• Energy Research

The analysis of molecular states of individual cells, as defined by their mRNA expression profiles and protein composition, has gained widespread interest in studying biological phenomena ranging from embryonic development to homeostatic tissue function and genesis and evolution of cancers. Although the molecular content of individual cells in a tissue can vary widely, their molecular states tend to be constrained within a transcriptional landscape partly described by the canonical archetypes of a population of cells. In this study, we sought to characterize the effects of an acute (partial hepatectomy) and chronic (alcohol consumption) perturbation on the molecular states of individual hepatocytes during the onset and progression of liver regeneration. We analyzed the expression of 84 genes across 233 individual hepatocytes acquired using laser capture microdissection. Analysis of the single-cell data revealed that hepatocyte molecular states can be considered as distributed across a set of four states irrespective of perturbation, with the proportions of hepatocytes in these states being dependent on the perturbation. In addition to the quiescent, primed, and replicating hepatocytes, we identified a fourth molecular state lying between the primed and replicating subpopulations. Comparison of the proportions of hepatocytes from each experimental condition in these four molecular states suggested that, in addition to aberrant priming, a slower transition from primed to replication state could contribute toward ethanol-mediated suppression of liver regenerative response to partial hepatectomy.

Impaired liver regeneration has been considered as a hallmark of progression of alcohol-associated liver disease. Our previous studies demonstrated that in vivo inhibition of the microRNA (miRNA) miR21 can restore regenerative capacity of the liver in chronic ethanol-fed animals. The present study focuses on the role of microRNA regulatory networks that are likely to mediate the miR-21 action. Rats were chronically fed an ethanol-enriched diet along with pair-fed control animals and treated with AM21 (anti-miR-21), a locked nucleic acid antisense to miR-21. Partial hepatectomy (PHx) was performed and miRNA expression profiling over the course of liver regeneration was assessed. Our results showed dynamic expression changes in several miRNAs after PHx, notably with altered miRNA expression profiles between ethanol and control groups. We found that in vivo inhibition of miR-21 led to correlated differential expression of miR-340-5p and anticorrelated expression of miR-365, let-7a, miR-1224, and miR-146a across all sample groups after PHx. Gene set enrichment analysis identified a miRNA signature significantly associated with hepatic stellate cell activation within whole liver tissue data. We hypothesized that at least part of the PHx-induced miRNA network changes responsive to miR-21 inhibition is localized to hepatic stellate cells. We validated this hypothesis using AM21 and TGF-β treatments in LX-2 human hepatic stellate cells in culture and measured expression levels of select miRNAs by quantitative RT-PCR. Based on the in vivo and in vitro results, we propose a hepatic stellate cell miRNA regulatory network as contributing to the restoration of liver regenerative capacity by miR-21 inhibition.