Knockout Of FTO Gene In Piec Cells And Its Metabolic Effects Based On CRISPR/Cas9 Technology

Severe obesity seriously affects the health of livestock and humans.Fat deposition is not only regulated by fat cell metabolism but also highly related to endothelial cell function.Exploring the regulatory role of obesity-related factors in endothelial cells is an effective means to study the correlation between endothelial cells and lipid metabolism,and to answer questions about the relationship between blood vessels and fat.The obesity-related gene FTO,as an important candidate factor,has become a hot spot in fat metabolism research in recent years.FTO has m RNA m6 A demethylation modification function and can greatly regulate fat metabolism.Previous studies have shown that specific deletion of the FTO gene in endothelial cells had no effect on mouse fat metabolism,but the specific mechanism has not been reported.Pigs have highly similar physiological functions and nutritional intake methods to humans,making them an excellent model for studying human metabolism mechanisms.In this study,using the CRISPR/Cas9 gene editing system and the PX459 vector,the FTO gene was knocked out in porcine iliac artery endothelial cells(PIEC)to obtain two stable PIEC cell strains-one with a frameshift mutation and the other with a whole-coding mutation of the FTO gene-for FTO function research.Analysis of cell proliferation ability showed that the two FTO mutations did not affect cell proliferation ability.To investigate the function of FTO,we used RT-PCR to analyze the effects of FTO on methylation and lipid metabolism-related genes.The results showed that complete depletion of FTO gene function led to decreased expression levels of methyltransferase METTL3 and peroxisome proliferator-activated receptor gamma(PPARγ),but did not affect the expression of IRX3.In cell strain 2,a deletion of amino acids 170-237 from FTO resulted in a similar degree of decrease in PPARγexpression as observed in complete FTO depletion,but did not affect the expression of METTL3,and significantly reduced IRX3 expression.Metabolite analysis of the three types of cells(wild-type PIEC,cell strain 1,and cell strain 2)indicated that the structural-functional properties of FTO amino acids 170-237 mainly regulate glucose metabolism pathways,lipids and some amino acids,but do not affect the Fe2+/α-ketoglutarate dependent dioxygenase involved in the citric acid cycle within this amino acid range.Transcriptome data suggested that FTO is highly associated with cellular movement,microtubule formation,and cilia motility.The FTO gene may regulate microtubule stability by degrading certain RNAs based on ELP3 complex,thereby altering cell skeleton bundling,microtubule construction,and interaction with endothelial cells.Therefore,FTO may participate in vascular dilation through microtubule formation and cilia motion.In summary,we successfully constructed two types of FTO mutation cells,and our differential analysis of wild-type cells provided a more detailed interpretation of the functional implications of FTO amino acids.This study provides a theoretical basis for studying FTO function in endothelial cells related to obesity.