| Objective: PAK4 is a serine/threonine protein kinase that is highly expressed in many tumor tissues and cells,and is relative to cytological behaviors such as cell proliferation,cell cycle and migration,but there is no research about autophagy.As a tumor suppressor,p53 is mutational in more than 50% of tumors and few studies suggest that it is related to the role of PAK4.In this study,we examined cell proliferation,cell cycle,migration,and related molecules in PAK4-knockdown Hep G2 cells to investigate whether PAK4 carries out functions through p53 and the underlying mechanism.Methods: Use the NCBI website to compare and select PAK4-RNAi target sequences,construct plasmids and lentiviruses.Transfect or infect Hep G2 cells with sh Control and sh PAK4 plasmids or lentiviruses,respectively to construct PAK4-knockdown cell lines.MTT assay,Transwell assay and PI staining flow cytometry were used to detect cell proliferation,migration and cell cycle.Immunoblotting and immunofluorescence were used to detect expressions of target proteins.Results: Hep G2 cells emerge into the logarithmic growth phase within day 1 to 4.5 after transfer of culture.For the plasmid transfection experiment,72 h post-transfection,the results showed that expression of PAK4 in group sh PAK4(0.53±0.28)was lower than that in the sh Control group(1.33±0.05,p<0.01).The OD values in group sh PAK4 at 72 h,96h and 120h(0.40±0.01,0.49±0.01,0.54±0.01)were lower than those in group sh Control(0.52±0.01,0.75±0.00,0.91±0.00,p<0.05).72 h post-transfection,the expression of LC3-II in group sh PAK4(1.78±0.17)was higher than that in group sh Control(0.65±0.07,p<0.0001),but group sh Control was also higher than that in group control(0.06±0.01,p<0.01).For the lentivirus infection experiment: the GFP is the strongest when 96 h post-infection by lentivirus.And the expression of PAK4 in group sh PAK4(0.08±0.03)was significantly lower than that in group sh Control(0.76±0.06,p<0.0001).At 48 h,72h,96 h and 120 h,the OD values in the sh PAK4 group(0.23±0.01,0.27±0.01,0.33±0.01,0.35±0.01)were lower than those in the sh Control group(0.44±0.01,0.44±0.01,0.56±0.04,0.63±0.01,0.80±0.02,p<0.05).Compared with the sh Control group(221.66±12.12),the number of cell migration in the sh PAK4 group(41.00±4.51)were decreased(p<0.0001).Theresults of cell cycle detection showed that the cell in the G2/M phase of group sh PAK4 at72 h,96h,120h(22.84%,18.19%,11.7%)was higher than group sh Control(3.04%,2.37%,1.97%,p<0.05).At 48 h,there was no significant difference between sh PAK4 and sh Control groups.96 h post-infection by lentivirus,the expression of p53 or LC3-II(0.89±0.04,1.28±0.12)in the sh PAK4 group was higher than the sh Control group(0.72±0.04,0.05±0.02,p<0.05).The expression of m TOR or p-Akt in group sh PAK4(0.86±0.04,0.63±0.06)was lower than group sh Control(1.11±0.10,0.76±0.04,p<0.05),and there were no statistical significances in p-p53 or Akt(p>0.05).There were no statistical significances in group sh Control and group Control all above indexes(p>0.05).Conclusion: The best period is 1~4.5 days after passage to intervent cells.Plasmid and lentivirus with PAK4-RNAi target sequence could knock PAK4 down in protein level,and construct Hep G2 cell line with stably low-expressed PAK4.The infective effection is the most efficient when 96 h post-infection by lentivirus.Low expression of PAK4 could upregulate p53,inhibit m TOR or inhibit Akt/m TOR signaling pathway,thereby inhibiting proliferation,arresting cell cycle in G2/M phase,inhibiting migration and activating autophagy in Hep G2 cells.It is suggested that PAK4 is a promising anti-tumor target whose mechanisms was related to p53. |
PDF Full Text Request