Establishment of a novel hepatic steatosis cell model by Cas9/sgRNA-mediated DGKθ gene knockout

摘要

To investigate the role of diacylglycerol kinase θ (DGKθ) in lipid metabolism and insulin resistance, the present study generated an in vitro hepatic steatosis cell model by knockout of the DGKθ gene in liver cancer cell line HepG2 using CRISPR/Cas9 technology. The cell line was characterized by Oil Red O staining and shown to exhibit increased intracellular lipid accumulation, compared with that in wild-type liver cancer cell line HepG2. The gene expression levels of signaling proteins in pathways involved in lipid metabolism, insulin resistance and gluconeogenesis were also examined. The DGKθ-knockout HepG2 cells showed increased mRNA and protein expression levels of lipid synthesis-related genes, fatty acid synthase, peroxisome proliferator-activated receptor-γ and sterol regulatory element-binding protein-1c, and decreased expression levels of the lipolysis-related gene, carnitine palmitoyltransferase1A. These changes may account for the increased intracellular lipid content of this cell line. The DGKθ-knockout HepG2 cells also exhibited an increased phosphorylation level of protein kinase Cε and decreased phosphorylation levels of insulin receptor substrate 1, mechanistic target of rapamycin and protein kinase B (also known as Akt). These changes have been reported to mediate insulin resistance. Taken together, an in vitro hepatic steatosis cell model was established in the present study, providing a valuable tool for understanding the pathogenesis of nonalcoholic fatty liver disease and associated insulin resistance, and for developing treatment strategies for this disease.