Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a spectrum ranging from steatosis to steatohepatitis (MASH), to advanced liver cirrhosis, and affects up to 40% of the global population. MASH is characterised by steatosis, hepatocyte ballooning, inflammation and fibrosis. MASH has become the leading indication for liver transplant and is projected to become the top cause of hepatocellular carcinoma by 2030, making it a pressing clinical concern. Prior studies show key components of RNA splicing machinery are dysregulated in MASLD livers, raising the possibility RNA splicing is a driver of MASLD development. However little is known about the RNA isoform changes caused by this splicing dysregulation and which might drive disease progression.
To examine MASLD’s impact on splicing, immortalised human liver hepatocytes (THLE-2) and stellate cells (LX-2) were treated with or without a cocktail of stress- and fibrosis-inducing factors. RNA and protein were collected for Oxford Nanopore long-read sequencing and mass spectrometry to identify expressed and translated isoforms in vehicle and cocktail-treated cells. We identified 495 and 367 up- and downregulated RNA isoforms mapping to 141 genes in THLE-2, and 1337 and 1089 up- and downregulated isoforms mapping to 751 genes in LX-2. These genes were enriched in MASLD-associated pathways, such as ‘lipid metabolism’ and ‘cholesterol metabolism’ in THLE-2, and ‘extracellular matrix production’, ‘cell adhesion’, and ‘pathways in cancer’ in LX-2. Using proteogenomics, we also identified translated novel isoforms in both cell lines, which may also be relevant to MASLD.
Despite widespread transcript-level isoform differences, expression of splicing factors and spliceosome machinery was relatively unchanged. This highlights the importance of studying downstream isoforms generated by dysregulated splicing, rather than changes in the splicing machinery itself. Altogether, we have created a database of isoform expression changes in human cells dysregulated in MASLD. Future work will examine whether these isoforms contribute to MASLD development and if they can be targeted for treatment or disease prevention in preclinical models.