TM6SF2/PNPLA3/MBOAT7 Loss-of-Function Genetic Variants Impact on NAFLD Development and Progression Both in Patients and in In Vitro Models
| Autor: | Giacomo P. Comi, Amalia Gastaldelli, Roberto Piciotti, Anna Ludovica Fracanzani, S. Sabatini, Marica Meroni, Paola Dongiovanni, Massimiliano Ruscica, Giorgio Soardo, Anna Alisi, Luca Miele, Fabrizia Carli, Francesco Fortunato, Chiara Macchi, Veronica Erconi, Miriam Longo, Dario Ronchi, Erika Paolini, Luca Valenti |
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| Jazyk: | angličtina |
| Rok vydání: | 2022 |
| Předmět: |
BMI
body mass index PGC1α Peroxisome proliferator-activated receptor-γ (PPARγ) coactivator-1α RC799-869 Mitochondrion DMSO dimethyl sulfoxide Mitochondrial Dynamics ApoB apolipoprotein B-100 Liver disease PC phosphatidylcholine MT-COX1 mitochondrially encoded cytochrome c oxidase subunit 1 PCR polymerase chain reaction Non-alcoholic Fatty Liver Disease HCC ANOVA analysis of variance Original Research GFP green fluorescent protein Liver injury LDH lactate dehydrogenase Fatty liver Liver Neoplasms Gastroenterology Single Nucleotide Diseases of the digestive system. Gastroenterology mRNA messenger RNA Hepatocellular carcinoma Phospholipases A2 Calcium-Independent ER Stress SNP single-nucleotide polymorphism NASH nonalcoholic steatohepatitis Carcinoma Hepatocellular ATP adenosine triphosphate Genotype LD lipid droplet TAG triacylglycerol Settore MED/12 - GASTROENTEROLOGIA NAFLD TM6SF2 Calcium-Independent Cer ceramide ORO Oil Red O T2D type 2 diabetes mTOR mammalian target of rapamycin SDHA succinate dehydrogenase complex flavoprotein subunit A Biology Polymorphism Single Nucleotide PI phosphatidylinositol ER endoplasmic reticulum FBS fetal bovine serum ORF open reading frame NADH nicotinamide adenine dinucleotide medicine Gene silencing Humans Genetic Predisposition to Disease Polymorphism sgRNA small guide RNA TEM transmission electron microscopy PCA principal component analysis Hepatology Carcinoma Membrane Proteins VLDL very-low-density lipoprotein Hepatocellular Lipase medicine.disease digestive system diseases OR odds ratio Phospholipases A2 Anaerobic glycolysis lyso lysophosphatidylinositol Akt protein kinase B CRISPR-Cas9 Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 Unfolded protein response Cancer research BSA bovine serum albumin DMEM Dulbecco’s modified Eagle medium NAFLD nonalcoholic fatty liver disease HCC hepatocellular carcinoma DAG diacylglycerol Acyltransferases |
| Zdroj: | Cellular and Molecular Gastroenterology and Hepatology Cellular and Molecular Gastroenterology and Hepatology, Vol 13, Iss 3, Pp 759-788 (2022) |
| Popis: | 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. Graphical abstract |
| Databáze: | OpenAIRE |
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