| Nonalcoholic fatty liver disease (NALFD) is an emerging health problem worldwide, partly due to the lifestyle changes. NAFLD represents a spectrum of liver damage in individuals encompassing simple steatosis, steatohepatitis (NASH), cirrhosis and even hepatocellular carcinoma in the absence of alcohol abuse. Population studies show that NALFD is strongly associated with obesity, type2diabetes and hypertension, which are the main features of the metabolic syndrome. As a genetic-environmental-metabolic stress related disease, the exact mechanisms underlying NAFLD are so far poorly understood. The pathogenesis may explained by the ’two-hit’ hypothesis. However, a growing body of evidence now strongly suggests that there are many other "hits" play a role in the liver injury occuring in NAFLD. The term hepatic steatosis refers to an intracellular accumulation of lipids and when further accompanied by inflammation, the condition is termed steatohepatitis. Altered lipid accumulation may provide a molecular rational for NAFLD.In deed, the intrahepatic lipid accumulation may arise from increased uptake of lipids, elevated de novo synthesis of fatty acids, impaired lipoprotein synthesis or secretion as well reduced fatty acid oxidation. Caveolin-1acts as a scaffold to organize multiple molecular complexes that regulates a variety of cellular events. Genetic evidence from the study of caveolin-1knockout mice, along with our previous work, indicate that caveolin-1plays a role as a negative modulator of lipid synthesis and cytokine-induced liver injury.To gain mechanistic insights into caveolin-1, we studied the expression of caveolin-1on NAFLD mouse and cell model.Applying with the newest gene edit technology-CRISPR/Cas9system, we built liver specificly caveolin-1knockout mice and caveolin-1null stable cell line. Besides, we used the recombined-lentivirus technology to overexpress caveolin-1. There is evidence for caveolin-1level to be positively correlated with hepatic lipid accumulation and grade of steatosis in NAFLD. We have demonstrated that caveolin-1deficiency accerated NAFLD progression by upregulating fatty acid synthase (SREBP-1,FASN, ACC) and activating COX-2. Thus, caveolin-1may act as a protective factor on NAFLD pathophysiology.The ubiquitin proteasome pathway (UPP) is the principal mechanism for protein catabolism and the key issue to guard normal protein and clear error one. Ubiquitin ligase (E3) is the most critical factor in the ubiquitin hydrolase pathway. By specifically identificating different substrate, E3shows a high selective protein degradation effect and is involved in the regulation of lipid metabolism. Lipid accumulation is crucial for progression of NAFLD. The trend of some E3ligases observed in the modulation of lipid accumulation has made it essential to get a total screen of the whole E3family for novel possibilities of preventive and effective treatment of NAFLD.Owing to previous work, we construncted a human E3sgRNA lentivirus libraryapplying the CRISPR/Cas9system. It targets2,196E3ligases. Besides, we construncted the firstknown human E3knockout cell library. And we built thefluorescence-based lipid accumulation report system by using BODIPY(?)493/503staining. Scrrened by FACS, we got24functional E3ligases. WWP2and the other9E3ligases have positive effect on lipid accumulation, while ING2, UBE2D1and the other12E3ligases act as a negative regulator. These E3ligases lead to further study on NAFLD and may be the possible targets for drug development.We explore the possible effect of caveolin-1on lipid accumulation in NAFLD, and got a number of functional E3ligases through screen. Here is the first time we show the apply of CRISPR/Cas9system on NAFLD mechanistic research.Together, these results help clarify the pathogenesis of NAFLD and is of great importance for further research and therapeutic interventions. |
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