TY - JOUR
T1 - Establishment of a novel hepatic steatosis cell model by Cas9/sgRNA-mediated DGKθ gene knockout
AU - Zhang, Jingjing
AU - Zhao, Junli
AU - Zheng, Xiaojing
AU - Cai, Kai
AU - Mao, Qinwen
AU - Xia, Haibin
N1 - Funding Information:
This study was supported by the Fundamental Research Funds for Innovation Founds of Graduate Programs, Shaanxi Normal University (grant no. 2015CXS024), research grants to Dr Haibin Xia and Dr Kai Cai from the National Natural Science Foundation of China (grant nos. 81272543, 81471772 and 31470058) and the Natural Science Foundation of Shaanxi Province, China (grant nos. 2014JZ005 and 2015JQ8302).
Publisher Copyright:
Copyright © 2017 Spandidos Publications. All rights reserved.
PY - 2018/2
Y1 - 2018/2
N2 - 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.
AB - 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.
KW - CRISPR/Cas9
KW - Diacylglycerol kinase θ
KW - Insulin resistance
KW - Lipid accumulation
KW - Nonalcoholic fatty liver disease
KW - Type 2 diabetes
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U2 - 10.3892/mmr.2017.8140
DO - 10.3892/mmr.2017.8140
M3 - Article
C2 - 29207074
AN - SCOPUS:85039713803
VL - 17
SP - 2169
EP - 2176
JO - Molecular Medicine Reports
JF - Molecular Medicine Reports
SN - 1791-2997
IS - 2
ER -