The Mechanism Of MicroRNA-361 On Regulation Of Hepatic Lipid Metabolism And Glucose Hemostasis

Non-alcoholic fatty liver disease(NAFLD)has become one of the most common public health problems worldwide and is the most common cause of chronic liver disease.NAFLD is characterized by aberrant hepatocellular accumulation of lipids.It can trigger a progressive cascade of lipid disorders,ranging from hepatosteatosis to non-alcoholic steatohepatitis,liver fibrosis,cirrhosis and eventually hepatocellular carcinoma.Nonalcoholic fatty liver disease is closely related to insulin resistance and metabolic risk factors(i.e.,abdominal obesity,hypertension,dyslipidemia,hyperglycemia).Furthermore,NAFLD has been associated with an increased risk of cardiovascular disease independent of metabolic risk factors.However,information regarding the molecular mechanism underlying the initiation and progression of NAFLD still remains unknown.Micro RNAs(mi RNAs or mi Rs)are endogenous,small non-coding RNAs which possess a central role in the regulation of both m RNA and protein expression of the target genes.The micro RNAs act at post-transcriptional level targeting the 3'-untranslated regions(3'-UTRs),which typically contain defined stability elements(including micro RNAs binding sites).It is now extensively accepted the role of micro RNAs as potent regulators of a wide range of specific pathways or physiological processes controlling multiple biological functions.Until few years ago the biological functions usually assigned to micro RNAs control consisted of developmental timing,apoptosis,proliferation,differentiation and organ development.To date,several studies have shown that micro RNAs are not only implicated in the regulation of cellular growth and differentiation,but even in the control of energy andhepatic metabolic functions regulating fatty acid(FA)and cholesterol metabolism.A large number of studies indicate that micro RNAs could act as novel biomarkers and potential therapeutic targets in the management of NAFLD.We performed a clustering analysis of micro RNA arrays using livers of mice fed a normal diet or high-fat-diet.We found that a number of micro RNAs were significantly altered in the livers of mice of two groups.Our screen revealed a pronounced up-regulation of mi R-361 in the livers of high-fat-diet mice compared to normal diet mice,which was further confirmed by q PCR in high-fat-diet,db/db and ob/ob mice.Next,to elucidate the role of mi R-361 in the liver,adenovirus containing mi R-361 or negative control(NC)were administered into C57BL/6 mice via tail vein injection.As expected,overexpression of mi R-361 resulted in a marked increase in hepatic TG contents,as well as serum TG levels.The observed alteration in hepatic TG content was further confirmed by Oil red O staining.In addition,elevated blood glucose and insulin resistance were associated with overexpression of mi R-361.However,body weight and cholesterol levels were not changed.On the contrary,the inhibition of the expression of mi R-361 was associated with decreased blood glucose,serum TG,serum insulin levels and hepatic TG contents.In agreement,the m RNA levels of lipogenic genes,SCD1 and Fasn,were elevated after the overexpression of mi R-361.The m RNA levels of CPT1? and MCAD genes,which involve fatty acid oxidation in the liver,were decreased with overexpression of mi R-361.In addition,the m RNA levels of gluconeogenesis genes,PEPCK and G6 Pase,were elevated.We next sought to determine the molecular mechanism of regulation of mi R-361 on lipid metabolism in mice.Using a stringent bioinformaticsapproach,we identified several putative murine mi R-361 target genes,among which the gene encoding Sirt1 harbored a mi R-361 binding site.Previous studies have shown that Sirt1 inhibit lipogenesis and hepatic TG accumulation,and play a pathological role in the hepatic TG homeostasis.We hypothesized that mi R-361 may regulate TG metabolism through the Sirt1 pathway.As predicted,protein levels of Sirt1 were decreased in the liver with overexpressing mi R-361.Similarly,overexpression of mi R-361 also led to a decrease in Sirt1 expression in Hepa1-6 cells.To further determine whether Sirt1 is a direct target gene of mi R-361,we constructed a luciferase reporter containing the Sirt1 3'-UTR and co-transfected with mi R-361 mimics or the control into Hepa1-6 cells.As expected,mi R-361 markedly repressed the luciferase reporter activity,while mutation of the binding sites resulted in abolished repression in luciferase activity.Overall,our data supported that Sirt1 is a direct target of mi R-361.In conclusion,our present study for the first time revealed that mi R-361 play a role in pathological role in the hepatic TG homeostasis.In addition,Sirt1 is a direct target gene of mi R-361 in the liver.Therefore,our data support the notion that mi R-361 mediated Sirt1 down-regulation plays a critical role in hepatic lipogenesis and TG accumulation.