Activation Of Cystic Fibrosis Transmembrane Regulator Suppressed Glioblastoma Cell Proliferation,Migration And Invasion

Background:Glioblestoma(GBM)is higly malignancy with poor prognosis.The discovery of novel target for chemotherapy has been a research hotspot in this field.Ion channels are one of the most important targets for drug developments.Cystic fibrosis transmembrane regulator(CFTR)is one of the vital chloride ion channels to regulate cellular chloride ion movement,mutation of which leads to cystic fibrosis.The mutation of CFTR was found to be related with the tumorogenesis of gastrointestinal tumors,while the relationship between CFTR and biological functions of GBM cells is unknown.The Cancer Genome Atlas(TCGA)and the relevant bioinformatics analysis website are free and open to public access.Researcher can mine the information between the target gene and diseases.The prelimary results of mining from these database indicated that low expression of CFTR mRNA is closely related to the poor prognosis of GBM patients.Thus,it is essential to carried out experiments to illustrate the effects of CFTR on biological functions of GBM cell and the underlying molecular mechanisms.Objective:The aims of this study are to elucidate the role of CFTR in GBM cell proliferation,migration and invasion,and subsequently reveal the relevant underlying molecular mechanisms.Methods and Results:1.The data from "Wanderer" database showed that CFTR mRNA expression was down-regulated in GBM tissues comparing with that in normal control.The data from "Human Protein Atlas" database showed that low CFTR mRNA expression was closely related with poor prognosis of GBM patients.2.Western bloting results showed that CFTR protein expression increased in U87 cells and U251 cells comparing with that in human normal glia cells.3.MTT assay was used to determine cell viability.The cAMP-dependent and non-cAMP-dependent CFTR activator,Forskolin and Cact-A1 were used in this study.Forskolin concentration-and time-dependently reduced U87 and U251 cell viability.Forskolin at the range from 0 to 100 ?M had not cytotoxic effects on HEB cells.Cact-A1 treatment for 72h also reduced U87 cell viability in a concentration-dependent manner.In contrast,CFTR inhibitor CFTR inh-172 significantly increased cell viability and cell density,while partially restored inhibitory effects of Forskolin on U87 cell viability and cell density.4.CFTR activator significantly reduced GBM cell proliferation.CFTR activators significantly restrained GBM cell proliferation,decreased PCNA protein expression,and significantly reduced the positive rate of Ki67.Pretreatment with CFTR inh172 significantly attenuated Forskolin/Cact-Al-induced decrease of Ki67 positive rate.CFTR activation significantly inhibited GBM cell colony formation.In contrast,CFTR inhibitor CFTR inh172 treatment significantly increased comparing with control group.5.GBM cell migration was determined by using wound healing.Forskolin treatment significantly reduced the wound closure percentage.While pretreated with CFTR inh 172 follwing incubation with Forskolin,the wound closure significantly increased compring with Forskolin group.6.GBM cell invasion was measured using transwell assay.CFTR activation significantly inhibited GBM cell invasion.Both Forskolin and Cact-A1 significantly reduced the invasive cell number of GBM cells.In contrast,CFTR inh 172 treatment significantly increased invasive cell number.Forskolin treatment for 24h significantly reduced MMP2 protein expression,which was significantly attenuated by CFTR inh172 pretreatment.CFTR inh172 treatment alone also significantly increased MMP2 protein expression.7.CFTR overexpression reduced U87 cell viability and sensitized Forskolin against U87 cell viability.8.CFTR overexpression significantly inhibited U87 cell migration.9.CFTR activation significantly inhibited JAK3/STAT3 signaling pathway.Both Forskolin and CFTR overexpression significantly reduced the phosphorylation of JAK2 and STAT3 without significant change of their total proteins.When Forskolin was given in CFTR oxpression U87 cells,these ratioes were further significantly reduced.Conclusion:CFTR activation suppressed GBM cell proliferation,migration and invasion likely through dowregulation of JAK2/STAT3 signaling pathway.