The Effects Of Toll Like Receptor4Signaling On The Gene Expression Network Of Hepatic Stellate Cells And The Cellular Interaction Of Hepatic Stellate Cells And Hepatocellular Carcinoma Cells

Part I:The Effects of Toll like Receptor4Signaling on the Gene Expression Network of Hepatic Stellate CellsObjective:The aim of this study was to investigate the effects of Toll like receptor4(TLR4) signaling on the gene expression network and the function of hepatic stellate cells (HSCs).Methods:High throughput genome-wide mRNA expression microarray was used to detect the differential gene expression of wild type (JS1) and TLR4knockout (JS2) HSCs with or without the stimulation by LPS, the exogenous ligand of TLR4, and high mobility group box1(HMGB1), the endogenous TLR4ligand and a damage associated molecular pattern molecule (DAMP). Limma algorithm was used to screen the differentially expressed genes and Fisher test was carried out for the analysis of significant gene ontology (GO-Analysis). For the pathway analysis, Fisher exact test was used to calculate the significance of each pathway and the result of multiple hypotheses was adjusted with false discovery rate (FDR). The differentially expressed genes were then combined and analysed based on KEGG database for the significant pathways and function that they participated in, to build the gene interaction networks (Gene-Act-Net) and signal transduction processes, and obtain the central core regulatory genes. The co-expression networks in the control and case groups were established on the key intersecting genes with both the significant GO analysis and pathway analysis. Topology analysis was used to obtain the main functional modules of TLR4-dependent differentially expressed genes in HSCs under the stimulation of TLR4ligands.Results:1. The gene expression spectrums of JS1and JS2cells were significantly different under basal condition. A total of5421different probes were tested with2748up-regulated and2673down-regulated. Within them there are genes related to fibrogenesis (Col Ⅰ, Col Ⅲ, FN1), matrix remodeling (TIMP2, TIMP3, MMP2), growth factors and their receptors (VEGFD, FGF7, IGF1and IGF1R, PDGFα and PDFGRα、PDGFR), chemokine and chemokine receptors (CXCL12, CXCL11, of CXCR7), inflammation and immune (IL6), transcription factors and some important signaling molecules (Jun, Stat3, MAPK1). Gene ontology and signaling pathway analysis showed that these genes are associated with fibrogenesis, inflammation and immunity, cell cycle, apoptosis, the regulation of tumor, and participate in NOD-like receptor, mTOR, JAK-STAT, chemokines, focal adhesion and immune-related signalings. The analysis of gene interaction network showed that Pik3r3, multiple molecule of histocompatibility complex2(H2), MAPK1, PDGFRa and β,JAK2, PTK2, PRKCA, integrin α and β, IGF1and IGF1R, Jun were the central regulatory factors. The co-expression analysis showed that TLR4, Selp, GSK3β, Ephb3, SDC3and Atp6ap1, FZD4, integrinβ5, PAK3, FN1, DAPK1, Rps6kal, Sema4f, caspase3, Fcgr3and Piasl played a central role for the differentially expressed gene spectrums in JS1and JS2cells.2. The gene transcriptional profiles between JS1and JS2cells are significantly different under LPS or HMGB1stimulation. GO-analysis showed that the differentially expressed genes function in cell metabolism, proliferation and apoptosis, and immune. Signaling pathway analysis showed that LPS upregulated genes that belong to Toll-like receptor, neurotrophic factor, immune, the spliceosome and nucleotide excision repair related signaling, and downregulated PPAR signaling pathway in JS1cells. In JS2cells, PPAR and renin-angiotensin signaling pathways were down-regulated by LPS treatment. Gene interaction network analysis indicated that in JS1cell with LPS stimulation, a variety of multiple histocompatibility complex2molecules (e.g., H2-B1, H2-Q2, H2-Q10, H2-T10), molecules of the MAPK family, Pik3r3, PRKCA, Iκbκb are central regulatory factors, while under HMGB1stimulation, molecules of the MAPK family, TRAF6, IGF1R, GPX4, Cyp2el, Gstp, Gsst, Mgst3were the core regulatory factors. The gene interaction networks in JS2cell under either LPS or HMGB1stimulation were simple and lack of core regulatory factors.3. Co-expression analysis demonstrated that molecules of the MAPK family, PDGFa, Hercl, Rb1, FGF18, CDK4, CDK7and PRKCA played a central role in the regulation of gene expression in JS1cells after LPS stimulation, wherea the key regulatory factors post HMGB1stimulation in JS1cells inlcuded Herd, JAK1, ODC1,Hist4h4, Traf6, molecules of the MAPK family and PRKCA. The co-expression networks of JS2cells with LPS or HMGB1stimulation were lack of similarities when compared with JS1cells.4. Topology analysis on the common differentially expressed genes induced by LPS and HMGB1resulted in29major functional modules. Genes in these modules may impact on cytoskeleton formation, fat metabolism, integrin signaling pathway, oxidative stress, chemokine and chemokine receptor, transmembrane receptor signal transduction and immune function of HSCs.Conclusions:TLR4signaling pathway significantly may impact on the gene transcriptional spectrum of HSCs under both basal and stimulated condition by TLR4ligands. It participates in the regulation of many functional genes expression in HSCs that are associated with fibrogenesis, inflammation and chemotaxis properties, as well as growth, apoptosis and metabolism processes.Part Ⅱ:The Effects of Toll like Receptor4Signaling on the Cellular Interaction of Hepatic Stellate Cells and Hepatocellular Carcinoma CellsObjective:The aim of this study was to explore the effects and underling mechanisms of Toll like Receptor4(TLR4) signaling on the interaction of hepatic stellate cells (HSCs) and hepatocellular carcinoma (HCC) cells.Methods:Real-time quantitative PCR, Western Blot, enzyme-linked immunosorbent assay (ELISA) were used to varify the mRNA transcription and protein translation of the differentially expressed genes in wilde type HSC (JS1) when compared with those in TLR4konkout HSC (JS2). The effect of HSCs on the proliferation of HCC cells was investigated by indirect co-culture of JSl and JS2cell culture supernatants with HCC lines (i.e., HepG2, Hepa1-6), followed by CCK8assay.Results:1. The mRNA expression level of some key fibrogenic genes (Col Ⅰ, Col Ⅲ and FN1), transcription factors (SP1, STAT3and Fas), growth factors (FGF7, FIGF, IGF1), chemokines and their receptors (CXCL12, CXCR7), infalmmatory cytokines (IL6) were significantly higher in JS1cells when compared to JS2cells, whereas no significant changes were found in the level of PDGFa, CXCL11, and CXCR4mRNA expression. In line with the mRNA expression profile, the protein level of Col I, MMP2, DLK1, IGF1, FIGF and CXCL12were also upregulated in JS1cells.2. Both JSl and JS2promote the proliferation of HepG2、Hepal-6cells, with a much stronger effect observed in JS1cells.Conclusions:TLR4signaling mediates the important biological properties of HSC including fibrogenesis and their inflammatory phenotype. It mediates the expression of chemotactic cytokines CXCL12and receptors CXCR7and growth factor VEGFD in TLR4intact HSC. These tumor associated secretory factors may mediate the promoting effect of HSCs on HCC cell proliferation.