Construction of Fsp27 Gene Silencing Vector and Its Effect on Cell Lipolysis

doi:10.13560/j.cnki.biotech.bull.1985.2019-0369

Abstract

Abstract: This work is aimed to construct the fat-specific protein of 27（Fsp27）gene silencing vector and to investigate the effect of Fsp27 gene expression on the lipolysis of 3T3-L1 cells as well as explore its mechanism. RNAi technology was used to construct the eukaryotic interference vector of Fsp27 gene to down-regulate the expression of the Fsp27 gene. The “cocktail” method induced differentiation of 3T3-L1 preadipocytes into mature adipocytes. Liposomes were transfected into adipocytes,oil red O stained lipid droplets,and enzymatic method was employed to determine the content of glycerol and triglyceride in cells. Western blot analysis was used to detect the protein expression of Fsp27,HSL,ATGL and PPARγ in cells. The results from Western blot showed that the positive sh-Fsp27 vector down-regulated the expression of Fsp27,while the expression of ATGL and PPARγ increased（P<0.05）,and the silencing results by sh-Fsp27-2 was the best. The results of enzymology demonstrated that the content of triglyceride in the positive sh-Fsp27 interference group decreased,and the content of glycerol increased（P<0.05）. The results of oil red O staining showed that there were large lipid droplets accumulated in the blank control group and the negative control group,while the distribution of small lipid droplets in the positive sh-Fsp27 group was major,and no obvious big fat drops was observed. In conclusion,the silencing effect by sh-Fsp27-2 is the best,Fsp27 gene silencing may accelerate the lipolysis rate of 3T3-L1 cells,which is mainly achieved by inhibiting the fusion among small lipid droplets and increasing the ATGL-mediated hydrolysis .

Key words: Fsp27, RNAi, 3T3-L1 preadipocytes, lipolysis

XU Xiang, DONG Wei-peng, ZHANG Shao-hua, FENG Chen-yi, LIU Tian-fu, YAN Jiong. Construction of Fsp27 Gene Silencing Vector and Its Effect on Cell Lipolysis[J]. Biotechnology Bulletin, 2020, 36(1): 88-94.

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