoise module is the most significant module related to the tumor trait. According the KEGG results, the turquoise module, enriched in DNA replication, p53 signaling pathway, cell cycle, especially HTLV-1 infection associated pathway, increased in HBV-associated HCC tissues. The acceleration of genome DNA replication, the misregulation of tumor suppressor p53, and the abnormal cell cycle-associated pathway, all played critical roles in the initiation and development of HCC. Furthermore, the enrichment of HTLV-1 infection associated pathway indicated that some signaling pathways associated with viral infections were also significantly activated in HBV-associated HCC tissues.
According to the topological network analysis from the turquoise model, 7 key genes were identified playing critical roles in the network. They are CCNB1, GINS1, PRC1, KIF20A, NUSAP1, NEK2, and BUB1B. CCNB1 is the gene, which encodes a regulatory protein involved in mitosis, and it is expressing predominantly during G2/M phase 35. It was reported that CDK1-CCNB1 enables MPS1 kinetochore localization to create a spindle checkpoint-permissive state36.GINS1 encodes subunit 1 of the GINS complex, and the complex is essential for the initiation of DNA replication 37. It has been reported that the high expression of KIF20A is associated to poor prognosis of glioma patients, and KIF20A can be a potential immunotherapeutic target for glioma 38, 39. PRC1 encodes a protein involved in cytokinesis process, and the protein maintains a high level during the S and G2/M phases of mitosis 40. NUSAP1 encodes a nucleolar-spindle-associated protein playing a role in spindle microtubule organization 41. NEK2 has shown it is involved in some different cancers: NEK2promotes aerobic glycolysis in multiple myeloma42; targeting NEK2 attenuates glioblastoma growth and radioresistance43; NEK2 can be a prognostic biomarker of hepatocellular carcinoma 44. BUB1B encodes a kinase playing a role in spindle checkpoint function. The kinase localizes to the kinetochore and plays a role in the inhibition of the anaphase-promoting complex/cyclosome (APC/C) 45. It has been reported that individuals having biallelic TRIP13 or BUB1B mutations are prone to having embryonal tumors, and their cells display severe spindle assembly checkpoint (SAC) impairment 46. In our expression and survival analyses, all 7 key/hub genes, which were identified in our selected module,can be the biomarkers and potential therapeutic targets of HBV-associated HCC.
In summary, we provide a systematic biological interpretation of gene expression data derived from HBV associated HCC tissues and adjacent normal tissues. Based on WGCNA, there were 21 modules identified, and the turquoise module which was the most significant module relating to tumor trait was selected to be analyzed in detail. Our results showed that the turquoise module, enriched in DNA replication, p53 signaling pathway, cell cycle, and HTLV-1 infection associated pathway, was activated in HBV-associated HCC tissues. 7 hub/key genes were identified. All of our finding may provide new perspectives to the understanding of pathways and genes underlying HBV-associated HCC, and further analysis are needed.