The Key Role Of Elp3 In HCC Development Depends On Its HAT Activity

Objective:In this study,we construct several deletion sequences of Elp3,compared with the overexpression and interference of Elp3 sequence,we investigate that the function of Elp3 in HCC relies on it’s HAT groups and it participating the formation of the Elongator complex.Methods:1. Sequencing and double enzyme identification of recombinant plasmid was used to identify the PIRES2-EGFP-elp3 plasmid and the other four deletion plasmids.2. RT-PCR and western blot was used to identify the mRNA and protein expression level of the target genes after transient transfect every plasmid in 293 a cells and HepG2 cells.3. Real-time PCR and western blot was used to identify the mRNA and protein expression level of Elp3 after transient transfect every plasmid in HepG2 cells.4. CCK-8 assay was used to detect the cell growth under the different treated conditions.5. Wound healing assay was used to detect the cell migration ability under the different treated conditions.6. Transwell assay was used to detect the cell ability of invasion and migration under the different treated conditions.7. Comet assay was used to detect the change of DNA damage in different groups.8. Western blot was used to detect the change of protein expression level of Elp3、Elp4、AKT、p-AKT、MMP2、MMP9 in different groups.Results:1. Sequencing and double enzyme identification of recombinant plasmid results showed that the target genes which insert in the expression carrier is correct.2. RT-PCR results showed that the deletion sequences which insert in the expression carrier can express the correct mRNA.Western blot results showed that the deletion sequences which insert in the expression carrier can express the correct protein in 293 a cells and Hep G2 cells, which showed our construction is correct.3. Real time PCR and Western blot results showed that the deletion sequences didn’t affect the endogenous expression of Elp3,and the expression level of Elp3 didn’t change compared with the normal HepG2 cells.The Elp3 group induced the expression of Elp3 protein and the Elp3 i group reduced it.4. CCK-8 results showed that the Elp3-d1、Elp3i groups ruduced the cell growth, the Elp3-d3、Elp3 groups increased the cell growth obviously, and the Elp3-d2、Elp3-d4 groups had no difference.5. Wound healing results showed that the Elp3-d1、Elp3i groups inhibited cell migration,the Elp3-d3、Elp3 groups promoted that ability,and the Elp3-d2、Elp3-d4 groups had no difference.6. Transwell results showed that the Elp3-d1、Elp3i groups inhibited cell migration and invasion,the Elp3-d3、Elp3 groups promoted that ability,and the Elp3-d2、Elp3-d4 groups had no difference.7. Comet assay results showed that the Elp3-d1、Elp3i groups inhibited the DNA damage repair ability,the Elp3-d3、Elp3 groups promoted that ability,and the Elp3-d2、Elp3-d4 groups had no difference.8. Western blot results showed that the Elp3-d1、Elp3i groups reduced the protein expression level of p-AKT、MMP2、MMP9,the Elp3-d3、Elp3 groups induced them,and the Elp3-d2、Elp3-d4 groups had no difference.Conclusion:1. Four deletion plasmids are successfully constructed,and can correctly express the target genes.2. The insert of deletion plasmids didn’t affect the endogenous expression of Elp3.3. Elp3 promoted cell proliferation、invasion and migration and promoted DNA damage repair relyed on it’s HAT groups.4. The promotion of Elp3 in AKT signal pathway may rely on the completion of Elongator complex and the HAT groups.5. Elp3 participated in many cell activities and actived some genes might through the acetylize function.