DArT Markers Effectively Target Gene Space in the Rye Genome

Large genome size and complexity hamper considerably the genomics research in relevant species. Rye (Secale cereale L.) has one of the largest genomes among cereal crops and repetitive sequences account for over 90% of its length. Diversity Arrays Technology is a high-throughput genotyping method, in which a preferential sampling of gene-rich regions is achieved through the use of methylation sensitive restriction enzymes. We obtained sequences of 6,177 rye DArT markers and following a redundancy analysis assembled them into 3,737 non-redundant sequences, which were then used in homology searches against five Pooideae sequence sets. In total 515 DArT sequences could be incorporated into publicly available rye genome zippers providing a starting point for the integration of DArT- and transcript-based genomics resources in rye. Using Blast2Go pipeline we attributed putative gene functions to 1101 (29.4%) of the non-redundant DArT marker sequences, including 132 sequences with putative disease resistance-related functions, which were found to be preferentially located in the 4RL and 6RL chromosomes. Comparative analysis based on the DArT sequences revealed obvious inconsistencies between two recently published high density consensus maps of rye. Furthermore we demonstrated that DArT marker sequences can be a source of SSR polymorphisms. Obtained data demonstrate that DArT markers effectively target gene space in the large, complex, and repetitive rye genome. Through the annotation of putative gene functions and the alignment of DArT sequences relative to reference genomes we obtained information, that will complement the results of the studies, where DArT genotyping was deployed, by simplifying the gene ontology and microcolinearity based identification of candidate genes

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.ISSN:2165-049