Expression Of MiR-9 In NASH Mice And Its Effect On The Regulation Of Lipid Metabolism

Objective: Firstly,we established the nonalcoholic steatohepatitis(NASH)model in mice and determined whether the construction of the model was successful.Then the expression of miR-9 in liver and adipose tissue was detected.Finally,we studied on the effect of miR-9 on adipogenesis in vitro,and explored its possible mechanism.Methods:1.In vivo: Six to eight weeks male C57 BL/6J mice were divided into two groups,control diet group(CD)and high fat and high sugar group(HFHS).The weight,defecation,activity,hair changes and other general conditions were recorded during the modeling process.The mice were sacrificed after 12 and 28 weeks of feeding respectively.The blood,liver,as well as inguinal,epididymal and perirenal adipose tissues were collected.The weight of the tissues ware measured and serum ALT,AST,TC,TG,FPG and LDL-C were measured.He staining,oil red O staining,Sirius red staining and Masson staining were used to evaluate the pathological changes of liver.The pathological changes of epididymal adipose tissue were judged by HE staining.The expression of adipogenesis and inflammatory factors in epididymal white adipose tissue and liver were detected by qRT-PCR,as well as the expression of miR-9 in NASH model.2.In vitro: Firstly,the 3T3-L1 preadipocytes adipogenic differentiation was induced.Lipid droplets were stained with Oil Red O.TG content was detected by kits.miR-9expression level were determined by qRT-PCR.Then the effect of overexpression or inhibition of intracellular miR-9 expression on adipose differentiation of 3T3-L1 cells was determined.Bioinformatics analysis was performed to predicte that the target gene of miR-9 was PNPLA3.miR-9 mimics,inhibitor and PNPLA3 wild / mutant plasmids were designed and synthesized.After transfection,double luciferase assay was performed to confirm the regulation of miR-9 on target gene.In order to further explore the molecular mechanism of miR-9 affecting lipid metabolism,3T3-L1 preadipocytes were induced to differentiate in the presence or absence of compound C(AMPK inhibitor)for 14 days after transfection of miR-9 mimics or shPNPLA3,respectively.Oil red O staining and TG content were used to detect lipid production.The expression of AMPK and phosphorylated AMPK,PPAR-? and OXPAT were detected by Western Blot.Results:1.After 12 weeks of modeling,the body weight and the ratio of fat weight /body weight in the model group increased significantly,which were more obvious at the28 th week,but the liver index only increased significantly at the 28 th week.With the prolongation of modeling time,the mice in the model group appeared abdominal obesity,decreased activity,increased urination and less smooth hair.In addition,serum FPG,ALT,AST,TC,and LDL-C level were increased,but serum TG level did not increase.HE staining and oil red O staining showed hepatic steatosis and infiltration of inflammatory cells.At the 28 th week,steatosis and inflammation were further aggravated.Sirius red and Masson staining suggested liver perisinusoidal mild fibrosis.Therefore,the NASH model was successfully constructed after 12 weeks of high fat feeding,and developed NASH with mild fibrosis after 28 weeks.qRT-PCR showed that the expression levels of liver lipogenic factors SREBP-1c,ACC and inflammatory factors MCP-1 and CD68 mRNA were increased at 12 weeks,while the expression of FASN,TNF-? and fibrogenic factors ?-SMA,COL3A1 and TIMP-1mRNA were not increased until 28 weeks.HE staining of epididymal adipose tissue in the model group showed adipocytes hypertrophy,and SREBP-1c PPAR?,and MCP-1 mRNA levels were not significantly increased until 28 weeks.The level of miR-9 in liver and epididymal adipose tissue of the model group was significantly higher than that of the control group at the same time.2.3T3-L1 cells were successfully induced differentiation,the lipid droplets and TG content were increased.miR-9 was up-regulated during adipogenesis by qRT-PCR.After transfection of 3T3-L1 cells and induction of differentiation for 14 days,it was found that the overexpression of miR-9 reduced the formation of lipid droplets in adipocytes,while the inhibition of miR-9 increased the formation of lipid droplets.Double luciferase assay confirmed that miR-9 acted on PNPLA3 3' UTR and inhibited its expression.Interference of PNPLA3 or overexpression of miR-9 resulted in the decrease of PNPLA3 protein level and lipogenesis,but the lipid level recovered after treatment with compound C at the same time.In addition,shPNPLA3 or miR-9overexpression promoted the phosphorylation of AMPK protein and down-regulatedthe expression of PPAR? and OXPAT protein.And this effect was blocked by compound C.Conclusions: High fat and high sugar diet successfully induced NASH model with obesity,and the expression of miR-9 in liver and adipose tissue was up-regulated.miR-9 activated the AMPK signaling pathway by targeting PNPLA3 and inhibited the expression of PPAR? and OXPAT,resulting in the inhibition of the 3T3-L1 preadipocytes differentiation and adipogenesis.These results indicated that miR-9played a negative role in the process of adipocyte differentiation and adipogenesis.