Corticotropin-releasing Factor(CRF) Promote The Apoptosis Of U87MG Cell Via Enhancing The Phosphorylation Of GSK3β

Background: Glioma is the most common tumor in the central nervous system neoplasms,showing invasive growth,infringing easily the important nerve structure,and has a poor prognosis,the characteristics of high recurrence rate and mortality.Although current surgical procedures,radiotherapy and chemotherapy have greatly improved,but the treatment and prognosis of glioma was still far from reaching an ideal effect.Depending on biological treatment（target therapy）to extend survival time of the patients gives us a new way of thinking.The results show that increased intracellular levels of cAMP and activated cAMP signal transduction pathway can promote the survival of the central and peripheral nerve tissue.The study of CRF acts on cerebellar granule cell neurons have shown that activation of CRF-CRFR-cAMP-PKA resulting in GSK3 beta phosphorylation.The phosphorylation of GSK3 beta has an important role in the neuroprotective effect.The biological function of GSK3 beta is more complex,such as gene transcription in the cancer cell,accelerating cell cycle,participating in the process of tumor cell invasion and metastasis and apoptosis.For the diversification of GSK3 beta plays different roles in different tumors,so it may become a potential target for tumor therapy.The previous experimental studies confirmed that CRF can promote the apoptosis of glioma U87 MG,and the apoptosis related protein capase-3expression are consistent with and cell apoptosis trend.Further studies have found that CRF can significantly increase the levels of cAMP and PKA in U87 cells and promote apoptosis.Overall,when CRF acts on two kinds of cells,the expression trends of upstream signaling molecules are consistent.But the result is quite the opposite.So it is necessary to further study the expression ofGSK-3 under the action of CRF and elucidate downstream signaling pathway of CRF promotes apoptosis of U87 MG cells.Objective: To research the expression of p GSK3β in U87 MG cell cytoplasm and nucleus stimulated by CRF,and to explore the role of GSK3βin U87 MG cell apoptosis initiated by CRF.Methods:1 Immunofluorescence microscopy was used to detect the expression of pGSK3 β of U87 MG cells in control group and experimental group intervened with CRF.2 Cultured U87 MG cells,then intervention with different concentrations of CRF(10^-7 and 10^-8 10^-9 mol / L),intervention time points for 12 h,24h,36 h,the corresponding control groups were set in each CRF concentration and each time point.Application of cytoplasmic/nuclear protein extraction kit to extract the cytoplasmin and nuclear protein.Western blot was used to further detect the expression and distribution of pGSK3β in cytoplasm and nucleus.Results:1 Immunofluorescence microscopy showed that the control group and the experimental group had the expression of pGSK3β.2 The results of pGSK3β in cell cytoplasm and nuclear detected by Western blot showed that: When the CRF concentration is 10^-7 mol/L,there was no significant difference between the experimental group and the control group in the expression of cell cytoplasm pGSK3β and nuclear pGSK3β in 12 hours and 24 hours(cytoplasm Z_12h=-1.091,P=0.1375,Z_24h=-0.218,P=0.4135,nuclear Z_12h=-0.655,P=0.2565,Z_24h=-0.655,P=0.2565),but in 36 hours,the expression of pGSK3β in the experimental group was significantly higher than that in the control group,both in cytoplasm and nucleus(cytoplasm Z_36h=-1.964,P=0.025,nuclear Z_36h=-1.964,P=0.025).When the CRF concentration is 10^-7 mol/L,there was no significant difference between the experimental group and the control group in the expression of cell cytoplasm pGSK3β and nuclear pGSK3 in 12 hours and 24hours(cytoplasm Z_12h=-1.107,P=0.134,Z_24h=-1.528,P=0.0635,nuclearZ_12h=-0.655,P=0.2565,Z_24h=-1.091,P=0.1375),but in 36 hours,the expression of pGSK3β in the experimental group was significantly higher than that in the control group,both in cytoplasm and nucleus(cytoplasm Z_36h=-1.964,P=0.025,nuclear Z_36h=-1.964,P=0.025).When the CRF concentration is 10^-9 mol/L,there was no significant difference between the experimental group and the control group in the expression of cell cytoplasm pGSK3β and nuclear pGSK3β in 12 hours and 24 hours 36 hours(cytoplasm Z_12h=-0.655,P=0.2565,Z_24h=-0.218,P=0.4135,Z_36h=-0.218,P=0.4135,nuclear Z_12h=-0.218,P=0.2135,Z_24h=-1.091,P=0.1375,Z_36h=-1.091,P=0.1375).3 There was a significant difference in the ratio of experimental group/control group,both in the cytoplasm and nucleus between CRF 10^-7mol/L group,CRF 10^-8 mol/L group and CRF 10^-9 mol/L group(cytoplasm Chi-square Z_{experiment group/control group} =7.200,p =0.027,nuclear Chi-square Z_{experiment group/control group}=7.200,p =0.027).Conclusions:1 Immunofluorescence microscopy showed that the expression of pGSK3β was present in U87 MG cells.2 The results of pGSK3β in cell cytoplasm and nuclear detected by Western blot showed that: CRF can promote the phosphorylation of GSK3β,and showed a concentration dependence,the higher the concentration,the stronger the effect.