HBsAg/2GPI activates the NF-B pathway via the TLR4/MyD88/IB axis in hepatocellular carcinoma

Chronic hepatitis B virus (HBV) infection remains one of the leading causes of hepatocellular carcinoma (HCC) globally. However, the mechanism underlying the mediation by HBV surface proteins (HBsAgs) of the early steps in the virus life cycle and following HCC development is unclear. -2-glycoprotein I (2GPI) specifically interacts with HBsAg and demonstrates high expression during the earliest stages of hepatitis B virus infection. In the present study, the assessment of HCC and adjacent tissues revealed that the levels of mRNA and protein of 2GPI were highly expressed in HBV-related HCC. Previous studies have reported that HBsAg activates the nuclear factor (NF)-B pathway via interaction with 2GPI in HCC. However, the underlying mechanism of how the interaction between HBsAg and 2GPI confers activation of the NF-B pathway is still unclear. The HBsAg is comprised of three carboxyl-co-terminal HB proteins. In the present study, immunofluorescence assay and EMSA consistently revealed that a combination of recombinant small HBV surface antigen (rSHB) and 2GPI can significantly activate the NF-B signaling pathway. Another study from our team revealed that high expression of 2GPI enhanced HBsAg binding to cell surfaces and its interaction with Annexin II. However, Annexin II is not a transmembrane protein. Therefore, by a knockdown experiment with TLR2, TLR4 or MyD88 siRNAs using cells with co-incubated HBsAg/2GPI, certain aspects of the mechanism through which the HBsAg/2GPI complex activates the NF-B pathway through the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/IB axis were explained. In the present study, we identified the functional domain of HBsAg co-interaction with 2GPI for the activation of NF-B and revealed the mechanism of the HBsAg/2GPI-activated NF-B pathway which could contribute to the treatment of HBV-related HCC. A novel finding of the present study is that HBsAg can bind to 2GPI. We first identified the functional domain of HBsAg with 2GPI to activate NF-B. Second, by siRNA knockout experiments, we identified the downstream molecules involved in the activation of NF-B induced by 2GPI/HBsAg. In addition, we found that HBsAg/2GPI activated the NF-B pathway through the phosphorylation of Ser32/36 by IB.