# Integrated Bioinformatics Analysis Reveals the Impact of SHEV ORF3-Related LncRNA Network on Bile Secretion Pathway (ko 04976) in HepG2 Cells

## TL;DR

This study identifies lncRNAs linked to bile secretion disruption in HepG2 cells infected with SHEV, offering new insights into the virus's infection mechanism.

## Contribution

The study reveals a novel lncRNA network regulated by SHEV ORF3 that impacts the bile secretion pathway.

## Key findings

- Three lncRNAs associated with the bile secretion pathway were identified in HepG2 cells expressing SHEV ORF3.
- Molecular docking suggests specific binding sites between lncRNA UBC and UBC protein.
- Six lncRNA-mRNA regulatory networks were predicted, involving two downregulated UBC mRNA transcripts.

## Abstract

Swine hepatitis E (SHE), caused by the swine hepatitis E virus (SHEV), is a significant zoonotic disease which is often associated with disrupted bile secretion. This study focused on the role of the SHEV ORF3 protein in this pathological process. Using bioinformatics approaches on HepG2 cells expressing genotype IV SHEV ORF3, we identified three specific long non-coding RNAs (lncRNAs) linked to the bile secretion pathway. We further constructed their potential regulatory networks with target mRNAs and characterized putative molecular binding sites. These findings could offer novel insights into the functions of the ORF3 protein and the infection mechanism of SHEV.

(1) Background: Swine hepatitis E (SHE) is an emerging zoonotic disease caused by the swine hepatitis E virus (SHEV). The open reading frame 3 (ORF3) protein is a recognized virulence factor of SHEV. Jaundice, the typical clinical sign of SHE, primarily results from disruptions in bile production, secretion, and excretion. However, the mechanism by which SHEV ORF3 influences bile metabolism remains unclear. (2) Methods: Building on our previous work involving adenovirus-mediated overexpression of genotype IV SHEV ORF3 in HepG2 cells and subsequent high-throughput lncRNA/transcriptome sequencing, this study performed KEGG enrichment analysis on differentially expressed lncRNAs. Candidate lncRNAs were validated via qRT-PCR. Cis-regulated target genes were predicted by integrating differentially expressed mRNA data. Furthermore, AlphaFold 3.0 was employed to analyze the molecular binding sites between lncRNA UBC (MSTRG.6881.4) and its target, UBC protein. (3) Results: We identified three lncRNAs associated with the bile secretion pathway (ko 04976) in HepG2 cells expressing genotype IV SHEV ORF3, which were further confirmed by qRT-PCR: lncRNA UBC (MSTRG.6881.4), lncRNA UBC (MSTRG.6881.9), and lncRNA UBC (MSTRG.6881.12). Bioinformatics prediction suggested six lncRNA-mRNA regulatory networks involved these lncRNAs and two downregulated UBC mRNA transcripts (ENST00000540700 and ENST00000536769). Molecular docking indicated that nucleotides 395U and 41C of lncRNA UBC (MSTRG.6881.4) could potentially bind to residues 82Lys, 88Thr, and 90Thr of the UBC protein, with predicted binding energies ranging from −4.73 to −0.75 kcal/mol. (4) Conclusions: The successful identification of bile secretion-related lncRNAs, coupled with the prediction of their regulatory networks and molecular interaction sites, has advanced our understanding of SHEV ORF3 function and the pathogenesis of SHEV infection.