Molecular Mechanism Of Diacylglycerol Kinase Theta Involved In Lipid Metabolism

Nonalcoholic fatty liver disease(NAFLD)is caused by the destruction of the balance of lipid metabolism in the liver cells,which res?lted in triglyceride(TG)acc?m?lation.In vivo TG and diacylglycerol(DAG)can be converted to each other,and diacylglycerol co?ld be converted to phosphatidic acid(PA)by its phosphorylation mediated by diacylglycerol kinase(DGKs).Therefore,DGKs play important roles in the process of diacylglycerol acc? m? lation.DGKs are divided into five types,and DGK? is the only subtype of type V.DGK? can participate in a variety of intracell?lar signaling pathways by reg?lating the concentration of DAG and PA.Studies have been reported that DGK? was involved in glycolipid metabolism and other pathways,but its specific molec?lar mechanism and physiological function remain unclear.In this study,the DGK? gene knockout HepG2 cell line was established by CRISPR/Cas9 genome editing technology,and the in vitro function of DGK? gene was studied.In addition,the adenovirus vector carrying the CRISPR/Cas9 system was used to study the function of DGK? in mice.In this study,the molec?lar mechanism involving lipid metabolism of DGK? was studied in vitro and in vivo.The main contents of this research include the following aspects.1.Establishment of DGK? gene knockout HepG2 cell line based on CRISPR/Cas9 technology.The best activity sgRNA4 was screened by T7E1 digestion.PCR was used to amplify the sequence of the upstream and downstream homologous arm of DGK? targeting region for constructing donor vector.This donor vector was co-transfected into HepG2 cells with Cas9 expression vector by screening the cells with dr?g,DGK? gene knockout cell was obtained and confirmed by PCR and Sanger sequencing.2.Characterization of DGK? gene knockout HepG2 cell line in vitro.The proliferation rate of WT HepG2 and DGK? KO HepG2 cells was measured by MTT assay.Using oil red O staining to analyze the intracell?lar lipid acc?m?lation,then the intracell?lar PA and DAG contents were also measured.RT-PCR and Western blot were used to detect the expression of glycolipid metabolism-related and ins?lin resistance-related factors in each group.3.Investigation on the molec?lar mechanism of DGK? gene involving lipid metabolism in mice.The best mouse DGK? sgRNA was screened by T7E1 digestion.Hepa1-6 cells were co-infected by adenovirus vector pAd5/U6-sgRNA4 and pAd5/tet-on-Cas9 adenovirus.T7E1 assay was used to test the in vitro targeting efficiency.These two adenoviruses were injected into the mice by tail vein,and the liver specific DGK? knockdown mice model was obtained thro?gh RT-PCR detection.Ser?m from each group of mice was obtained for lipoprotein content analysis.HE and oil red O staining were used to observe the acc?m?lation of lipid in hepatocytes.RT-PCR was used to detect the mRNA expression of DGK? and lipid-related factors in mice.The following res?lts are obtained in this study.1.The highest activity of sgRNA4 was successf?lly screened and the HepG2 cell line with DGK? knockout was obtained.Compared with the control group,the proliferation rate of DGK? KO HepG2 cells was significantly increased,and intracell?lar lipid acc?m?lation was obviously found.The content of PA in DGK?KO HepG2 cells was significantly decreased,while DAG was significantly increased.2.Compred with the control group,in the DGK? KO HepG2 cells,the mRNA and protein levels of lipid synthesis related factors(FAS,PPAR? and SREBP-lc)were up-reg?lated and the level of lipid catabolism related factors(CPT1a)was down-reg?lated.The phosphorylation level of PKC? was increased,while phosphorylation level of IRS-1 was decreased.The phosphorylation levels of mTOR and AKT were reduced.3.The mouse DGK? sgRNA4 with the highest target activity was screened and the mouse model of liver specific DGK? knockdown was obtained.4.Compared with the control group,in liver-specific DGK? knockdown mice,the levels of total cholesterol(CHOL)and TG in ser?m lipoprotein were significantly increased.HE staining of liver tissue showed significant vacuolar structure in liver cells,and intracell?lar orange droplets were increased by oil red O staining.5.Compared with control group,the mRNA expression of SREBP-1c,FAS and PPAR?was significantly up-reg?lated in liver-specific DGK? knockdown mice,but there was no significant difference in mRNA expression of CPT1a and HADHa.