The Study Of Proteomics And Transcriptomics Associated With SIRT6 In The Disorders Of Liver Lipid Metabolism

Background: Scientific reports indicate that Sir2 can prolong the lives of lower organisms like yeast by restriction of calories, which depends on the deacetylation activity of Sir2. The Sirtuins, orthologues of Sir2 in mammals, are a cluster of proteins which have the deacetylation. SIRT6, one of Sirtuins, can maintain the normal physical structure and function by its deacetylation, regulate the metabolism of sugar and lipid, and resist aging. However, the definite mechanisms of sugar and lipid metabolism regulated by SIRT6 are complicated and not exclusive. Our work places the emphasis on the exploration of new key targets associated with SIRT6 at the regulation of glucose and lipid metabolism by the methods of proteomics and transcriptomics..Aims: 1. To construct the mice of liver-specific SIRT6 knockout and estabolish the L02 cell lines with different expression levels of SIRT6. 2. To screen the interactors of SIRT6 and estabolish the protein-protein network based on proteomics.3. To screen and identifythe transcriptional differencial genes involved metabolism of glucose and lipid based on genomics, and identify the molecules screened by proteomics and genomics.Methods: 1. Construct L-02 SIRT6-Flag cell line and control group, Construct L-02 SIRT6-KO cell line by TALEN knockout technique; identifying the formation of lipid droplet by oil red O staining and BODIPY staining in L-02 SIRT6-KO cell line and control. Construct the liver specific SIRT6 knockout mice by Cre-loxp system,and identify the genotype of SIRT6-LKO mice by DNA agarose gel electrophoresis and Western-Blot; establish the mice model of NAFLD by high fat diet(60% calories) and observe the degree of liver steatosis by Haematoxylin and eosin staining. 2. Pull down the interactors of SIRT6 by IP; screen and identify the potential interactors by label-free protein quantitative technology; construct the protein-protein interaction network.. 3. Screen the transcriptional differential genes by c DNA microarray between L-02 SIRT6-KO cell line and control group and analyse the result by bioinformatics.Results: 1. Successfully construct L-02 SIRT6-Flag cell line and L-02 SIRT6-KO cell line; oil red O staining and BODIPY staining show that the capacity of lipid synthesis in L-02 SIRT6-KO cell line is higher than control group.High content screening system shows that the average fluoresence density in L-02 SIRT6-KO cell line is stronger than control group.Successfully breed SIRT6 liver specific knockout mice: SIRT6-LKO mice; by high fat diet the NAFLD model of SIRT6-LKO mice and control group is estabolished. 2. Pull down a group of molecules interacted with SIRT6 potencially by IP and identify them by Label-free LC-MS/MS;IP experiment shows that SIRT6 can pull down GFAT1? 3. Obtain a group of transcriptional differential genes by c DNA array based on L-02 SIRT6-KO cell line; identify these molecules by q RT-PCR and Western-Blot andfind a series of target molecules associated with the metabolism of glucose and lipid, such as STEAP4?CEBPA?KYNU?PDE1A?HMGA2.Conclusions: 1. Firstly construct L-02 SIRT6-KO cell line by TALEN. Function assays demonstrate that SIRT6 knockout can contribute to the formation of lipid droplet in L02 cell line. Successfully construct SIRT6 liver specific knockout mice. 2. A group of molecules such as G3BP1?GFAT1?COASY?PARP1 are obtained by proteomics;not only find G3BP1?PARP1 which are reported, also find GFAT1?COASYwhich are new and involved metabolism of glucose and lipid. 3. Obtain a group of molecules associated with SIRT6. It can be analysed by GO and KEEG. There are multiple pathways associated with SIRT6 which are in the pathways of metabolism of glucose and lipid.