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DNA-encoded library versus RNA-encoded library selection enables design of an oncogenic noncoding RNA inhibitor.

ABSTRACT: Nature evolves molecular interaction networks through persistent perturbation and selection, in stark contrast to drug discovery, which evaluates candidates one at a time by screening. Here, nature's highly parallel ligand-target search paradigm is recapitulated in a screen of a DNA-encoded library (DEL; 73,728 ligands) against a library of RNA structures (4,096 targets). In total, the screen evaluated ∼300 million interactions and identified numerous bona fide ligand-RNA three-dimensional fold target pairs. One of the discovered ligands bound a 5'GAG/3'CCC internal loop that is present in primary microRNA-27a (pri-miR-27a), the oncogenic precursor of microRNA-27a. The DEL-derived pri-miR-27a ligand was cell active, potently and selectively inhibiting pri-miR-27a processing to reprogram gene expression and halt an otherwise invasive phenotype in triple-negative breast cancer cells. By exploiting evolutionary principles at the earliest stages of drug discovery, it is possible to identify high-affinity and selective target-ligand interactions and predict engagements in cells that short circuit disease pathways in preclinical disease models.

SUBMITTER: Benhamou RI

PROVIDER: S-EPMC8833215 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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DNA-encoded library versus RNA-encoded library selection enables design of an oncogenic noncoding RNA inhibitor.

Benhamou Raphael I RI Suresh Blessy M BM Tong Yuquan Y Cochrane Wesley G WG Cavett Valerie V Vezina-Dawod Simon S Abegg Daniel D Childs-Disney Jessica L JL Adibekian Alexander A Paegel Brian M BM Disney Matthew D MD

Proceedings of the National Academy of Sciences of the United States of America 20220201 6

Nature evolves molecular interaction networks through persistent perturbation and selection, in stark contrast to drug discovery, which evaluates candidates one at a time by screening. Here, nature's highly parallel ligand-target search paradigm is recapitulated in a screen of a DNA-encoded library (DEL; 73,728 ligands) against a library of RNA structures (4,096 targets). In total, the screen evaluated ∼300 million interactions and identified numerous bona fide ligand-RNA three-dimensional fold ...[more]

PMID: 35110406

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Project description: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.

Dataset Information

DNA-encoded library versus RNA-encoded library selection enables design of an oncogenic noncoding RNA inhibitor.

Publications

DNA-encoded library versus RNA-encoded library selection enables design of an oncogenic noncoding RNA inhibitor.

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