The progression-related sub-network deregulated in hepatitis C infection stage was constructed with four nodes (HLA-DQA1, HLA-DQB1, HLA-DPA1 and CD74) and interactions among them (). We proposed that this sub-network is potentially related to cirrhosis-HCC-risk, considering that those genes are differentially expressed in hepatitis C stage. It could be seen that many important functions, including positive regulation of B cell proliferation, negative regulation of apoptosis, etc., are enriched significantly (P value<0.01) in this sub-network (). In this sub-network, CD74 is the major histocompatibility complex, class II invariant chain. HLA-DQA1 and HLA-DPA1 belong to the HLA class II alpha chain paralogues while HLA-DQB1 belongs to the HLA class II beta chain paralogues.
The class II molecule is a heterodimer consisting of an alpha (DPA, DQA) and a beta (DPB, DQB) chain, both anchored in the membrane. It plays a central role in the immune system by presenting peptides derived from extracellular proteins. Within the DP and DQ molecules both the alpha chain and the beta chain contain the polymorphisms specifying the peptide binding specificities, resulting in up to 4 different molecules.
DQB1*0201 allele has been confirmed to be closely correlated with the progression of liver injury in chronic HCV infection 
. Meanwhile, DQA1*0103 appears to provide protection against chronic active infection with hepatitis C virus 
. Moreover, HLA-DQA1 and DQB1 are associated with development of cirrhosis, and DQB1 might be a risk factor for the occurrence of HCC 
. Since full function of major histocompatibility complex needs both the invariant chain and variant chain, the PPIs among DQA1, DQB1 and CD74 may participate in the induction of hepatitis C and the development and progression from hepatitis C to HCC.
Our network-based view shows that three progression-related sub-networks appear deregulated initially in cirrhosis stage. One of the sub-networks is composed of four nodes (VWF, LUM, COL1A1 and COL1A2) with four interactions among them (), and it can be considered an HCC-risk related sub-network. Certain functions, like, cell adhesion, are significantly enhanced (P value<0.01) in this sub-network (). Within the same sub-network, VWF functions as an antihemophilic factor carrier and a platelet-vessel wall mediator in the blood coagulation system. LUM is a member of the small leucine-rich proteoglycan (SLRP) family that includes decorin, biglycan, fibromodulin, keratocan, epiphycan, and osteoglycin. In these bifunctional molecules, the protein moiety binds collagen fibrils and the highly charged hydrophilic glycosaminoglycans regulate interfibrillar spacings. Lumican is the major keratan sulfate proteoglycan of the cornea but is also distributed in interstitial collagenous matrices throughout the body. Lumican may regulate collagen fibril organization and circumferential growth, corneal transparency, and epithelial cell migration and tissue repair. COL1A1 is the pro-alpha1 chains of type I collagen whose triple helix comprises two alpha1 chains and one alpha2 chain. COL1A2 is the pro-alpha2 chain of type I collagen whose triple helix comprises two alpha1 chains and one alpha2 chain. Type I is a fibril-forming collagen found in most connective tissues and is abundant in bone, cornea, dermis and tendon.
Significant functions (P value<0.01) of the progression-related sub network deregulated initially from cirrhosis.
COL1A1 and COL1A2 are associated with liver fibrogenesis 
. Moreover, COL1A2 is involved in the development or progression of hepatoma 
. VWF mRNA has been shown to be significantly upregulated in both fibrosis and HCC 
. In patients with fulminant hepatic failure and liver cirrhosis, circulating plasma VWF antigen levels are extremely high 
. Many fibrin thrombi have been found in the hepatic sinusoids in acute liver failure, suggesting a role for intravascular coagulation in the pathogenesis of hepatic necrosis 
. In cirrhotic liver tissue 
and even tissue from patients in early stages of alcoholic liver diseases 
, VWF immunostaining shows positive cells predominantly at the scar–parenchyma interface, within the septum, and in the sinusoidal lining. Portal or hepatic vein thrombosis is often observed in advanced cirrhosis 
and microthrombi formation has been found in one or multiple organs in half of autopsied cirrhotics 
. This hypercoagulable state in liver diseases may be involved in hepatic parenchymal extinction, the acceleration of liver fibrosis, and disease progression. Lumican has diverse biologic roles but has been thought to be primarily involved in fibrosis of the extracellular matrix through the binding of collagen fibrils and regulation of their lateral growth 
. Lumican expression is also increased with progression of hepatic fibrosis in rats 
. Decreased sulforylation of lumican side chains stimulates macrophage adhesion and the cellular inflammatory response 
, suggesting that changes in the structure of lumican may promote the inflammatory process that precedes and enhances collagen deposition during the process of hepatic fibrosis. Taken together, it implies that PPIs among COL1A1, COL1A2, VWF and LUM may participate in the induction of cirrhosis and play roles in the progression from cirrhosis to HCC.
