2,3,7,8-Tetrachlorodibenzio-p-dioxin (TCDD) is a persistent environmental contaminant known for aryl hydrocarbon receptor (AHR)-mediated liver effects, including metabolic dysfunction-associated steatotic liver disease (MASLD)-like pathologies such as steatosis, inflammation, and fibrosis. Although previous studies have focused on AHR-mediated regulation of protein-coding genes, recent attention has turned to long non-coding RNAs (lncRNAs) because of their potential roles in the progression of steatotic liver disease (SLD). Using bulk and single-nuclei (sn)RNAseq datasets, we compared the dose-dependent AHR-mediated induction of lncRNA and mRNA expression by TCDD in the mouse and rat liver. This study also investigated cell-specific lncRNA-gene regulation within the murine liver to identify divergent lncRNA expression patterns across different hepatic cell types. Lastly, differentially expressed (DE) lncRNAs associated with human liver diseases were examined to investigate potential mechanistic roles. Comparative analysis of gene expression identified 2,386 mouse and 916 rat DE lncRNAs, with 203 common to both species. In contrast, mice had 6,071 compared to 3,056 rat DE mRNAs, with 1,492 in common. Integration of AHR genomic enrichment and putative dioxin response elements (pDRE) data with DE lncRNAs revealed regulation patterns similar to mRNA-coding genes, with both exhibiting greater frequency proximal to the transcription start site in both mice and rats. snRNAseq analysis also revealed 5,495 DE lncRNAs across all liver cell subtypes. Pericentral and periportal hepatocytes exhibited the most significant changes, with 3,339 and 3,550 DE lncRNAs respectively, followed by macrophages with 2,116. Among all DE genes, 52 previously annotated lncRNAs in hepatocytes were differentially expressed by TCDD, many of which are associated with steatosis, fibrosis, and hepatocellular carcinoma. Collectively, these results suggest AHR-mediated differential expression of lncRNAs may play a significant role in the progression of steatosis to steatohepatitis with fibrosis elicited by TCDD.
Non-coding RNAs (ncRNAs) are transcribed RNA molecules that are not subsequently translated into proteins. Mammalian genomes are primarily comprised of non-coding regions, with up to 76-98% of the human genome encoding ncRNA sequences that vary in length, structure, and regulatory function. Long non-coding RNAs (lncRNAs) are a subset of ncRNAs defined by their length of greater than 200 nucleotides and include antisense, intergenic (lincRNAs), intronic, enhancer RNAs (eRNAs), pseudogene-derived, and circular RNAs (circRNAs), with other ncRNAs identified with advances in sequencing technologies. Although not translated into protein, lncRNAs serve pivotal roles in regulating gene expression, translation, chromatin structure, enzyme activity, cell differentiation and development, and can be differentially expressed by environmental stimuli. Unlike messenger RNAs (mRNAs), which serve as templates for protein synthesis, lncRNAs exert effects through interactions with other biomolecules.
lncRNAs have emerged as key contributors in the progression and development of various diseases and disorders. These non-coding RNAs are reported to silence tumor suppressor genes, act as oncogenes, interact directly with transcription factors to enhance cell proliferation and survival, modulate epigenetic machinery, and promote cancer progression. Beyond cancer, lncRNAs are linked to nephropathy, cardiovascular disease, rheumatoid arthritis, Alzheimer's disease, Huntington's disease, and spinal muscular atrophy as well as associated with developmental disorders, including craniofacial defects, brain and limb malformations, impaired adipogenesis, disrupted hematopoiesis, male infertility, and compromised liver function. On a cellular level, lncRNAs are also involved in chromosome modification, nuclear transport and transcription activation. In addition to these diverse roles and pathologies, lncRNAs regulate various metabolic pathways, including the metabolism of amino acids, cholesterol, glucose, and lipids. Consequently, dysregulated lncRNA expression may also contribute to the adverse effects elicited by environmental contaminants.
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates gene expression associated with various physiological and pathological processes, including xenobiotic metabolism, tissue development, immune response, and cellular proliferation. AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and related compounds, leads to differential gene expression and subsequent pathobiological effects. Most, if not all, of these responses are AHR-mediated, as these effects are absent in Ahr-null mice. In mice, TCDD elicits diverse liver pathologies including steatosis (lipid accumulation), inflammation, and fibrosis, reminiscent of metabolic dysfunction-associated steatotic liver disease (MASLD), a broad term used to characterize liver pathologies associated with metabolic dysfunction in humans. More specifically, TCDD has been shown to disrupt hepatic lipid metabolism, glycolysis, amino acid metabolism, the TCA cycle, the urea cycle, and nucleoside metabolism. Although most studies examining the effects of TCDD have focused on the differential expression of protein-coding genes, a recent study reported the differential expression of more than 4,000 lncRNAs, affecting various cell types, transcriptional zonal distributions, and intercellular communication patterns related to fibrosis. lncRNA dysregulation has also been implicated in metabolic-associated steatohepatitis (MASH), hepatocellular carcinoma (HCC), and cholestatic liver disease.
Considering TCDD induces similar liver pathobiologies, we explored AHR-mediated dysregulation of lncRNA expression as a possible contributing factor in the dose-dependent progression of steatosis to steatohepatitis with fibrosis which are risk factors for more complex metabolic diseases including MASLD, diabetes, and hepatocellular carcinoma. Bulk RNA-sequencing (RNAseq) in mice and rats was re-assessed to determine species-specific differences in lncRNA expression following TCDD treatment, while single-nuclei RNA-sequencing (snRNAseq) in mouse liver was also re-examined for cell type- and zone-specific lncRNA expression. This included re-analysis of the same 28-day TCDD exposure snRNAseq dataset previously referenced, which originally reported > 4,000 differentially expressed lncRNAs. Re-analysis uncovered dose-dependent, cell-specific lncRNA expression, with hepatocytes exhibiting distinct zonal lncRNA expression patterns. Integration with AHR ChIPseq and putative DRE (pDRE) data indicated that lncRNAs, like protein-coding mRNAs, were differentially expressed using canonical and non-canonical AHR pathways. Functional annotation of differentially expressed lncRNAs further implicated some in liver homeostasis and pathologies such as steatosis and fibrosis. These findings support a role for AHR-mediated lncRNA regulation in TCDD-elicited progression of steatosis and hepatotoxicity.