A Study On G Protein Coupled Receptor Kinase(GRK) 4-induced Proliferative Inhibition Of Human Osteosarcoma Cells And Its Mechanism

Objective: To investigate the inhibitory effect of G protein coupled receptor kinase(GRK)4 on the proliferation of human osteosarcoma cells.Methods: 1.Human osteosarcoma U2OS(p53 + / +)cells were transiently transfected with p EGFP-GRK4 plasmid.After transfection,cells were sorted into U2OS/GRK4(-)and U2OS/GRK4(+)cell populations by of fluorescent-activated cell sorting(FACS)technique;2.MTT assay was used to detect the proliferation of U2OS/GRK4(-)and U2 OS / GRK4(+)cells;3.U2 OS was transfected with p EGFP-GRK4 plasmid and then detected by flow cytometry at 24 hours after thransfection.The cells of U2OS/GRK4(-),U2OS/GRK4(+)and cell cycle distribution were gated up and analyzed by FITC channel and PI channel respectively.4.The kinase dead mutant p EGFP-GRK4 K216M/K217 M was constructed and transfected into U2 OS and Saos-2(p53-/-)cells respectively.Twenty-four hours after transfection,flow cytometry was used to detect distribution of cell cycle in U2OS/GRK4K216M/K217M(-)and U2OS/GRK4 K216M/K217M(+)?Saos-2/GRK4K216M/K217M(-)and Saos-2/GRK4 K216M/K217M(+);5.Lentivirus-mediated GRK4 gene expression technology to construct U2 OS cells(strains)with high or stable expression and infect U2 OS and Saos-2 cells;6.GRK4 gene mediated by lentivirus,LV5-GRK4(wild type)and LV5-GRK4K216M-K217M(mutant type)infect U2 OS and Saos-2 cells.After 72 hours,cells were collected,and the cells were planted in 96-well cell culture plates respectively.The cell proliferation was detected by the MTT method,and a growth curve was drawn;7.After 72 hours,the cells infected with lentivirus were collected,and the cells were planted in cover glass.In the 12-well cell culture plate of the slide,Ki-67 immunofluorescence staining was performed the next day;8.72 hours later,the lentivirus-infected cells were collected,and the cells were planted in the 12-well cell culture plate on the 6th and 9th Daily aging-related ?-galactosidase activity(SA-?-gal)staining;Results: 1.U2 OS /GRK4(-)(purity of 98.2%)and U2 OS / GRK4(+)(purity of 94.1%)were obtained by FACS;2.MTT assay showed that cell proliferation in U2 OS /GRK4(-)cells was significantly inhibited compared with that in U2 OS / GRK4(-);3.Cell cycle analysis demonstrated that,compared with U2 OS / GRK4(-)cells,U2 OS / GRK4(+)cells showed a significantly G1/G0 phase arrest(47.0±4.8% and 83.4±6.7% p<0.01,respectively);4.In U2 OS and Saos-2 cells,compared with GRK4 K216M/K217M(-)cells,the cell cycle progression of GRK4 K216M/K217M(+)cells was significantly arrested in G1/ G0(U2OS cells were 50.9±10.0% and 80.7±13.2% p<0.05,Saos-2 cells were 39.8±6.9%and 71.7±2.3% p<0.01,respectively);5.Lentivirus-mediated overexpression of GRK4 gene infects U2 OS and Saos-2 cells with an infection rate of> 70%;6.MTT experimental results show that U2 OS cells and Saos-2 cells are overexpressed with GRK4 compared to the NC control group The proliferation of wild-type and mutant U2 OS and Saos-2 cells was significantly inhibited;7.Immunofluorescence staining results showed that Ki-The number of 67 positive cells was significantly reduced;8.The results of cell senescence test showed that the blue staining rate of U2 OS / GRK4-Wt and Mut groups on the sixth day were(54.7 ± 7.4%,51.0 ± 17.5% p <0.05),which was significantly higher thanGRK4-NC The control group(5.0 ± 3.5% p <0.05),the blue staining rate of U2 OS / GRK4-Wt and Mut groups on day 9 were(69.7 ± 8.6%,74.7 ± 19.9% p<0.05),which was significantly higher than that of GRK4-NC control group(7.0± 3.5% p <0.05).Conclusion: GRK4 significantly inhibits the growth of human osteosarcoma U2 OS and Saos-2 cells through p53-independent signaling pathway,and its biological effect of inhibiting cell proliferation has nothing to do with its kinase activity.The results of this study provide valuable information for further exploring the molecular mechanism of GRK4 inhibition of tumor cell proliferation.