The progression-related sub-networks deregulated from BCLC stage 0 HCC included 11 sub-networks. The biggest sub-network in this category was comprised of 34 nodes with 42 PPIs and 2 TRIs (). Many functions are significantly enriched in this sub-network (), including induction of apoptosis, cell cycle arrest, etc. A core protein of this sub-network is ESR1, which interacts with most nodes in this sub-network. This protein regulates the expression of FOS, and subsequently FOS regulates HBA1. HBA1 participate in a smaller sub-network that constructed by four nodes (HBA1, HBB, HBA2, HBG2) with interactions among them. ESR1 is an estrogen receptor, a ligand-activated transcription factor composed of several domains important for hormone binding, DNA binding, and activation of transcription. FOS can dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP-1. As such, the FOS proteins have been implicated as regulators of cell proliferation, differentiation, and transformation. In some cases, expression of the FOS gene has also been associated with apoptotic cell death. The alpha (HBA) and beta (HBB) loci determine the structure of the 2 types of polypeptide chains in adult hemoglobin, Hb A. The normal adult hemoglobin tetramer consists of two alpha chains and two beta chains. Mutation in beta globin is the cause of sickle cell anemia. Absence of beta chain causes beta-zero-thalassemia. Reduced amounts of detectable beta globin causes beta-plus-thalassemia.
Significant functions (P value<0.01) of the progression-related sub network deregulated initially from BCLC stage 0 HCC.
Animal models and human epidemiologic studies have suggested that estrogens act as tumor promoters and might induce hepatocarcinogenesis 
. The estrogens exert the effects by binding to estrogen receptors (ESR). The genetic polymorphisms within ESRs could influence the effects of estrogens, which in turn results in genotype-dependent differences in risk for hepatocellular carcinoma. Indeed, the polymorphisms in the 5′ end of the ESR a (ESR1) gene have been shown to be associated with an increased hepatocellular carcinoma risk, supporting the involvement for the estrogen-ESR axis in the estrogen-induced hepatocarcinogenesis 
. The oncogene c-fos (FOS), which is required for quiescent cells to enter the cell cycle 
, also is up-regulated in HCC 
. HBV X peptide has been shown to activate the c-fos gene, which is postulated to contribute to hepatocarcinogenesis 
. Our result implies that the association between ESR1 and FOS may participate in the process of HCC initiation and may play roles in the progression of carcinogenesis from early to advanced HCC. Moreover, the hemoglobin (Hb A) level may be deregulated by this association in the progression of carcinogenesis from early HCC.
Previous studies have indicates that when hepatitis C patients establishes cirrhosis, the HCC incidence will increase largely. Cirrhosis is an important stage in the progression from hepatitis C to HCC. In the cirrhosis-related network, we can recognize some interesting components and relationships (). The nodes that appeared in cirrhosis-HCC-risk sub-network are located close to the nodes belonging to HCC-risk sub-networks. They are connected by CD44. The CD44 is a cell-surface glycoprotein. The CD74, a protein of cirrhosis-HCC-risky related sub-network, is also membrane protein and the rest of the sub-network proteins are located extracellularly. Similarly, the proteins in HCC-risk related sub-networks are also located in extracellular region. It could be anticipated that the synergistic action between deregulated cirrhosis-HCC-risky related sub-network and HCC-risky related sub-networks could be one of the reasons for the dramatic increase of HCC incidence when hepatitis C patients develop cirrhosis. As we mentioned before, the dysfunction of cirrhosis-HCC-risk related sub-network emerges from hepatitis C, while the dysfunction of HCC-risk related sub-networks gets started from cirrhosis. Those relationships and results supported by the experimental evidences imply that the dysfunction of HCC-risk related sub-networks is under the influence of the dysfunction of the cirrhosis-HCC-risk related sub-network.
Cirrhosis-related network mentioned in discussion.
Although we have identified progression-related sub-networks for HCV induced hepatocarcinogenesis and recognized some potential synergistic actions of proteins and genes in the sub-network, our approach has certain limitations. First, we only use experimentally confirmed interaction data in our analysis. So some potential associations among DEGs may be lost. Second, our analysis depends not only on biological reality but also on sampling, experimental conditions, and reported results; there could be other genes and proteins that significantly contribute to network functions not yet addressed.
In conclusion, we constructed the disease-related biological networks through the integration of those DEGs, PPIs and TRIs data for different stages of hepatitis C infected disease, including liver cancer. By comparison of the disease-related biological networks between each stage, the dynamic characteristics of the networks show that they mostly reflect the important features of the disease development and progression, which provides important information for us to explore the underlying mechanisms of the diseases. We identified progression-related sub-networks in the development and progression through dynamic biological network analysis and annotated significant functions of these sub-networks. Text mining results from published literature confirmed our hypothesis largely in the examples randomly chosen. It implies that these progression-related sub-networks, especially the ones which are annotated with significant functions, can be helpful in the understanding of molecular mechanism that underlies the progression of HCV induced hepatocarcinogenesis.