| Selenoprotein F is an important member of the selenoprotein family,in the past years of researches SelenoF was found to be a key gene for eyeball development and promotes the development of prostate disease,Ig M secretion increases in mice after knocking out SelenoF,and the SelenoF knockout could reduce the occurrence of colonic crypts and the development of colon cancer.Liver get high abundance of SelenoF expression.In the mice liver that knockout of SelenoF,the activity of enzymes related to glucose and lipid metabolism was found to increase.Disorder of glucose and lipid metabolism is one of the pathogenesis of metabolic dysfunction associated fatty liver disease(MAFLD).Increased glycolysis,decreased glycogen synthesis,and increased fatty acid synthesis co-promote the disease process of MAFLD.The regulation theory of ceRNA has provided a new direction for the research of many diseases.MicroRNA can inhibit the expression of target genes through specific binding of the effect region,and in the cytoplasm,LncRNA can also specifically adsorb microRNA,degrade the activity of microRNA,and play a role in affecting the function of target genes.There are no reports of microRNA or LncRNA that regulate SelenoF.In this study,SelenoF knockout C57BL6 male mice were used as the research animals,and the mouse fatty liver model was replicated by feeding 35%calorie-fat diet(HFD),palmitic acid stimulation was used to establishe AML-12 cell steatosis model in vitro.H&E staining,oil red O staining,transmission electron microscopy,TUNEL staining,realtime fluorescent quantitative PCR,western blotting,immunofluorescence technology,flow cytometry,dual luciferase reporter system,bioinformatics were carried out to observe the pathological changes of liver tissue,detect the related genes expression of glucose and lipid metabolism pathway in liver tissue,construct the lncRNA-miRNA-mRNA regulatory network of SelenoF and verify the ceRNA regulated-network in AML-12 cells,analyze the influence of ceRNA regulated-network on the glucose and lipid metabolism pathway.The purpose of this experiment is to explore the pathogenesis of SelenoF knockout in reducing HFD-induced fatty liver,and to clarify the role of the constructed ceRNA regulatory network in the disorders of glucose and lipid metabolism.The main results are as follows:(1)The histopathological results showed that there was obvious accumulation of lipid droplets in the liver of the HFD-fed wild-type mice,the nucleus were shrunk,squeezed to the edge or rupture,the hepatocyte cord was disordered,the steatosis was serious,and the cell volume increased.However,although a small amount of fatty vacuoles can be seen in the liver cells of the SelenoF knockout mice that fed with HFD,the cell morphology is mostly normal,and there is a slight hepatocyte cord disorder.The results show that SelenoF knockout can improve HFD-induced fat accumulation in mouse liver tissue.(2)Under the HFD environment,the activity of the IRS1/AKT pathway in the liver of wildtype mice was significantly inhibited,while in the SelenoF knockout mice was not inhibited.This indicated that SelenoF knockout can improve the inhibitory effect of HFD on IRS1/AKT pathway.(3)Under the HFD environment,the expression of glycolysis-related enzymes(HK2,PKM2,LDHA)in the liver of wild-type mice increases,and the expression of glycogen synthase(p GSK3?,GSK3?)decreases,while the expression of glycolysis-related enzymes in the SelenoF knock-out mouse liver were decreased compared with the wild type,and the expression of glycogen synthase increased.It shows that SelenoF knockout can improve the abnormal glucose metabolism of mice liver tissue induced by HFD.(4)Under the HFD environment,the expression of fatty acid synthase(ACC?,FASN,ACLY)in the liver of wild-type mice increased,and the expression of lipolytic enzymes(PPAR?,Lipase)decreased.However,the expression of fatty acid synthase in the liver of SelenoF knockout mice decreased compared with that of wild-type mice,lipolytic enzymes are increased compared with wild type.It shows that SelenoF knockout can improve HFD-induced abnormal lipid metabolism in mouse liver tissue(5)Under the HFD environment,the expression of apoptosis-related genes(Capase-3,Capase-9)in the liver of wild-type mice increased,and the expression of the anti-apoptotic gene Bcl-2decreased,however,the exptession of apoptosis in the liver of SelenoF knockout mice was decreased,the expression of the anti-apoptotic genes increased when compared with that of wild-type.It shows that SelenoF knockout can improve HFD-induced hepatocyte apoptosis in mice(6)In AML-12,sodium palmitate is used to establish steatosis model in vitro.The AKT activator SC-79 and inhibitor MK2206 were used to clarify the regulation of the IRS1/AKT pathway on glucose and lipid metabolism when SelenoF knocked out.The use of inhibitors reversed the inhibitory effect of SelenoF knockdown on glycolysis and fatty acid synthesis,and the use of activators rescued the promotion of glycolysis and fatty acid synthesis by SelenoF overexpression(7)Bioinformatics predicts potential interaction between LncNATug1/miR-1934-3p/SelenoF.in the liver with SelenoF knockout,when Tug1 expression decreased,miR-1934-3p expression increased,and then the dual luciferase reporter gene system verified the prediction results in AML-12 cells.Overexpression or knockdown of miR-1934-3p in AML-12 cells inhibited or promoted the expression levels of SelenoF and Tug1,knockdown of Tug1 in AML-12 cells can inhibit the expression of SelenoF and promote the transcription of miR-1934-3p.The ceRNA regulatednetwork also affected the expression of the IRS1/AKT pathway and downstream glucose and lipid metabolism in AML-12 cells,miR-1934-3p mimic inhibited the increase in glycolysis and fatty acid synthesis,promoted glycogen synthesis and lipolysis,and this effection can be reversed by SelenoF overexpression,inhibitor of miR-1934-3p was consistent with the overexpression of SelenoF.SiTug1 can inhibit glycolysis and fatty acid synthesis,promote glycogen synthesis and lipolysis,but this effection can be rescued by miR-1934-3p knockdown or SelenoF overexpression.This result indicated that Tug1 can regulate the target gene SelenoF by binding miR-1934-3p,and affect the expression of IRS1/AKT pathway and glucose and lipid metabolism related enzymes.In summary,SelenoF knockout can improve HFD-induced liver steatosis in mice,reduce lipid accumulation in liver cells,reduce the expression of glycolysis-related and fatty acid synthase enzymes,and promote the expression of lipolytic enzymes and glycogen synthesis enzymes.LncRNATug1 can affect the expression of SelenoF by competitively binding miR-1934-3p,and the constructed ceRNA regulated-network can regulate the expression of downstream glucose and lipid metabolism-related enzymes through the IRS1/AKT pathway.That is,the Tug1/miR-1934-3p/SelenoF pathway activates the IRS1/AKT pathway to improve the HFD-induced glucose and lipid metabolism disorder in mouse hepatocytes.The research results will provide a theoretical basis for the prevention and treatment of fatty liver,and provide a new reference for the invention of targeted drugs for fatty liver. |
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