MiR-27a Induces Hepatic Steatosis Through Down-regulation NFE2L2

Objective:Fatty liver is a reversible disease.The decrease of mitochondrialβ-oxidative capacity and the increase of ROS are the important causes of the disease.Mitochondrial biosynthesis is a highly malleable cellular process that responds to energy requirements,growth and development signals and environmental stimuli.It plays an important physiological role in mitochondrial metabolism,quality and function.When mitochondrial biosynthesis declines,which leads to mitochondrial dysfunction and steatosis as a mutual cause.ROS produced by oxidative stress will trigger inflammation,bring about endoplasmic reticulum stress,lead to mitochondrial dysfunction and loss of the ability of liver cells to synthesize endogenous antioxidants,which will further aggravate liver steatosis and accelerate the process of liver disease.Recent researches have shown that NFE2L2 is participated in the regulation of mitochondrial biosynthesis and oxidative stress,and NFE2L2 plays a crucial part in liver lipid metabolism and the pathogenesis of non-alcoholic fatty liver.Our previous study found that mi R-27a secreted by adipose tissue into blood is involved in the remote regulation of obesity insulin resistance.However,it hasn’t yet been determined whether mi R-27a can induce liver steatosis through the down-regulation of NFE2L2 by impaired liver mitochondrial biosynthesis and oxidative stress.This study focused on exploring the effect of mi R-27a down-regulation of NFE2L2 on hepatic steatosis in the state of obesity,and clarifying the role of this pathway in the occurrence of fatty liver by reducing mitochondrial biosynthesis and inducing the accumulation of reactive oxygen species,so as to provide a new target and new strategy for the pathogenesis and prevention of obesity-induced hepatic steatosis.Methods:Male C57BL/6J mice aged 3-4 weeks were purchased.They were randomly assigned to two groups and fed low-fat or high-fat diets.After 4 weeks,half of low-fat fed mice were randomly injected with mi R-27a overexpressing lentivirus through tail veins,and half of high-fat fed mice were randomly injected with mi R-27a knockdownIIIlentivirus through tail veins.After feeding for another 12 weeks,the mice were detected for oral glucose tolerance,fasting blood glucose,and fasting insulin,triglyceride and cholesterol in liver tissue.At the same time examined the levels of mi R-27a in serum and liver,and observed liver histomorphology to clarify the relationship between mi R-27a and liver steatosis.We detected NFE2L2 protein,regulatory factors of mitochondrial biosynthesis of NRF1 and TFAM,protein expressions of mitochondrial complex III,IV and V,and oxidative stress related indicators in mice liver,and confirmed that mi R-27a induced liver steatosis was related to down-regulation of NFE2L2 induced mitochondrial biosynthesis damage and oxidative stress.The overexpression of mi R-27a and the overexpression of NFE2L2 were interfered in Hep G2 cells,tested the mitochondrial biosynthesis key indicators,protein expressions of mitochondrial complex III,IV and V,reactive oxygen species related indicators.And we examined the mitochondrial function,number and morphology,tested the triglyceride content of the treated Hep G2 cells,and has carried on the oil red O staining to investigate whether mi R-27a impaired mitochondrial biosynthesis and induced oxidative stress to induce liver steatosis by down-regulating NFE2L2.Results:In vivo studies,compared with low-fat fed mice,mi R-27a levels were significantly increased in the serum and liver of mi R-27a mice overexpressed and high-fat fed mice.The blood glucose,blood lipid,glucose tolerance and insulin levels of mi R-27a overexpressed mice and high-fat fed mice were markedly increased.While after mi R-27a is silenced,the blood sugar,blood lipids,glucose tolerance,insulin and the levels of mi R-27a in the serum and liver of obese mice were decreased significantly.Compared with low-fat fed mice,the liver triglyceride and cholesterol levels of mi R-27a overexpressed mice and high-fat fed mice were increased.HE staining of the liver indicated hepatic cell disorder,and there were small lipid droplets in the cytoplasm,presenting hepatic steatosis.While after mi R-27a is silenced,high-fat fed mice had lower liver triglyceride and cholesterol levels,hepatocytes were arranged neatly,fatty vacuoles were reduced,and steatosis was significantly improved.Compared with low-fat fed mice,mi R-27a overexpression and high-fat fed mice liver NFE2L2 protein expression decreased;IVmi R-27a silencing reversed this situation.Compared with low-fat fed mice,mi R-27a overexpression and high-fat fed mice liver mitochondrial biosynthesis related factors,mt DNA copy number and mitochondrial complex protein expression decreased,and MDA content was significantly increased;while compared with high-fat fed mice,the combination of high-fat and mi R-27a silencing reversed this situation,increased the related indicators of mitochondrial biosynthesis,increased the expression of mitochondrial complex protein,and decreased the MDA content.In vitro studies,mi R-27a down-regulated NFE2L2,reduced the expression of mitochondrial biosynthesis related factors,mt DNA copy number,cellular ATP content,and mitochondrial membrane potential in Hep G2 cells,which indicated that mitochondrial biosynthesis decreased,the number of mitochondria decreased,and the function declined;The fluorescence intensity of H₂DCFDA was increased,which indicated that ROS increased;The lipid droplets of Hep G2 cells increased and became larger,the lipid deposition increased,and the triglyceride content increased significantly.However,overexpression of NFE2L2 reversed these changes,reduced lipid deposition,improved mitochondrial biosynthesis,and reduced peroxidation levels.Conclusion:mi R-27a can impair mitochondrial biosynthesis and increase ROS to cause liver steatosis by down-regulating NFE2L2.