The Mechanisms Of Downregulation MICA/B Expression By HBV On Hepatocytes To Escape From NK Lysis

ObjectiveIt is reported that hepatocellular carcinoma (HCC) is the fifth morbidity cancer all over the world and HBV carrier are more likely to progress HCC than people without HBV. HBV is a kind of hepadnavirus, its genome includes four opening reading frames (ORF):HBx, HBc, HBs and HBp. Now we are rather clear about how these four ORFs contribute to HBV replication, but we are not exactly know what role they play in the progression of HBV+liver cancer. In addition, the immune system possesses the inherent capacity to resist HBV infection. NK cells represent the main effector cells involved in innate immune responses to the viral infections and are particularly enriched in the liver. In the process of clearing HBV-infected hepatocytes, NK cells could secrete lots of cytokines, such as IFN-y and TNF-α, and they also kill HBV-infected hepatocyes by recognition the ligands of NK cell receptors. It is demonstrated that HBV infection leads to down-regulation of NK cell activating receptors NKG2D, NKp30and NKp44, as well as decreased secretion of IFN-y and TNF-a. At the same time, the expression of corresponding ligands of activating receptor MICA/B and ULBPs on hepatocytes decreased and the production of IL-10and TGF-β enhanced.In this report, we constructed four plasmids expressing HBx. HBs, HBc, HBp of HBV genome separately to test which one contributes to the HBV-induced down-regulation of MICA/B. Then, we try to explore the mechanisms that may lead to the regulation of MICA/B expression. Firstly, which is the most important in our experiment, we tried to discover new transcription factors that bind to MICA/B promoter and examine whether HBV infection influences the expression of the new transcription factors. In addition, we also examined whether STAT3, which has been confirmed as a kind of inhibitive transcription factor, contributes to the regulation of MICA/B expression. Secondly, we analyzed several miRNAs that bind to MICA/B to test whether they are involved in the HBV-induced down-regulation of MICA/B. Methods1MTT and CFSE/7AAD methods were used to test the sensitivity of HepG2, HepG2-HBV, HepG2.2.15cells and HepG2cells-transfected with HBx, HBc, HBs and HBp to NK92lysis.2Q-PCR and FACS were applied to confirm the expression of MICA/B, GATA-2and GATA-3on HepG2cells transfected with HBx, HBc, HBs and HBp or after silencing of GATA-2or GATA-3.3Luciferase reporter assay was used to test the activity of MICA/B promoter in HepG2cells transfected with HBx, HBc, HBs and HBp or after silencing of GATA-2or GATA-3.4Western Blot was performed to determine the expression of GATA-2, GATA-3and STAT3on HepG2. HepG2.2.15cells and HepG2cells transfected with HBx, HBc, HBs and HBp.5Chromatin Immunoprecipitation (ChIP) was used to test the binding of GATA-2or GATA-3to MICA promoter in HepG2and HepG2.2.15cells.6Immunoprecipitation was applied to confirm whether HBx can bind to GATA-2or GATA-3.Results1HBV+HepG2.2.15cells are less susceptible to NK92lysis than HepG2cells. Compared with HepG2cell, the expression of NKG2D ligands on HBV+HepG2cells decreased.2HepG2cells transfected with HBx or HBc were less susceptible to NK92lysis than HepG2cells transfected with negative control.3Both HBx and HBc gene downregulated the MICA/B expression on HepG2cells. HBx gene downregulated the expression of ULBP2on HepG2cells. HBx and HBp gene downregulated Fas expression on HepG2cell.4Sliencing of HBx or HBc increased MICA/B expression on HepG2.2.15cells.5Sliencing of transcription factors GATA-2or GATA-3up-regulated MICA/B expression on HepG2and HepG2.2.15cells. Luciferase reporter assay showed that the activity of MICA/B promoter increased after silencing of GATA-2or GATA-3.6The expression level of GATA-2and GATA-3was higher in HepG2.2.15cells than that in HepG2cells, the binding of GATA-2or GATA-3to MICA/B promoter also increased. HBx protein could bind to GATA-2or GATA-3to regulate MICA/B expression. After silencing of HBx, the binding of GATA-2or GATA-3with MICA/B promoter decreased.7miRNA20a, miRNA106b and miRNA106b were changed in different level in HepG2cells transfected with HBx, HBs, HBc and HBp. The inhibitor of miRNA106b could reverse the down-regulation of MICA/B caused by HBx and HBc transfection.8Transfection of HBx and HBc could lead to the activation of STAT3and STAT3-decoy reverse the MICA/B down-regulation caused by HBx and HBc.Conclusions1HBV+HepG2.2.15cells are less susceptible to NK92lysis than HepG2cells.2HBx and HBc caused the down-regulation of MICA/B expression and further reduced the susceptible to NK lysis.3GATA-2or GATA-3could bind to MICA/B promoter and inhibit MICA/B expression.4GATA-2and GATA-3are inhibitory transcription factors that could bind to MICA/B promoter. HBx protein acts as a transactivator by binding to GATA-2or GATA-3to suppress the expression of MICA/B.5HBx, HBc, HBs and HBp could activate miRNA20a, miRNA93and miRNA106b to inhibit the expression of MICA/B.6HBx and HBc could activate STAT3to regulate the expression of MICA/B.