Acetylation Modification Of Triose Phosphate Isomerase And Its Effect On The Proliferation Of A549 Cells And HFL1 Cells

Background:Idiopathic pulmonary fibrosis(IPF),known as a tumor that is not a tumor,is prone to occur in the elderly.There is a lack of data on the incidence of IPF in China,but the number of patients is increasing each year.Lung cancer(LC)has become one of the most threatening tumors due to its high morbidity and mortality.At present,the pathogenesis and etiology of IPF and LC have not been fully elucidated.A series of studies have shown that the body’s metabolism is related to the occurrence and development of the two.For example,fibrosis tissues are in a metabolically active state,and high glucose environment can promote the proliferation and migration of lung cancer cells.Studies have shown that some key enzymes in the glycolysis pathway,such as LDH5 and PFK-2,may be related to IPF and LC.Therefore,there may be potential targets for IPF and LC therapy in the glycolytic pathway.Triose-phosphateisomerase(TPI)is a catalytic enzyme in the process of glycometabolism,which can catalyze the conversion of dihydroxyacetone phosphate(DHAP)to glyceraldehyde 3-phosphate(G3P).Glycolysis pathway can be related to lipid metabolism,pentose phosphate pathway,gluconeogenesis pathway,etc.through DHAP and G3 P.Therefore,TPI plays an important role in regulating the body’s metabolic balance.At present,studies have shown that TPI expression is up-regulated in gastric cancer,prostate cancer,colorectal cancer,and breast cancer cells.The post-translational modification of proteins is an important regulatory mechanism for many cell life activities.Studies have shown that histone acetylation is involved in the transcriptional regulation of genes.There are also acetylation modification sites in the TPI protein molecules,but there are few studies on the mechanism of acetylation and its function.Purpose:Explore the acetylation modification mechanism of triose phosphate isomerase,including screening acetylation modification sites and enzymes that catalyze their acetylation and deacetylation modification.And preliminarily explored the effect of inhibiting the acetylation of TPI on the proliferation of A549 cells and HFL1 cells.Methods:1.The survival rates of HEK293 T cells,A549 cells and HFL1 cells treated with different concentrations of TSA and NAM for 24 hours were determined by CCK-8method.2.The expression and acetylation level of TPI in HEK293 T cells,A549 cells and HFL1 cells treated with TSA or NAM were analyzed by Western Blot.3.Using PCR technology,agarose gel electrophoresis,plasmid extraction and other methods to construct wild-type TPI and site-directed mutation TPI eukaryotic overexpression vector.4.Transfection and Western Blot methods were used to analyze the protein expression level and acetylation level of TPI after site-directed mutation.5.The CCK-8 method was used to determine the survival rate of HEK293 T cells,A549 cells and HFL1 cells after the acetylation modification of interference TPI molecules.6.Using PCR technology,agarose gel electrophoresis,plasmid extraction and other methods to construct eukaryotic overexpression vectors for acetyltransferase and deacetylase.7.The expression of TPI and the level of acetylation after acetyltransferase and deacetyltransferase overexpression were analyzed by transfection and Western Blot.Results:1.CCK-8 results showed that the cell survival rate in HEK293 T,A549 and HFL1 cells decreased with the increase of inhibitor concentration.2.Western Blot results showed that the NAM group had no effect on the acetylation level of TPI protein,while the TSA group significantly increased the acetylation level of TPI.3.The wild-type TPI and the mutant TPI eukaryotic expression vectors were Purpose:successfully constructed by enzyme digestion and sequencing.The concentration and purity of the plasmid were proved to meet the requirements of transfection after extraction.4.The results of Western Blot showed that the high expression of tag proteins in each transfection group proved successful transfection.Compared with the wild-type plasmid group,after the mutation of lysine 275 of TPI,the degree of acetylation of TPI protein molecules decreased significantly.The results are statistically significant.5.CCK-8 results showed that lysine 275 mutation of TPI promoted the proliferation of A549 cells and HFL1 cells.6.The eukaryotic expression vectors of acetyltransferases and deacetylases were successfully constructed by enzyme digestion and sequencing.After plasmid extraction,the concentration and purity of the plasmid were proved to be in line with the transfection requirements.7.Western Blot results showed that the high expression of the tag proteins in each transfection group proved successful transfection.Compared with the transfection empty vector group,it was found that the acetylation level of TPI increased after the overexpression of TAF1 and CREBBP.The HDAC3,HDAC4,HDAC5,HDAC7,and HDAC11 of the Ⅰ,Ⅱ,and Ⅳ deacetylase families have a tendency to decrease the acetylation level of TPI.Conclusions:1.TPI can undergo acetylation modification,and the deacetylase family of class I,II,and IV are enzymes that catalyze its deacetylation.2.Lysine 275 of TPI may be its main acetylation modification site,and its mutation will promote the proliferation of A549 cells and HFL1 cells.3.The HAT1,KAT2 A,KAT2B,and KAT8 of the HATs family are not the main enzymes that catalyze the acetylation of TPI.CREBBP may have a certain effect on the acetylation of TPI.TAF1 may be the main enzyme for the acetylation of TPI.HDAC1,HDAC2,HDAC6,HDAC8,HDAC9,and HDAC10 are not the main enzymes that catalyze the deacetylation modification of TPI.HDAC3,HDAC4,HDAC5,and HDAC11 have a certain degree of deacetylation modification of TPI.HDAC7 may be the main enzyme for acetylation modification of TPI.