| Objective: In this study, we investigate that overexpression and depletion of Elp3 or Elp4 could affect cell migration and invasion, and explore the mechanism of Elp3 and Elp4 in regulating the ability of cell migration and invasion.Methods:1. Identified the vectors of pFLAG-CMV4-Elp3 o, p FLAG-CMV4-Elp4 o, GV248-Elp3 i and GV248-Elp4 i.2. Identified the suitable G418 and Puror concentration in the selection of stable transfection.3. Obtained the Elp3 or Elp4 overexpression and depletion in HepG2 cell lines by G418 and Puror selection method. Transfected HepG2 cell was divided into different groups: Elp3 overexpression group, Elp4 overexpression group, Elp3 depletion group and Elp4 depletion group.4. After different treatment, we verified the intergrated plasmid in genomic DNA by RT-PCR. Elp3 and Elp4 expression level in each group was detected by Real-time PCR and Western blot.5. Cell ability of migration and invasion was measured by wound healing assay and transwell assay.6. The mRNA expression level of MMP-2, MMP-9 and Paxillion was detected by real-time PCR.7. The protein expression level of PI3 K, AKT, p-AKT, MMP-2, MMP-9 and Paxillion was detected by western blot.8. The protein expression level of PI3 K, AKT and p-AKT was detected by western blot followed by transfection with different does of pFLAG-CMV4-Elp3 o, pFLAG-CMV4-Elp4 o, GV248-Elp3 i and GV248-Elp4 i.9. Identified the suitable LY294002(the inhibitor of PI3K/AKT signal pathway) concentration in the treatment of HepG2 cells.10. After the treatment of LY294002, cell ability of migration and invasion was measured by wound healing assay and transwell assay.11. After the treatment of LY294002, The mRNA expression level of MMP-2, MMP-9 and Paxillion was detected by real-time PCR.12. After the treatment of LY294002, The protein expression level of PI3 K, AKT, p-AKT, MMP-2 and MMP-9 was detected by western blot.Results:1. We successfully detected 1601 bp band in p FLAG-CMV4-Elp3 plasmid, 1613 bp band in pFLAG-CMV4-Elp3 plasmid, 44 bp in GV248-Elp3 i and GV248-Elp4 i plasmid.2. HepG2 was cultured with different concentrations of G418 and Puror for 14 days. After observation we conformed that the 600ng/μl is the best concentration of G418 and the 50ng/μl is the best concentration of Puror for this experiment.3. HepG2 was cultivated in medium which contained G418 and Puror after transfction, we obtained the stable transfection cell line after single clone selection and identification.4. Elp3 and Elp4 interference inhibited cell migration and invasion, but overexpression groups promoted the ability.5. The overexpression groups increased the mRNA expression of MMP-2, MMP-9 and Paxillin, but the depletion groups decreased the expression level.6. The overexpression groups increased the protein expression of p-AKT, MMP-2, MMP-9 and Paxillin but the depletion groups decreased the expression level.7. Western blot analysis demonstrated that the expression of Elp3 and Elp4 promoted the protein expression level of p-AKT in does-dependent manner.8. After the treatment of LY294002, the ability of migration and invasion was inhibited.9. The overexpression groups increased the mRNA expression of MMP-2 and MMP-9 after the treatment of LY294002, but the depletion groups didn’t.10. The protein expression level of p-AKT, MMP-2 and MMP-9 has been reduced.Conclusion:1. In vitro, Elp3 and Elp4 can promote metastasis and invasion of hepatocellular carcinoma. However, Elp3 and Elp4 depletion restrain such phenomenon.2. Elp3 and Elp4 can regulate cell migration and invasion partly through PI3K/AKT signal pathway. |
