SIRT6对肝细胞脂质合成的影响及其转录基因分析

The Effect of SIRT6 on Lipid Synthesis of Hepatocytes and Analysis of Its Transcriptional Genes

目的:在肝脏L-02 SIRT6-KO细胞系中,观察SIRT6在肝脏脂滴形成中的作用,初步研究在肝脏脂质代谢中SIRT6相关的转录差异基因发挥作用的分子机制。方法:利用基因敲除技术TALEN技术,构建肝脏L-02 SIRT6特异性敲除细胞系,并经q PCR和Western blot检测其表达水平;利用油酸刺激L-02 SIRT6-KO细胞及其对照组,体外模拟肝脏脂肪变的条件,并经高内涵和油红染色验证其脂滴形成能力;利用基因芯片检测L-02 SIRT6-KO及其对照细胞系中相关差异基因,并经生物信息学分析筛选脂代谢相关差异基因。结果:建立人肝脏SIRT6特异性敲除细胞系,q PCR显示m RNA下降50%,但Western blot证明SIRT6完全敲除;BODIPY实验及油红O染色发现,L-02 SIRT6-KO细胞比对照组其脂滴形成数量增多,高内涵荧光检测显示L-02 SIRT6-KO细胞中脂滴荧光强度比对照组增强(176.38±2.55 vs 104.26±2.08);通过对差异转录基因分析,发现了一组在脂质代谢中和SIRT6相关的关键基因如STEAP4、HMGA2、PDE1A、INSL4等。结论:成功建立人肝脏L-02 SIRT6-KO细胞系,并经实验证明SIRT6缺失能够促进脂滴形成;筛选到一组和SIRT6相关参与脂质代谢的关键基因。

Objective： To observe the roles of SIRT6 in the formation of liver lipid droplet, and preliminarily elucidate the mechanisms of SIRT6 and its transcriptional genes in the dysfunction of liver lipid metabolism. Methods： Construct L-02 SIRT6-KO stable cell line and control cell line by TALEN technique provided by Gene Tech, and then detect the expression level of SIRT6 by q PCR and Western blot. Oleic acid treatment is uesd in L-02 SIRT6-KO cell line and control cell line to mock the condition of fatty liver development in vitro and identify the formation of lipid droplet by oil red O staining and BODIPY staining. The intensity of lipid droplet is measured by High Content Screening System. The transcriptional differential genes are screened by c DNA microarray between L-02 SIRT6-KO cell line and control group and analysed by bioinformatics. Results： Successfully establish the SIRT6-KO cell line in human liver L-02 cell line and the result is validated by q PCR and Western blot. The number of lipid droplet formation in L-02 SIRT6-KO cell line is much more than control group exhibited by oil red O staining and BODIPY staining. High Content experiment indicates that the fluorescence intensity of lipid droplets is much higher in L-02 SIRT6-KO cell line than control group（176.38±2.55 vs 104.26±2.08）. A group of key genes（STEAP4, HMGA2, PDE1 A, INSL4）associated with lipid metabolism from c DNA array are analysed by informatics and validated by q PCR and western blot. Conclusions： Human specific SIRT6 knockout cell line in liver is established successfully. Loss of function mutation in SIRT6 can contribute to the formation of lipid droplet in L-02 cell. A group of key genes associated with SIRT6 involving in liver lipid metabolism are discoveried by c DNA microarray and bioinformatics, which may participate in the process of liver lipid development.