Melatonin Selectively Activates CREB-dependent Genes Through Src And PKA Parallel Pathways To Inhibit Osteosarcoma Cell Proliferation And Migration

Objective: To observe the effect of melatonin on the proliferation and migration of osteosarcoma cells,and to investigate the role of the PKA pathway and Src pathway in mediating CREB-dependent gene transcription in the above-mentioned regulatory process.Methods: Mg-63 osteosarcoma cells were cultured,and Transwell assay,Celigo staining assay and cck-8 assay were performed 24 hours after the effect of melatonin at different concentrations to verify the effect of melatonin on the proliferation and migration of osteosarcoma cells.24 hours after the effect of melatonin and H89 at the optimal concentration of 4m M,western blot experiments were conducted to detect the phosphorylation level of p-ERK1/2 / p-p90RSK/p-CREB pathway to verify the relationship between PKA and CREB-dependent pathway.Western blot assay was conducted 24 hours after the effect of 4m M melatonin and PP2 to detect the phosphorylation level of p-ERK1/2 / p-CREB pathway to verify the relationship between Src and the CREB-dependent pathway.Western blot assay was performed 24 hours after the effects of melatonin and PP2 on the c AMP-PKA were applied to detect the expression level of c AMP to verify the relationship between the c AMP-PKA and the Src pathway.Results:(1)Compared with the control group,the proliferation of osteosarcoma cells in the treatment group with different concentrations of melatonin was reduced.Compared with the control group,the Transwell metastasis rate in the treatment group with different concentrations of melatonin was analyzed by T-test P<0.05.T-test analysis of scratch mobility at 48 hours P<0.05.It was confirmed that melatonin at different concentrations had an inhibitory effect on cell migration in a concentration-dependent manner.(2)the expression of p-ERK1/2 in cells pretreated with H89 and melatonin only decreased slightly.On the contrary,H89 treatment alone can cause up-regulation(P<0.05),indicating that PKA has a significant inhibitory effect on ERK1/2 cascade.H89 reduced the phosphorylation of CREB,and this effect was even more pronounced in cells treated with melatonin other than H89.In addition,we also tested the effect of H89 on the transcriptional activity of CRE-dependent genes,and found that H89 caused a slight decrease in the transcriptional activity of CRE-dependent genes,but the inhibition was more obvious after melatonin was added.(3)the effects of PP2 on melatonin treated cells and untreated control group were compared,and it was found that the phosphorylated ERK expression was significantly reduced in the melatonin group(P<0.01),and the p-ERK1/2 level was also significantly reduced.Similar results were found for p-CREB expression(p <0.01).CRE-luciferase assay showed the same trend of CRE-dependent gene transcriptional activity.(4)melatonin reduced the level of c AMP in osteosarcoma,combined treatment with PP2 had no further effect.Conclusion: melatonin inhibits the proliferation and migration of osteosarcoma cells in a concentration-dependent manner,and its mechanism of action is related to the parallel involvement of the PKA pathway and the Src pathway in mediating CREB dependent gene transcription.