The Effect And Mechanism Of FTL On The Migration And Invasion Of Glioma

Background and objective:Gliomas were originated in the neuroectodem and they accounted for approximately 75% of primary malignant brain tumor.Despite of the current standard therapy regimen(temozolomide combined with simultaneous radiotherapy),there were still many patients suffered tumor recurrence within several months after surgery because of the highly invasive behavior of glioma cells.Glioma cells always invaded normal brain tissue along the long fibers or blood vessels,which made it difficult to resect tumor completely and to achieve satisfactory radiotherapy effect.Ferritin light chain(FTL)was one of the most important iron metabolism related proteins.Few studies were performed to investigate the role of FTL in regulating migration and invasion in glioma.So in this study,we tried to investigate the effect of FTL on migration and invasion of glioma cells and to elucidate its possible molecular mechanism.Methods:FTL m RNA expression microarray data and accompanying clinical data were downloaded from Gliovis,UCSC platform and Chinese glioma genome altas(CGGA).We used the RNA-Seq to explore the differential expression of FTL between low grade glioma(LGG)and high grade glioma(HGG).Besides,the correlation between FTL and molecular pathological features(IDH1,subtypes)was analyzed.Glioma samples from our insitituion were used to valdivated the expreesion of FTL in glioma.Then we used sh RNA and plasimid to knockdown and overexpresse the expression of FTL in U87 and U251 cells,repectively.Wound healing and transwell were used to investigated the effect of FTL on migration and invasion of glioma cells.Western blot(WB)and Immunofluorescence(IF)were used to detect the expression of migration related markers(MMP2,Vimentin,E-cadherin and Snail1)and signaling related proteins.Finally,we subcutaneously implanted NC-sh RNA or FTL-sh RNA transfected U87 cells into nude mice.Then WB and immunochemistry were used to detect expression of migration related marker and signaling related proteins.Results:Gene expression level of FLT was higher in HGG patients than LGG patients according to TCGA and CGGA datasets(PartⅠ Figure1A-1B).In order to verify this result,we used tissue microarrays which contained 142 glioma tissues and the results of IHC revealed that HGG patients had higher level of FTL expression than LGG patients(PartⅠ Figure2C-2E).IDH1 mutation was an important marker in predicting the prognosis of glioma patients.Our analysis found that patients with IDH1 mutant had lower level of FTL than HGG patients(PartⅠ Figure3A-3B).Besides,FTL was high in mesenchymal subtype both in TCGA and CGGA datasets(PartⅠ Figure3C-3D).The prognostic role of FTL in glioma hadn’t been explored before,so we used Kaplan-Meier method to investigate the relationship between FTL and patients’ survival.Based on TCGA dataset,we found that patient with higher FTL expression had shorter overall survival(OS)time than patients with low FTL expression(PartⅠ Figure4A-4F).sh RNA was used to knockdown the expression of FTL in U87 and U251.The results showed that knockdown FTL inhibited the migration and invasion of glioma cells,while overexpression FTL was able to enhanced the migration and invasion of glioma cells(PartⅡ Figure1&2).Besides,the results showed that knockdown FTL could reduce the expression of migration related markers(MMP2,Vimentin,Snail1,E-cadherin)and overexpression FTL could increase the expression of migration related markers(PartⅡ Figure3&4).In order to elucidated the mechanism of FTL regulating glioma cell migration and invasion,we found that FTL expression in tissues was positively correlated with the expression of β-catenin(PartⅢ Figure1A-1C).What’s more,knockdown FTL reduced the expression of β-catenin and subsequent IF and WB revealed that the nuclear accumulation of β-catenin was reduced in FTL-sh RNA than NC-sh RNA glioma cells(PartⅢ Figure2A-2C).It is widely recognized that β-catenin can be a downstream molecular of AKT/GSK3β signaling.After knockdown the expression of FTL in U87 and U251,we found that the level of p-AKT(ser473)and p-GSK3β(ser9)were decrease,while p-β-catenin was increased.Overexpression FTL increase p-AKT(ser473)and p-GSK3β(ser9)expression,but reduced the expression of p-β-catenin(PartⅢ Figure2A).Furthermore,U87 and U251 cells transfected with either NC-sh RNA or FTL-sh RNA were treated with IM-12,an GSK3β inhibitor.We found that the effect of knockdown FTL on migration and invasion could be partly eliminated by treating with IM12(PartⅢ Figure3).Finally,we found that knockdown FTL in U87 could inhibit the growth of glioma cells in vivo(PartⅣ Figure1A-1C).Then we tried to find the influence of FTL in migration and invasion,we used IHC to detect the expression of migration related markers.The results showed that knockdown FTL could significantly reduce the expression of MMP2,Vimentin and Snail1 in vivo.Also the level of p-GSK3β and β-catenin were decreased in vivo(PartⅣ Figure2A-2B).Conclusion:FTL increased with the increasing of glioma grade and patients with higher FTL expression had poorer clinical prognosis that patients with low FTL expression.Furthermore,FTL promote the migration and invasion of glioma by regulating the AKT/GSK3β/β-catenin in vitro and in vivo.These indicated that FTL could be used as an important prognostic marker,as well as a novel therapeutic target.