The Study Of GRP78 Maintains Self-renewal And Radioresistance Of Glioma Stem Cells

BackgroundsGlioma is the most common primary tumor in human brain.And for the patients who suffered from the more malignant glioma(WHO grade ?/?),the recurrence and poor prognosis were quite often,even though with the treatment combined with surgery,irradiation and chemotherapy.Recently,experts had classified glioma as three distinct subclasses in the light of disparity of gene expressing pattern.And two subclasses of them,proneural(PN)and mesenchymal subclasses(MES),closely relate to the treatment efficacy and prognosis.Commonly,the MES subclass was characterized by radiaresistance and recurrence.Therefore,exploring the mechanisms of these malignant behaviors from the aspect of molecular subclass is essential to the progression of treatment and clinical translation.Glucose-regulated protein 78(GRP78)has been proved that highly expressed in glioma and related to the malignant properties,such as treating resistance.But the study for prognosis determined by GRP78 expression in glioma is still short in Chinese journals so far,and the correlation between GRP78 and MES subtype is poorly understood.Fulfilling these researches helps us to understand the molecular mechanisms of glioma and provide ingenious ideas for clinical translation.Objectives:To study the correlation between the expression of GRP78 and poor prognosis in glioma,and determine the specific functions of GRP78 in MES-subtype glioma stem cells.Methods:1.The mRNA profiling data and corresponding clinical information were acquired from the TCGA database,then the Prism software was used to visualize differential expression of GRP78 in different WHO grades and molecular subtype.The prognosis data was used to indicate correlation between GRP78 expression and survival.2.The glioma specimen was collected in the Department of Neurosurgery,Qilu Hospital of Shandong University,with the pathological diagnosis as high-grade glioma(WHO ?/? grade).High-throughput sequencing was applied to profile mRNA expression of each sample.And the standardized data was used to run GSEA,analyzing the correlation between GRP78 expression and mesenchymal subtype.3.Four different kinds of GSC were used to detect expression of GRP78 protein in GSCs.The lentivirus expressing shGRP78 was applied to construct the mesenchymal GSCs with constant knockdown of GRP784.With the knockdown of GRP78 using siRNA,western blot was performed to detect the change of mesenchymal signatures and related signal pathways.5.Sphere formation assay and in vivo limiting dilution assay were applied to determine the impact of knock-down GRP78 on self-renewal of GSCs.6.Nude mice were used to established the GSC-derived xenograft model.The bioluminescence imaging and survival data were applied to evaluate the capacity of tumorigenesis in vivo of GRP78-knockdown GSCs.7.Delivering some dose of radiation to knock-down GRP78 and control groups,TUNEL fluorescence staining and Annexin V-PI dual staining for flowcytometry were performed to indicate the apoptosis,which is coincide with the radioresistance of GSCsResults:1.TCGA database revealed that GRP78 mRNA level in glioma positively relates to WHO grades,as the high-grade glioma express increasing GRP78.And GRP78 expression of mesenchymal glioma is significantly higher than it of other subtypes.Meanwhile,in the GBM patients,high expression of GRP78 companied with the poor prognosis,which has shorter lifecycle.2.Analyzing sequencing data of glioma samples collected in Qilu Hospital of Shandong University,GRP78 mRNA expression has the significant positive correlation with Phillips-MES gene set.3.Knockdown of GRP78 in MES-subtype GSCs downregulated expression of MES signatures CD44 and YKL40 as well as MES-related pathways STAT3 and NF-?B.4.Comparing to the control group,constant knockdown of GRP78 in GSCs inhibited self-renewal ability significantly,as it led to a remarkable decrease in sphere's diameter and a significant ascent of the probability that wells without sphere formation5.GSC cells succeed to grow in mice after the intracranial implantation.Comparing with the control group,GRP78 knockdown suppressed tumorigenesis in vivo significantly,corresponding to the less tumor volume and prolonged survival6.GRP78 knockdown decreased the radioresistance of MES-subtype GSCs,appearing the higher property of apoptotic cells than the control group hadConclusions:1.High-grade(WHO ?/? grade)and mesenchymal-subtype gliomas expressed higher level of GRP782.GRP78 silencing decreased the expression of MES-related proteins in GSCs.3.Silencing GRP78 significantly suppressed the self-renewal and radioresistance of mesenchymal GSCs.