Background & aims: MicroRNAs (miRNAs) act as a regulatory mechanism on a post-transcriptional level by repressing gene transcription/translation and play a central role in the cellular stress response. Osmotic changes occur in a variety of diseases including liver cirrhosis and hepatic encephalopathy. Changes in cell hydration and alterations of the cellular volume are major regulators of cell function and gene expression. In this study, the modulation of hepatic gene expression in response to hypoosmolarity was studied.
Methods: mRNA analyses of normo- and hypoosmotic perfused rat livers by gene expression arrays were used to identify miRNA and their potential target genes associated with cell swelling preceding cell proliferation. Selected miR-141-3p was also investigated in isolated hepatocytes treated with miRNA mimic, cell stretching, and after partial hepatectomy. Inhibitor perfusion studies were performed to unravel signalling pathways responsible for miRNA upregulation.
Results: Using genome-wide transcriptomic analysis, it was shown that hypoosmotic exposure led to differential gene expression in perfused rat liver. Moreover, miR-141-3p was upregulated by hypoosmolarity in perfused rat liver and in primary hepatocytes. In concert with this, miR-141-3p upregulation was prevented after Src-, Erk-, and p38-MAPK inhibition. Furthermore, luciferase reporter assays demonstrated that miR-141-3p targets cyclin dependent kinase 8 (Cdk8) mRNA. Partial hepatectomy transiently upregulated miR-141-3p levels just after the initiation of hepatocyte proliferation, whereas Cdk8 mRNA was downregulated. The mechanical stretching of rat hepatocytes resulted in miR-141-3p upregulation, whereas Cdk8 mRNA tended to decrease. Notably, the overexpression of miR-141-3p inhibited the proliferation of Huh7 cells.
Conclusions: Src-mediated upregulation of miR-141-3p was found in hepatocytes in response to hypoosmotic swelling and mechanical stretching. Because of its antiproliferative function, miR-141-3p may counter-regulate the proliferative effects triggered by these stimuli.
Lay summary: In this study, we identified microRNA 141-3p as an osmosensitive miRNA, which inhibits proliferation during liver cell swelling. Upregulation of microRNA 141-3p, controlled by Src-, Erk-, and p38-MAPK signalling, results in decreased mRNA levels of various genes involved in metabolic processes, macromolecular biosynthesis, and cell cycle progression.
Keywords: Camsap1, calmodulin-regulated spectrin-associated protein 1; Cdc25a, cell division cycle 25A; Cdk8, cyclin dependent kinase 8; Colchicine; Cpt2, carnitine palmitoyl transferase 2; Dmxl1, Dmx Like 1; Dstyk, dual serine/threonine and tyrosine protein kinase; Duoxa2, dual oxidase maturation factor 2; ECM, extracellular matrix; Eml4, EMAP like 4; GO, gene ontology; Gucy2c, guanylate cyclase 2C; Hepatocytes; Hypoosmolarity; Liver perfusion; Liver regeneration; Luc7l3, LUC7 like 3 pre-MRNA splicing factor; Msantd2, Myb/SANT DNA binding domain containing 2; Nags, N-acetyl glutamate synthase; Nfat5, nuclear factor of activated t cells 5; Osmosis; PHx; PHx, partial hepatectomy; PI, propidium iodide; Prmt1, protein arginine methyltransferase 1; RT, room temperature; Rab30, member RAS Oncogene Family; Rps6, ribosomal Protein S6; Scnn1a, sodium channel epithelial 1 subunit alpha; Slc39a10, solute carrier family 39 member 10; Tfeb, transcription factor EB; Timp1, tissue inhibitor of metalloproteinase 1; Zmpste24, zinc metallopeptidase STE24; miR-141-3p; miRNA, microRNA; microRNA; qPCR, quantitative PCR.
© 2022 The Author(s).