Application of network construction to estimate functional changes to insulin receptor substrates 1 and 2 in Huh7 cells following infection with the hepatitis C virus

Abstract

Hepatitis C virus (HCV) is closely associated with insulin resistance (IS), acting primarily by interfering with insulin signaling pathways, increasing cytokine-mediated (tumor necrosis factor α, interleukin 6) inflammatory responses and enhancing oxidative stress. In the insulin signaling pathways, the insulin receptor substrate (IRS) is one of the key regulatory factors. The present study constructed gene regulatory sub‑networks specific for IRS1 and IRS2 in Huh7 cells and HCV‑infected Huh7 (HCV‑Huh7) cells using linear programming and a decomposition algorithm, and investigated the possible mechanisms underlying the function of IRS1/2 in HCV‑induced IS in Huh7 cells. All data were obtained from GSE20948 of the Gene Expression Omnibus database from the National Center for Biotechnology Information. Genes which were significantly differentially expressed between Huh7 and HCV‑Huh7 cells were analyzed using the significance analysis of microarray algorithm. The top 50 genes, including IRS1/2, were used as target genes to determine the gene regulatory networks and next the sub‑networks of IRS1 and IRS2 in HCV‑Huh7 and Huh7 cells using Gene Regulatory Network Inference Tool, an algorithm based on linear programming and the decomposition process. The IRS1/2 sub‑networks were divided into upstream/downstream groups and activation/suppression clusters, and were further analyzed using Molecule Annotation System 3.0 and Database for Annotation, Visualization, and Integrated Discovery software, two online platforms for enrichment and clustering analysis and visualization. The results indicated that in Huh7 cells, the downstream network of IRS2 is more complex than that of IRS1, indicating that the insulin metabolism in Huh7 cells may be primarily mediated by IRS2. In HCV‑Huh7 cells, the downstream pathway of IRS2 is blocked, suggesting that this may be the underlying mechanism in HCV infection that leads to insulin resistance. The present findings add a further dimension to the understanding of the pathological mechanisms of HCV infection-associated insulin resistance, and provide novel concepts for insulin resistance and glucose metabolism research.

Figure 1

IRS1 and IRS2 network in Huh7 and HCV-infected Huh7 cells. (A) IRS1 network in Huh7 cells. (B) IRS2 network in Huh7 cells. (C) IRS1 network in HCV-Huh7 cell lines. (D) IRS2 network in HCV-Huh7 cell lines. Red circle with gene name indicates the genes upstream of IRS1 or IRS2. Green circle with gene name indicates the genes downstream of IRS1 or IRS2. Solid red line with red arrow indicates the activation of the gene in the upstream of IRS1 or IRS2 to IRS1 or IRS2. Dashed red line with red dot indicates the inactivation role of the gene upstream of IRS1 or IRS2 to IRS1 or IRS2. Solid green line with green arrow indicates the activation role of IRS1 or IRS2 to the gene downstream of IRS1 or IRS2. Dashed green line with green dot indicates the inhibitory role of IRS1 or IRS2 to the gene downstream of IRS1 or IRS2. IRS, insulin receptor substrate; HCV, hepatitis C virus; KLF10, kruppel-like factor 10; STC2, stanniocalcin 2; INHBE, inhibin βE; LYPD1, LY6/PLAUR domain containing 1; SOCS2, suppressor of cytokine signaling 2; RCN1, EF-hand calcium binding domain; FHL2, four and a half LIM domains 2; SLC16A14, solute carrier family 16 (monocarboxylic acid transporters), member 14; ASNS, asparagine synthetase (glutamine-hydrolyzing); ARRDC4, arrestin domain containing 4; SAMD5, sterile α motif domain containing 5; ATF3, activating transcription factor 3; PRNP, prion protein; PPARGC1A, peroxisome proliferator-activated receptor γ, coactivator 1α; OSMR, oncostatin M receptor; SFT2D3 WDR33, SFT2 domain containing3 WD repeat domain 33; INADL, InaD-like (Drosophila); TGFB1I1, transforming growth factor β1 induced transcript 1; BDNF, brain-derived neurotrophic factor; BHLHE41, basic helix-loop-helix family, member e41; RAB27B, RAB27B member RAS oncogene family; PCK2, phosphoenolpyruvate carboxykinase 2 (mitochondrial); PLD6, phospholipase D family, member 6; ZNF295, zinc finger protein 295; PLA1A, phospholipase A1 member A; SAMD5, sterile α motif domain containing 5; UAP1L1, UDP-N-acteylglucosamine pyrophosphorylase 1-like 1; LOC100506392, B3 domain-containing proteinLOC_Os12g40080-like.

Figure 2

Comparison with the hypothesis of previous literature and the present study. Solid lines with an arrow indicate strengthening effects, dashed lines with an arrow indicate inhibitory effects. Black text indicates the hypothesis of previous literature, red text indicates the hypothesis of the current study. HCV, hepatitis C virus; NS5A, non-structural protein 5A; TNFα, tumor necrosis factor α; PP2A, protein phosphatase 2A; JNK, c-Jun N-terminal kinase; ER, endoplasmic reticulum; IRS1/2, insulin receptor substrate 1/2; PI3K, phosphoinositide 3-kinase; PDK1, 3-phosphoinositide-dependent kinase 1; mTOR, mammalian target of rapamycin; Akt, protein kinase B; MAPK, p38 mitogen-activated protein kinase; FOXO1, forkhead box O1; G6Pase-α, glucose-6-phosphatase-α; PEPCK, phosphoenolpyruvate carboxykinase; JAK, janus kinase; STAT, signal transducer and activator of transcription; PTEN, phosphatase and tensin homolog deleted on chromosome 10.