Su1002 Pyrosequencing Analysis of the Synonymous Codon Usage Pattern of rtN236T Mutation in 1418 Cases of Chronic Hepatitis B Patients Treated With Adefovir Dipivoxil

Abstract

[Background] We previously reported that soluble factors produced from hepatocyte could contribute to the immune pathogenesis of HBV-related inflammation and progression of HCC. (JID2010, JG2012). It has been reported that the progression of fibrosis and HCC is different among the patients infected with various HBV genotypes and HCV. [Aim] The aim of this study is to analyze the effects of chemokines derived from HBV (genotype A, B and C) and HCV -infected hepatocytes and hepatoma cells for the various kinds of immune cells in vitro and in vivo. [Methods] Plasmid Construction: At least 5 clones of each fragment were sequenced and consensus sequence was identified and used as template for 1.24-fold the HBV genome of different genotypes (2 strains each for A, B and C). Cells: Huh7 and HepG2 cells were used for the experiment of transfection. cDNA array: cDNA array for 84chemokine-related genes were carried out. Flow-cytometry analysis: The expression of NKG2D and NKG2A on the NK cells, PD1 on CD8+ cells and MHC class I-related molecule A (MICA) on the HCC cells were analyzed by FACS canto-II. Migration Assay: various kinds of human primary lymphocyte were negatively isolated by MACS beads and used for the migration assay. NOG Mouse model: NOG mice were used for HBV-expressed HCC-transplanted-mouse model. Human primary lymphoid cells were introduced into this mouse. Immunohistochemistry: The liver samples obtained from 20 HBV related HCC patients, HCV-related HCC patients and non HBV/HCV-related HCC patients were stained with CX3CL1, CX3CR1, CD3 and CD56 antibodies. [Results] CCBP2, IL18, CMTM3, CMTM4, CX3CL1, CXCL12, TYMP, GPR81, LTB4R, SDF2 and VHL were well expressed (threshold cycle value range<28) in HBV-replicated-Huh7 cells and significantly up-regulated in comparison to mock transfected Huh7 cells (p<0.01). Among these chemokines-related genes, the expression level of CX3CL1 mRNA was significantly different among genotype A, B and C (relative expression: 1.41, 2.04 and 3.31) (p<0.05). The migration activity of CD16+CD56dimNK cells, CD4+ cells, CD8+ cells and CD14+ Monocyte but not CD19+ B cells or Tregs cells were significantly different among genotype A, B and C in vitro (p<0.05). Moreover, the neutralization of CX3CL1 could cancel these differences of migration activities. Significantly lower expression of NKG2D on CX3CR1+NK cells were observed in primary human peripheral blood mononuclear cells obtained from HBV-related HCC patients and the Tumor-infiltrating lymphocytes in HCC with HBV expression in NOG mouse model in comparison to those is non-HBV related HCC. (p<0.05). [Conclusion] The differential expre s s ion l eve l s o f CX3CL1 and MICA and the d i f f e r en t i a l a t t r ac t i on of NKG2DlowCX3CR1+NK cells and PD-1+ CX3CR1+ CD8 T cells might be involved in HBV persistent infection and hepatocellular carcinoma by affecting immune cells.