Study On The Mechanism Of IL-1β Mediated IL-6 Production And Promoting Glioma Growth

Objective:To investigate the expression of interleukin-1β(IL-1β)in glioma and its effect on the proliferation,invasion and migration of human glioma cells,and to clarify the role of interleukin-6(IL-6)in it and its related mechanism.Methods:1.The TCGA database and GEPIA analysis website was used to analyze the expression of IL-1β in glioma samples;The relationship between different expression of IL-1β and prognosis was studied by survival analysis,and the correlation between IL-1β and IL-6 in glioma samples was further analyzed by correlation analysis.2.The concentrations of IL-1β and IL-6 in the supernatants of human astrocytes NHA and glioma cell lines U87,U251 and A172 were detected by ELISA.Glioma cell lines with high expression of IL-1βand IL-6 were used as follow-up subjects.Furthermore,ELISA was used to detect the production of IL-6 under different concentrations of IL-1β stimulation.3.CCK-8,Transwell chamber and cell scratch test were used to detect the effects of different concentrations of IL-1β on the proliferation,invasion and migration of glioma cells 4.Under the stimulation of a certain concentration of IL-1β.recombinant human IL-6 protein and IL-6 neutralizing protein were used to change the content of IL-6 in glioma cell culture medium.The experiment was divided into four groups:control group(Ctrl group),IL-1β group,IL-1β+IL-6 group and IL-1β+Anti-IL-6 group.The changes of proliferation.invasion and migration of glioma cells were detected by CCK-8,Transwell chamber test and cell scratch test,and the levels of JAK2 and STAT3 phosphorylated proteins were detected by WB.5.Under the stimulation of a certain concentration of IL-1β,siRNA mediated by cationic liposome Lipofectamine 2000 was used to down-regulate the expression of JAK2 and STAT3 in glioma cells.The experiment were divided into four groups:IL-1β group,IL-1β+siNC group,IL-1β+siJAK2 group and IL-1β+siSTAT3 group.The changes of proliferation,invasion and migration of glioma cells were detected by CCK-8,Trans well chamber test and cell scratch test,and the levels of JAK2 and STAT3 phosphorylated proteins were detected by WB.6.The stable expression cell line was constructed by transfecting a kind of glioma cell line with lentivirus.The cells were divided into four groups:control group(Ctrl group),IL-1β group,IL-1β+Anti-IL-6 group and IL-1β+shSTAT3 group.The transfected cells were inoculated subcutaneously into nude mice,and the growth of tumor was observed After 25 days,the tumor was removed and the size and weight of the tumor were measured.Then the tumor was embedded in paraffin and made into sections,and the expression of proliferation index(Ki-67)was detected by immunohistochemical experiment.Results:IL-1β was highly expressed in gliomas,and the high expression of IL-1βled to shorter survival time(P<0.05),and there was a strong positive correlation between the expression of IL-1β and IL-6 in gliomas(P<0.05,R=0.66).Compared with NHA,the level of IL-1β in U87 and U251 culture medium was significantly increased(P<0.05).The level of IL-6 in culture medium of U87,U251 and A172 was significantly increased(P<0.05).In U87 and U251 cells,IL-1β promoted the production of IL-6,and the proliferation,invasion,and migration of glioma cells.Under the stimulation of IL-1β,IL-6 protein can further promote the proliferation,invasion and migration of glioma cells,while IL-6 antibody can significantly inhibit the tumor promoting effect of IL-1β,Mechanistic studies showed that IL-1β stimulation can promote the expression of JAK2 and STAT3 phosphorylated proteins in U87 and U251 cells,IL-6 protein can further promote the expression,while IL-6 neutralizing protein can significantly inhibit its expression.The down-regulation of JAK2 and STAT3 significantly inhibited the proliferation,invasion,and migration of U87 and U251 cells stimulated by IL-1β,The results of nude mice showed that IL-1β could promote the tumorigenesis of U87 cells and the expression of Ki-67 in nude mice,while down-regulating the expression of IL-6 and STAT3 significantly inhibited the tumorigenesis of IL-1β and the expression of Ki-67.Conclusion:IL-1β promotes the proliferation,invasion and migration of glioma cells through the IL-6/JAK2/STAT3 axis.