Molecular Mechanisms Of MiR-137-3p Regulating Adipocyte Differentiation

Objective:The purpose of this study was to investigate the effect of epididymal adipose tissue on miR-137-3p and autophagy in DIO(diet-induced obesity)mice after 14 weeks of high-fat feeding through in vivo experiments and to analyze the possible mechanism of these changes.The agonist and inhibitor of miR-137-3p were constructed and transfected into 3T3-L1 preadipocytes cell line in vitro.Through a series of experiments,the effect and mechanism of miR-137-3p on 3T3-L1 preadipocytes differentiation were clarified.Method:1.DIO mouse model was established.Six-week-old mice were randomly divided into two groups:(1)chow-fed(CF)group: mice were fed with standard laboratory chow diet;(2)high-fat(HF)group: mice were fed a high fat diet.they were fed with standard diet or high-fat diet.Food intake was monitored daily,body weight was monitored once a week.2.After 14 weeks of high-fat intervention,mice were killed and sampled.HE staining was used to observe the morphological changes of epididymal adipocytes and liver cells,to analyze the changes of serum lipid metabolism.The m RNA expression of adipose miR-137-3p was detected by RT-PCR.The expression of LKB1/AMPK/m TOR signal pathway and autophagy-related proteins in epididymal adipose tissue was detected by Western Blot.3.Through the construction of miR-137-3p agonist and inhibitor,white fat inducer was given to induce the differentiation of 3T3-L1 preadipocytes.CCK-8method and oil red O staining were used to detect the effect of miR-137-3p on the proliferation and differentiation of 3T3-L1 cells,as well as the generation of lipid droplets;the changes of lipid content were detected by enzyme labeling;RT-PCR was used to detect m RNA expression of over-expressed miR-137-3p,m RNA expression of genes related to adipogenesis and lipolysis;Western Blot was used to detect the protein levels of LKB1/AMPK ? m TOR/PPAR? signaling pathway andautophagy-related molecules.4.Dual luciferase assay predicted and verified the target gene of miR-137-3p.Results:1.DIO mice gained weight,dyslipidemia,epididymal adipocytes enlarged and hepatocyte steatosis.The expression of miR-137-3p total RNA increased in epididymal fat.2.The protein expression of LKB1/P-AMPK was inhibited in the epididymal adipose tissue of DIO mice.The expression of P-m TOR/PPAR? and autophagy signaling pathway gene and protein were significantly changed.3.miR-137-3p agonist(mimic)promoted the differentiation of 3T3-L1 preadipocytes,promoted lipid synthesis and inhibited lipid decomposition.The result of inhibitor transfection was opposite.4.When the cells were transfected with miR-137-3p agonist,the protein expression of LKB1/P-AMPK was decreased,while m TOR/PPAR ? was up-regulated,and autophagy was inhibited.The result of inhibitor transfection was opposite.5.Inhibition of miR-137-3p may target LKB1,regulate the dynamics of autophagy through LKB1/AMPK/m TOR signal pathway,thus inhibiting lipid production.Conclusion:1.In vivo experiment: when mice have a long-term high-fat diet and adequate nutrition,miR-137-3p may regulate autophagy and affect lipid production and differentiation through LKB1-AMPK-m TOR signal pathway.2.In vitro experiment: miR-137-3p agonist promoted the differentiation of3T3-L1 preadipocytes,promoted lipid synthesis and inhibited lipid decomposition.miR-137-3p inhibitor inhibited the differentiation of 3T3-L1 preadipocytes,lipid synthesis and decomposition.