Elp3 Inhibits Cell Apoptosis Induced By Cisplatin In HCC Cells

Objective:In this study,we construct stable transfected HepG2 cells,compared with 7701 cells,we found that overexpression of Elp3 can inhibit cell apoptosis which induced by cisplatin,while depletion of Elp3 can promote cell apoptosis.Then we explored the mechanism of Elp3 in regulating the cell apoptosis induced by cisplatin.Methods:1.The lentiviral vectors was used to transfect plasmids into HepG2 cells,and obtained the Elp3 overexpression and depletion cell lines by Puror selection method.2.Western blot?RT-PCR and Real-time PCR were used to identify the protein and mRNA expression level of Elp3 in table transfected HepG2 cells.3.Flow Cytometry assay was used to measure the apoptosis of HepG2 cells and 7701 cells which treated with different dose of cisplatin.4.Flow Cytometry assay was used to measure the apoptosis of HepG2-Elp3o and HepG2-Elp3i cells treated with cisplatin,and the apoptosis of 7701-Elp3o and 7701-Elp3i cells treated with cisplatin.5.Western blot was used to detect the change of protein expression level of Elp3?caspase3?cleaved caspase3?AKT?p-AKT?BAD?p-BAD in HepG2-Elp3o and HepG2-Elp3i cells which are treated or not-treated with cisplatin.6.Western blot was used to detect the protein expression level of Elp3?caspase3?cleaved caspase3?AKT?p-AKT?BAD?p-BAD which transfected with different does of PIRES2-EGFP-Elp3o and GV248-Elp3i plasmids.7.Western blot was used to detect the change of protein expression level of Elp3?AKT?p-AKT?BAD?p-BAD in 7701-Elp3o and 7701-Elp3i cells which are treated or not-treated with cisplatin.8.Flow Cytometry assay was used to measure the apoptosis change level of HepG2 cells which transfected with four deletion plasmids.Results:1.After 7 days of Puror selection,we can observe the green fluorescent protein expression level of stable transfected HepG2-Elp3o and HepG2-Elp3i cells.2.RT-PCR and Real-time PCR results showed that the lentiviral vectors can express the correct mRNA,and western blot results showed the correct protein expression in HepG2-Elp3o and HepG2-Elp3i cells.3.Flow Cytometry results showed that compared with 7701 cells,HepG2 cells can resist cell apoptosis induced by cisplatin.4.Flow Cytometry results showed that overexpression of Elp3 can inhibit cell apoptosis while deletion of Elp3 can promote cell apoptosis in HepG2 cells which treated by cisplatin.But in 7701 cells,the overexpression or deletion of Elp3 can not affect cell apoptosis.5.Western blot results showed that in cisplatin-treated HepG2 cells,overexpression of Elp3 can inhibit the dissociation of caspase3 and promote the expression of p-AKT and p-BAD,while deletion of Elp3 can do the opposite..6.Western blot results showed that the expression of Elp3 promoted the protein expression level of p-AKT and p-BAD in does-dependent manner.7.Western blot results showed that in cisplatin-treated 7701 cells,overexpression or deletion of Elp3 can not affect the expression level of p-AKT and p-BAD.8.Flow Cytometry results showed that transfection of four different Elp3 deletion plasmids can affect cell apoptosis induced by cisplatin.Compared with control cells,the Elp3-d3 and Elp3o cells can inhibit cell apoptosis similarly,while the Elp3-dl and Elp3i cells can promote cell apoptosis.Conclusion:1.Stable transfected HepG2-Elp3o and HepG2-Elp3i cells are successfully constructed,and can express the target genes correctly.2.Compared with 7701 cells,we found that overexpression of Elp3 can inhibit cell apoptosis induced by cisplatin,while depletion of Elp3 can promote cell apoptosis.3.In HepG2 cells,overexpression of Elp3 can activate AKT signal pathway and promote the expression of p-BAD,but in 7701 cells,Elp3 can not affect them.4.The inhibition of Elp3 in cell apoptosis may rely on it's HAT groups.