The Effect Of STAT1 Activation Induced By FRK On Glioma Cell Growth

BackgroundGliomas are the most common and fatal primary malignant tumors in the brain.Even with surgery and postoperative chemoradiotherapy,patients with glioblastoma multiforme(GBM)have a median survival of only 14 months and a 5-year mortality rate of 95%.The occurrence and development of glioma is a multiple progressive process that requires the ability of cell adhesion,migration,proliferation and angiogenesis.Studying the molecular mechanisms and important targets that regulate glioma progression will help develop new effective treatment strategies.Current studies have found that the family of signal transducers and activators of transcription(STATs)play an important role in gliomas.Tyrosine site phosphorylation of STATs is a key link in the cascade of STAT signaling pathways.Our previous study found that the Fyn-related kinase(FRK)gene,protein tyrosine kinase 5(PTK5),plays as a tumor suppressor in the development and progression of glioma.This study aimed to investigate the role of FRK in the activation pathway of STATs and its effect on the growth of glioma.Methods1.The U251 and U87 cells with stable FRK overexpression were generated by lentivirus technique.The effects of FRK overexpression on the related proteins of STAT signaling pathway were detected by Western blot(WB).SiRNA-FRK was designed by small interfering RNA(siRNA)technology,and transiently transfected into U251 and U87 cells to downregulate the expression level of FRK by lipofectamine method.The effects of FRK downregulation on the related proteins of STAT signaling pathway were detected by WB.2.The U251 and U87 cells stably expressing FRK were treated with AG490(the specific inhibitor of JAK2),and the changes of proteins such as the phosphorylated JAK2(p-JAK2)and the phosphorylated STAT1(p-STAT1)were detected by WB.3.The U251 and U87 cells stably expressing FRK were treated with Fludarabine(Flu,a specific inhibitor of STAT1),and the internal and external binding of FRK and STAT1 was detected by Co-Immunoprecipitation(CO-IP).4.The U251 and U87 cells stably expressing FRK were treated with Flu,the effect of FRK on the subcellular localization of p-STAT1 was observed by the immunofluorescence assay.The effects of FRK overexpression on the expression of downstream target genes of STAT1 were detected by WB.5.The STAT1 plasmid was transiently transfected by lipofectamine method,and the effects of STAT1 overexpression on the proliferation of U251 and U87 cells were detected by CCK8 or Edu cell proliferation assays.The STAT1-siRNAs were transiently transfected by lipofectamine method and the effects of STAT1 downregulation on the proliferation of U251 and U87 cells were detected by CCK8 or Edu cell proliferation assays.6.SiRNA-STAT1 was transiently transfected into U251 and U87 cells stably expressing FRK by lipofectamine method,and whether STAT1 was involved in the growth inhibition of glioma cells by FRK was detected by CCK8 or Edu cell proliferation assays.7.The expressions and correlation of FRK and STAT1 in non-tumor brain tissue and various grades of glioma were detectd by WB or immunohistochemistry(IHC).8.U87 cells overexpressing FRK were implanted into the right striatum of nude mice by stereotactic injection technique to construct a glioma orthotopic transplantation model of nude mice.The effect of FRK on the growth of human glioma was evaluated by analyzing the survival time of nude mice and calculating the tumor volume.The effect of FRK on the proliferation of glioma cells was detected by Ki67 cell immunofluorescence method.The effect of FRK on apoptosis of glioma cells was examined by cleaved caspase3 cell immunofluorescence method.Results1.We successfully constructed U251 and U87 cells stably overexpressing FRK.FRK overexpression significantly up-regulated the protein level of p-JAK2 and p-STAT1,but it had no significant effect on the phosphorylation level of STAT3.Down-regulation of FRK inhibited the protein levels of p-JAK2 and p-STAT1,but it still had no effect on STAT3 activation.2.U251 cells overexpressing FRK was treatead using AG490,and the results showed that the phosphorylation level of JAK2 was significantly inhibited,but the up-regulation of the phosphorylation level of STAT1 induced by FRK was not significantly changed.3.The results of CO-IP showed that both FRK and STAT1 could be combined to form a protein complex.The over-expressed U251 cells were treated with Flu,and the binding amount of FRK to STAT1 was significantly reduced.4.The results of cellular immunofluorescence showed a significant increase in the total amount and nuclear translocation of p-STAT1 in FRK overexpressing U251 cells showed.However,the nuclear amount of p-STAT1 was significantly reduced after Flu-treated U251 cells overexpressing FRK.WB results showed that FRK overexpression promoted the protein expression of Bax and inhibited the expression of Bcl-2.After treatment of FRK-overexpressing U251 cells with Flu,the increase of Bax was abolished,and the Bcl-2 inhibitory effect was also reversed.5.The results of CCK8 and EdU cell proliferation experiments showed that STAT1 overexpression significantly inhibited the proliferation of glioma cells.In contrast,down-regulation of STAT1 promoted proliferation of glioma cells.6.The results of CCK8 and EdU cell proliferation experiments showed that the down-regulation of STAT1 promoted the proliferation of glioma U251 and U87 cells in the presence of FRK over-expression.7.The results of WB and IHC showed that both FRK and p-STAT1 in human glioma tissues were significantly lower than non-tumor brain tissues,and they decreased with the increase of malignant grade of glioma.The expression between FRK and p-STAT1 proteins is positively correlated.8.The orthotopic transplantation model of glioma in nude mice was successfully constructed.The survival curve showed that the survival time of nude mice in the FRK overexpression group was significantly prolonged,and the HE staining results showed that the tumor volume was also significantly reduced.The results of cellular immunofluorescence showed that the positive rate of Ki67 in the FRK overexpression group was significantly decreased,and the positive rate of Cleaved caspase3 was significantly increased.Conclusions1.FRK can promote the activation of JAK2 and STAT1,but it cannot activate STAT3.The activation of STAT1 may not be dependent on JAK2.2.FRK can be combined with STAT1 to form a protein complex,promote the nuclear translocation of STAT1,and participate in the regulation of the expression of STAT1 target genes such as Bcl-2 and Bax.3.STAT1 can inhibit the proliferation of glioma cells,and play a role in mediating the growth inhibition of FRK on glioma.4.Both FRK and p-STAT1 were down-regulated in glioma tissues,showing a positive correlation.5.FRK can inhibit the growth of glioma in nude mice,and prolong the survival time of nude mice.