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Updated: Oct 12, 2025
Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis
Published on: March 11, 2017
TM6SF2/PNPLA3/MBOAT7 Loss-of-Function Genetic Variants Impact on NAFLD Development and Progression Both in Patients
Miriam Longo1, Marica Meroni2, Erika Paolini3
1General Medicine and Metabolic Diseases, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy; Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Milano, Italy.
Background & Aims:
The I148M Patatin-like Phospholipase Domain-containing 3 (PNPLA3), the rs641738 in the Membrane bound O-acyltransferase domain containing 7-transmembrane channel-like 4 (MBOAT7-TMC4) locus, and the E167K Transmembrane 6 Superfamily Member 2 (TM6SF2) polymorphisms represent the main predisposing factors to nonalcoholic fatty liver disease (NAFLD) development and progression. We previously generated a full knockout of MBOAT7 in HepG2 cells (MBOAT7-/-), homozygous for I148M PNPLA3. Therefore, we aimed to investigate the synergic impact of the 3 at-risk variants on liver injury and hepatocellular carcinoma (HCC) in a large cohort of NAFLD patients, and create in vitro models of genetic NAFLD by silencing TM6SF2 in both HepG2 and MBOAT7-/- cells.
Methods:
NAFLD patients (n = 1380), of whom 121 had HCC, were stratified with a semiquantitative score ranging from 0 to 3 according to the number of PNPLA3, TM6SF2, and MBOAT7 at-risk variants. TM6SF2 was silenced in HepG2 (TM6SF2-/-) and MBOAT7-/- (MBOAT7-/-TM6SF2-/-) through Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9).
Results:
In NAFLD patients, the additive weight of these mutations was associated with liver disease severity and an increased risk of developing HCC. In HepG2 cells, TM6SF2 silencing altered lipid composition and induced the accumulation of microvesicular lipid droplets (LDs), whereas the MBOAT7-/-TM6SF2-/- cells showed a mixed microvesicular/macrovesicular pattern of LDs. TM6SF2 deletion strongly affected endoplasmic reticulum and mitochondria ultrastructures, thus increasing endoplasmic reticulum/oxidative stress. The mitochondrial number was increased in both TM6SF2-/- and MBOAT7-/-TM6SF2-/- models, suggesting an unbalancing in mitochondrial dynamics, and the silencing of both MBOAT7 and TM6SF2 impaired mitochondrial activity with a shift toward anaerobic glycolysis. MBOAT7-/-TM6SF2-/- cells also showed the highest proliferation rate. Finally, the re-overexpression of MBOAT7 and/or TM6SF2 reversed the metabolic and tumorigenic features observed in the compound knockout model.
Conclusions:
The co-presence of the 3 at-risk variants impacts the NAFLD course in both patients and experimental models, affecting LD accumulation, mitochondrial functionality, and metabolic reprogramming toward HCC.
Insights
Three genetic variants (PNPLA3, MBOAT7, TM6SF2) significantly worsen nonalcoholic fatty liver disease (NAFLD) progression and hepatocellular carcinoma (HCC) risk. These variants impact lipid droplet accumulation and mitochondrial function in experimental models.
Area of Science:
- Hepatology
- Genetics
- Molecular Biology
Background:
- Nonalcoholic fatty liver disease (NAFLD) development and progression are influenced by key genetic factors.
- The Patatin-like Phospholipase Domain-containing 3 (PNPLA3) I148M, Membrane bound O-acyltransferase domain containing 7-transmembrane channel-like 4 (MBOAT7-TMC4) rs641738, and Transmembrane 6 Superfamily Member 2 (TM6SF2) E167K polymorphisms are major predisposing variants.
- Previous studies established MBOAT7 knockout cells (MBOAT7-/-) that are homozygous for the I148M PNPLA3 variant.
Purpose of the Study:
- To investigate the combined impact of PNPLA3, MBOAT7, and TM6SF2 risk variants on liver injury and hepatocellular carcinoma (HCC) in NAFLD patients.
- To create in vitro models of genetic NAFLD by silencing TM6SF2 in HepG2 and MBOAT7-/- cells.
Main Methods:
- Stratified 1380 NAFLD patients based on the number of PNPLA3, TM6SF2, and MBOAT7 risk variants (score 0-3).
- Utilized CRISPR/Cas9 to silence TM6SF2 in HepG2 cells (TM6SF2-/-) and MBOAT7-/- cells (MBOAT7-/-TM6SF2-/-).
Main Results:
- The additive effect of these variants correlated with NAFLD severity and increased HCC risk in patients.
- TM6SF2 silencing in HepG2 cells induced lipid droplet accumulation and altered lipid composition.
- Compound MBOAT7-/-TM6SF2-/- cells exhibited altered lipid droplet patterns, endoplasmic reticulum/mitochondrial stress, impaired mitochondrial function, and increased proliferation.
- Re-expression of MBOAT7 and/or TM6SF2 reversed these metabolic and tumorigenic changes.
Conclusions:
- The co-occurrence of these three risk variants significantly influences NAFLD progression.
- These variants affect lipid droplet accumulation, mitochondrial function, and metabolic reprogramming towards HCC in both patient cohorts and experimental models.
