The Roles Of Hbx Protein And CIITA Epigenetic Silence In Mastering HLA-DR Expression In HepG2and L02Cell Lines

Hepatitis B virus (HBV) infection is a serious public health problem worldwide.Hepatocellular carcinoma (HCC) is closely related with HBV infection, which is one of themain causes of HCC in China. However, the precise mechanism of HBV in the oncogenesisof HCC remains unclear.The lever of host immune response is one of the most important factors affectingoncogenesis, progression and prognosis of tumors. The immune escaping of HCC cells playsan important role in the development and progression of HCC. To explore the molecularmechanisms of HCC immune escaping helps to generate immunotherapy strategies. Majorhistocompatibility class II (MHC-II) molecules play an important role in anti-infection andantineoplastic immune responses by presenting the foreign antigens to Th cells. Someresearches suggest that the lack of human leukocyte antigen DR (HLA-DR) expression isconsidered to be a key mechanism that enables some gastrointestinal and breast cancer cells toescape from immune surveillance. Class II transactivator (CIITA) is considered to be a switchmolecule of mastering HLA-DR expression. It was reported that CIITA gene silence was thekey mechanism for the lack of induced HLA-DR expression in some gastrointestinal andbreast cancer cells. Our previous studies showed that CIITA and HLA-DR expression wereabsent in tumor cells of the HBV related HCC tissues, while different degrees of CIITA andHLA-DR expression were found on hepatocytes of paired adjacent non-tumor tissues. Thesefindings indicated that the lack of HLA-DR expression in HCC may be related with theCIITA gene silence and played an important role in the HCC immune escape. However, themechanism of CIITA gene silence in HCC has not been clarified.DNA methylation and/or histone deacetylation in promoters of host genes are believed tobe key mechanisms of gene silence. Some studies have indicated that DNA methylationand/or histone deacetylation of CIITA promoter IV were the key epigenetic mechanisms of CIITA and HLA-DR silence in some cancer cells. There are different epigenetic mechanismsfor CIITA gene silence in different tumor cells. In melanoma cells, the silence of CIITA genewas caused by histone deacetylation of its promoter IV, but not associated with DNAmethylation. In gastrointestinal tumor cells, CIITA gene silence was caused by DNAmethylation of its promoter IV alone. DNA methylation and histone deacetylationco-regulated its silence in leukemia cells. In HCC, HLA-DR related epigenetic mechanism ofCIITA silence is still unknown.HBV genome contains four open reading frames, S, C, X and P gene. HBx proteinencoded by X gene is a multi-functional regulator. HBx protein can activate multiple signaltransduction pathways, directly acts with transcription factors and participates in hostepigenetic regulations by upregulating the expression of DNA methyltransferases (Dnmt) andhistone deacetylases (HDAC). Many previous studies had indicated that the pathogenesis ofHCC was closely related with HBx protein. The silencing of tumor suppressor genes causedby epigenetic modifacations of HBx was considered to be the important mechanism for thedevelopment and progression of HCC. Thereby, we presumed that HBx may participate ininducing CIITA epigenetic silence by upregulating expression of Dnmt and HDAC in liver orHCC cells. However, the roles of HBx protein have not been clarified in the regulation ofCIITA epigenetic modification and HLA-DR expression in hepatocytes.Firstly, in order to study the relationship between the silence of CIITA gene expressionand absence of HLA-DR expression in hepatocarcinoma cell line (HepG2), constitutive andIFN-γ induced CIITA and HLA-DR expression were examined in human normal liver cellline (LO2) and HepG2, and the effect of CIITA cDNA transfection on HLA-DR expressionwas studied in HepG2cell lines. Secondly, in order to study the roles of DNA methylationand histone acetylation status of CIITA promoter IV in mastering the expression of CIITAand HLA-DR, DNA methylation and histone acetylation status of CIITA promoter IV wereexamined in L02and HepG2cell lines using Methylation specific PCR (MSP) and Chromatinimmunoprecipitation (ChIP) assay, and the effects of Dnmt inhibitor5-Aza-CdR and HDACinhibitor TSA on HLA-DR and CIITA expression were studied. Finally, HBV X eukaryoticexpression vector was constructed and transfected into L02and HepG2cell lines to study theinfluence of HBx protein on HLA-DR and CIITA expression. Results:1. Constitutive expression of CIITA and HLA-DR were not detected while IFN-γinduced expression were detected in L02cell lines, and the levels of CIITA and HLA-DRexpression were time and dose dependent with IFN-γ. Both constitutive and IFN-γ inducedexpression of CIITA and HLA-DR were not detected in HepG2cell lines. HLA-DRexpression was observed in the HepG2cell lines transfected with EBS-NPL-CIITA, but not inHepG2cell lines and HepG2cell lines transfected with empty vector EBS-NPL.2. CIITA promoter IV was hemimethylated in HepG2cell lines but not methylated inL02cell lines. The status of histone H3acetylation in CIITA promoter type IV was observedin L02cell lines, but not observed in HepG2cell lines. HepG2cell lines were treated with5-Aza-CdR, TSA alone, or their combination. The results showed that IFN-γ inducedexpression of CIITA were observed in HepG2cell lines treated with5-Aza-CdR, TSA, ortheir combination. The relative levels of CIITA mRNA expression were significantly higherin groups treated with combination of5-Aza-CdR and TSA(2.13±0.22) than in groups treatedwith5-Aza-CdR (1.15±0.27), and in groups treated with TSA(the relative value of which wasdefined as1), P＜0.05. The levels of HLA-DR expression significantly higher in groupstreated with combination of5-Aza-CdR, TSA and IFN-γ(28.5%±2.81%) than in groupstreated with combination of5-Aza-CdR and IFN-γ (11.9%±1.78%), and in groups treatedwith combination of TSA and IFN-γ (9.31%±2.24%),showed in P＜0.05.3. pHBx-IES2-EGFP plasmid was constructed and transfected into L02and HepG2celllines by the method of electroporation. CIITA and HLA-DR were detected in L02cell linestransfected with HBV X and HLA-DR was detected in HepG2cell lines transfected withHBV X.Conclusion:1. The silence of CIITA expression is key mechanism leading to the absence ofconstitutive and IFN-γ induced HLA-DR expression in HepG2cell lines.2. Both DNA methylation and histone deacetylation of CIITA promoter IV playimportant role in the CIITA epigenetic silence and absence of HLA-DR expression in HepG2cell lines, which may result in the tumor cells escaping from immune surveillance in thedevelopment and progression of HCC.3. HBx protein induces expression of CIITA and HLA-DR in L02and HepG2cell lines, prompting that HBx is not related with the absence of HLA-DR caused by CIITA epigeneticsilence in the HBV related HCC cells.