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

Zhang,Jingjing; Zhao,Junli; Zheng,Xiaojing; Cai,Kai; Mao,Qinwen; Xia,Haibin

doi:10.3892/mmr.2017.8140

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

Authors:
- Jingjing Zhang
- Junli Zhao
- Xiaojing Zheng
- Kai Cai
- Qinwen Mao
- Haibin Xia
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Affiliations: Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi 710062, P.R. China, Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
Published online on: November 22, 2017 https://doi.org/10.3892/mmr.2017.8140
Pages: 2169-2176
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

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.

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