Fig 2: HBsAg-induced NF-κB factors bind and upregulate LINC00665 expression. In the course of HBV infection, the virus enters hepatocytes and undergoes nucleocapsid disassembly to release the HBV genome into the nucleus. Here, the HBV genome is acted upon by host factors to form the transcriptionally active covalently closed circular DNA (cccDNA). HBV cccDNA transcribes the HBV mRNA, including the pre-S2/S 2.1-kb mRNA, which in turn is translated to HBsAg and pre-S2 protein. HBsAg is the major component of the HBV envelope and is secreted as both infective HBV particles and noninfective subviral particles. In the current study, we show that HBsAg induces nuclear translocation of the NF-κB factors NF-κB1, RelA, and c-Rel. These factors bind and activate the LICN00665 promoter, leading to increased transcription of the lncRNA. Using various cell-based assays, we show LINC00665 drives hepatocarcinogenesis by promoting cell proliferation, colony formation, and cell migration but inhibiting apoptosis.

Fig 3: HBsAg regulates LINC00665 through the NF-κB pathway. An immunofluorescence assay was performed to visualize (A) RelA, (B) NF-κB1, and (C) c-Rel in stable HepG2-HBsAg cells and control HepG2-Puro cells as described in Materials and Methods. Nuclear fluorescence intensity of (D) RelA, (E) NF-κB1, and (F) c-Rel was significantly greater in HepG2-HBsAg than in control HepG2-Puro cells. Bar, 10 μm. (G) Real-time PCR was performed to measure change in LINC00665 expression in HepG2-HBsAg and HepG2-Puro cells in the presence of BAY 11-7082 (2 μM) or vehicle control (DMSO). Fold change in LINC00665 expression was measured relative to HepG2-HBsAg cells treated with DMSO. (H) The LINC00665 minimal promoter was cloned upstream of the luciferase reporter gene in the PGL3-basic vector. This construct was used to transfect HepG2-HBsAg and HepG2-Puro cells treated with BAY 11-7082 (2 μM) and vehicle control (DMSO). A luciferase assay was performed to measure the change in LINC00665 promoter activity 48 h after transfection. Fold change in LINC00665 promoter activity was normalized to that of HepG2-HBsAg cells treated with DMSO. Addition of the NF-κB inhibitor BAY 11-7082 nullified the HBsAg-mediated upregulation of LINC00665. All data are means and SD from three independent experiments (n = 3). *, P < 0.05; **, P < 0.01; NS, not significant (paired Student’s t test).