DNMT1/PKR double knockdowned HepG2 (HepG2-DP) cells have high hepatic function and differentiation ability.

Area of Science:

• Hepatology and Cell Biology
• Pharmacology and Drug Metabolism

Background:

• HepG2 cells are a common human hepatocyte model but have limited drug metabolism functions compared to primary hepatocytes.
• Previous studies showed zebularine upregulated hepatic gene expression in HepG2 cells by inhibiting DNA methyltransferase 1 (DNMT1) and protein kinase R (PKR).

Purpose of the Study:

• To establish and characterize a new HepG2 cell subline (HepG2-DP) with enhanced hepatocyte functions through stable double knockdown of DNMT1 and PKR.
• To evaluate the functional improvements and differentiation potential of HepG2-DP cells for drug metabolism and pharmacokinetic studies.

Main Methods:

• Stable double knockdown of DNMT1 and PKR in HepG2 cells to create the HepG2-DP subline.
• Assessed albumin production, CYP1A2 gene expression, and lipid droplet accumulation.
• Performed comprehensive gene expression analysis of transcription factors and drug-metabolizing enzymes.
• Investigated HepG2-DP cell maturation via differentiation induction.

Main Results:

• HepG2-DP cells showed increased albumin production, CYP1A2 gene expression, and lipid droplet accumulation compared to parental HepG2 cells.
• Upregulation of key hepatic transcription factors (HNF1β, HNF4α, ONECUT1, FOXA family) and nuclear receptors was observed in HepG2-DP cells.
• Differentiation induction further increased the expression of major CYPs and Phase II, III drug-metabolizing enzymes in HepG2-DP cells.

Conclusions:

• The newly established HepG2-DP cell line exhibits significantly enhanced hepatocyte functions, serving as a more effective model than standard HepG2 cells.
• HepG2-DP cells demonstrate potential for further maturation through differentiation induction, expanding their utility in drug metabolism and pharmacokinetic research.