General 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.
General Medicine and Metabolic Diseases, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy.
Cell Mol Gastroenterol Hepatol. 2022;13(3):759-788. doi: 10.1016/j.jcmgh.2021.11.007. Epub 2021 Nov 23.
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.
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 (MBOAT7TM6SF2) through Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9).
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 MBOAT7TM6SF2 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 MBOAT7TM6SF2 models, suggesting an unbalancing in mitochondrial dynamics, and the silencing of both MBOAT7 and TM6SF2 impaired mitochondrial activity with a shift toward anaerobic glycolysis. MBOAT7TM6SF2 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.
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.
I148M 类脂肪酶结构域包含 3（PNPLA3）、MBOAT7-TMC4 跨膜通道样 4 中膜结合 O-酰基转移酶结构域中的 rs641738 以及 E167K 跨膜 6 超家族成员 2（TM6SF2）多态性是导致非酒精性脂肪性肝病（NAFLD）发展和进展的主要易感因素。我们之前在 HepG2 细胞中生成了 MBOAT7 的完全敲除（MBOAT7），该细胞纯合 I148M PNPLA3。因此，我们旨在通过对大量 NAFLD 患者进行研究，来探讨 3 种风险变异对肝损伤和肝细胞癌（HCC）的协同影响，并通过在 HepG2 和 MBOAT7 细胞中沉默 TM6SF2 来创建遗传 NAFLD 的体外模型。
根据 PNPLA3、TM6SF2 和 MBOAT7 风险变异的数量，将 1380 名 NAFLD 患者分为 0-3 分的半定量评分。通过簇状规律间隔短回文重复序列和 CRISPR 相关蛋白 9（CRISPR/Cas9）对 HepG2（TM6SF2）和 MBOAT7（MBOAT7TM6SF2）中的 TM6SF2 进行沉默。
在 NAFLD 患者中，这些突变的累加权重与肝病严重程度和发生 HCC 的风险增加相关。在 HepG2 细胞中，TM6SF2 沉默改变了脂质组成并诱导微泡状脂质滴（LD）的积累，而 MBOAT7TM6SF2 细胞则显示出微泡状/大泡状 LD 的混合模式。TM6SF2 缺失强烈影响内质网和线粒体超微结构，从而增加内质网/氧化应激。在 TM6SF2 和 MBOAT7TM6SF2 模型中，线粒体数量均增加，表明线粒体动力学失衡，并且沉默 MBOAT7 和 TM6SF2 会损害线粒体活性并向无氧糖酵解转移。MBOAT7TM6SF2 细胞的增殖率也最高。最后，MBOAT7 和/或 TM6SF2 的重新过表达逆转了在复合敲除模型中观察到的代谢和致瘤特征。
这 3 种风险变异的共存影响了患者和实验模型中的 NAFLD 进程，影响了 LD 积累、线粒体功能以及向 HCC 的代谢重编程。
