Hepatitis B Virus-Encoded HBsAg Contributes to Hepatocarcinogenesis by Inducing the Oncogenic Long Noncoding RNA LINC00665 through the NF-κB Pathway.

Clinical and in vivo studies have demonstrated a role for hepatitis B virus (HBV)-encoded HBsAg (hepatitis B surface antigen) in HBV-related hepatocellular carcinoma (HCC); however, the underlying mechanisms remain largely unknown. Here, we investigated the role of HBsAg in regulating long noncoding RNAs (lncRNAs) involved in HCC progression. Our analysis of microarray data sets identified LINC00665 as an HBsAg-regulated lncRNA. Furthermore, LINC00665 is upregulated in liver samples from HBV-infected patients as well as in HCC, specifically in HBV-related HCC liver samples. These findings were supported by our in vitro data demonstrating that HBsAg, as well as HBV, positively regulates LINC00665 in multiple HBV cell culture models. Next, we evaluated the oncogenic potential of LINC00665 by its overexpression and CRISPR interference (CRISPRi)-based knockdown in various cell-based assays. LINC00665 promoted cell proliferation, migration, and colony formation but inhibited cell apoptosis in vitro. We then identified the underlying mechanism of HBsAg-mediated regulation of LINC00665. We used immunofluorescence assays to show that HBsAg enhanced the nuclear translocation of NF-κB factors in HepG2 cells, confirming that HBsAg activates NF-κB. Inhibition of NF-κB signaling nullified HBsAg-mediated LINC00665 upregulation, suggesting that HBsAg acts through NF-κB to regulate LINC00665. Furthermore, the LINC00665 promoter contains NF-κB binding sites, and their disruption abrogated HBsAg-induced LINC00665 upregulation. Finally, HBsAg facilitated the enrichment of the NF-κB factors NF-κB1, RelA, and c-Rel in the LINC00665 promoter. Taken together, this work shows that HBsAg can drive hepatocarcinogenesis by upregulating oncogenic LINC000665 through the NF-κB pathway, thereby identifying a novel mechanism in HBV-related HCC. IMPORTANCE Hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC). Numerous reports indicate an oncogenic role for HBV-encoded HBsAg; however, the underlying mechanisms are not well understood. Here, we studied the role of HBsAg in regulating lncRNAs involved in hepatocarcinogenesis. We demonstrate that HBsAg, as well as HBV, positively regulates oncogenic lncRNA LINC00665. The clinical significance of this lncRNA is highlighted by our observation that LINC00665 is upregulated in liver samples during HBV infection and HBV-related HCC. Furthermore, we show LINC00665 can drive hepatocarcinogenesis by promoting cell proliferation, colony formation, and cell migration and inhibiting apoptosis. Taken together, this work identified LINC00665 as a novel gene through which HBsAg can drive hepatocarcinogenesis. Finally, we show that HBsAg enhances LINC00665 levels in hepatocytes by activating the NF-κB pathway, thereby identifying a novel mechanism by which HBV may contribute to HCC.

Biogenesis, characterization, and functions of mirtrons.

MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression. They base pair with the complementary target mRNA at the 3'UTR and modulate cellular processes by repressing the mRNA translation or degrading the mRNA. There are well-documented mechanisms of biogenesis of miRNA; however, a sizeable number of miRNAs are also produced by non-canonical pathways. Mirtrons represent a predominant class of non-canonical miRNAs. Mirtrons originate from intronic regions and are produced in a splicing-dependent and Drosha-independent manner. Mirtrons constitute about 15% of all miRNAs produced in a human body and have caught attention of researchers worldwide due to their unconventional origin, sequence characteristics, evolutionary dynamics, ability to regulate variety of cellular processes and their immense potential in disease therapeutics. In this comprehensive review we collate the research done in the past decade including biogenesis, sequence characteristics, regulation, and emerging therapeutic roles of mirtrons. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action.