| BackgroundAs the most common malignant tumor in adult,glioma is prone to relapse and thus has a poor prognosis.Tumor recurrence,the chief reason for poor prognosis of glioma,is largely attributed to glioma stem cells(GSCs)and the invasive growth characteristics resulted from epithelial-mesenchymal transition(EMT).However,the relationship between glioma stem cell and its invasiveness and the mechanisms in tumor recurrence are still unknown.Therefore,we determined whether leucine-rich repeat-containing G protein-coupled receptor 5(LGR5),proved as a stem cell marker for colon cancer and gastric cancer by recent studies,can serve as a novel GSC marker involved in EMT and a therapeutic target in glioma.ObjectiveTo explore whether the LGR5 can serve as a GSC marker and its regulation mechanism of invasiveness and EMT in GSCs.Further explore the role of LGR5 in predicting survival and recurrence time of gliomas so as to provide new targets for the treatment of malignant glioma.Methods1.Flow cytometry detection of LGR5 was performed in both parent cells and the enrichment cells by serum-free culture method of U87,U251 and A172 glioma cell lines and 8591,LHH and 7112 primary glioma cells,respectively.The enrichment levels was analyzed;Immunofluorescence of LGR5 and glial fibrillary acidic protein(GFAP)were detected in above cells.2.LGR5+ and LGR5-cells were obtained by fluorescence-activated cell sorting(FACS)in U251 and 8591 glioma cells.Cell proliferation,coloning formation,drug resistance,invasion and migration and tumor formation were performed to determine the stemness propertie of LGR5+and LGR5-cells.3.The expression levels of LGR5 and other reported cancer stem cell(CSCs)markers CD133,CD24,CD44,CD90 and EpCAM in LGR5+ and LGR5-cells were detected by flow cytometry detection.4.The stem cells enriched from U251 and 8591 cells were respectively conducted for LGR5 knockdown and overexpression.Western blot was used to examine the expression levels of above CSC markers and the sternness genes SOX2,Nanog and OCT4.Immunofluorescence of LGR5 and SOX2 were performed in GSCs.5.The invasion and migration assays were used to determine the ability of invasiveness and migration in both knockdown and overexpression groups and Wnt-C59 treatment group.The expression levels of Wnt/?-catenin signaling pathway and EMT markers in above groups were detected by Western blot.6.The intracranial orthotopic transplantation tumor was performed in LGR5 knockdown group(shLGR5),Wnt-C59 group and control group(shCtrl),and the growth rate of intracranial tumor and survival time was compared with each group.7.Immunohistochemical was used to detect the expression levels of LGR5,N-cadherin and Ki67 in glioma specimens and the levels and correlations were analyzed in different grades.Immunofluorescence was used to detect the expression level of LGR5 in glioma tumorous and peritumoral tissues.8.The overall survival(OS)and progression-free survival(PFS)of the low grade and high grade glioma patients were analyzed according to the expression levels of LGR5 by Kaplan-Meier method.Univariate analysis and multivariate survival analysis were performed in low grade and high-grade glioma patients by Cox proportional hazards model.9.The statistical methods used in above study included ANOVA,Student t test,Spearman correlation test?chi-square test(X2)and Fisher's exact test.The Kaplan-Meier,Log-rank test and Cox proportional hazards model were used to analyze theOS and PFS.P values less than 0.05 were considered indicative of statisticalsignificance.Results1.Flow cytometry displayed that the positive proportions of LGR5 were 2.46%,2.01%,5.76%,1.34%,1.79%and 1.45%in U251,U87,A172,8591,LHH and 7112 parent cells,respectively,and 21.50%,11.23%,16.04%,15.42%,11.41%and 4.53%in U251,U87,A172,8591,LHH and 7112 enriched cells,respectively.The positive rates of LGR5 in enriched cells were 8.7,5.6,2.8,11.5,6.4 and 3.1 times higher than those in parent cells for U251,U87,A172,8591,LHH and 7112,respectively.All of the abovementioned glioma cells were proved to be GFAP positive and possessed different expression levels of LGR5 which was expressed in the cell membrane and cytoplasm.2.Compared with LGR5-cells,LGR5+ U251 cells possessed a higher ability of proliferation,coloning formation,drug resistance,invasion and migration in vitro and tumor formation in vivo.Immunohistochemical results showed that LGR5 had significant correlation with N-cadherin and Ki67 in vivo.3.LGR5+ glioma cells possessed considerably enhanced expression of CD 133,CD44,CD90,CD24,and EpCAM,while the expression of CD90 remained unchanged.Western blot revealed that CD133,CD44,CD24 and EpCAM and stem cell genes Sox2 and Nanog in shLGR5 GSCs were significantly decreased compared with those of the shCtrl GSCs,while the expression of OCT4 remained unchanged.Immunofluorescence staining showed that LGR5 and SOX2 were co-expressed in both U251-GSCs and 8591-GSCs.4.The invasion and migration capacities of shLGR5 GSCs were weakened as a result of LGR5 knockdown,whereas the invasion and migration capacities of Lenti-LGR5 GSCs were enhanced as a result of LGR5 overexpression.The Wnt/?-catenin pathway and the expression levels of the EMT markers were inhibited in the shLGR5 GSCs compared with those in the shCtrl GSCs and promoted in the Lenti-LGR5 GSCs compared with those in the Lenti-GFP GSCs.Furthermore,the Wnt/?-catenin pathway and the expression levels of EMT markers were inhibited by Wnt-C59 in dose-dependent manner,as was the invasiveness and migration of GSCs.5.The tumor growth curve showed that tumors in the shLGR5 and Wnt-C59 groups were significantly inhibited,in contrast to those in the shCtrl group.A log-rank test of the three groups revealed that the OS of the shLGR5 and Wnt-C59 mice was significantly prolonged compared with that of the shCtrl mice.In addition,the expression levels of Ki67 and Active-?-catenin in both the shLGR5 and Wnt-C59 groups were lower than those in the shCtrl group,whereas the expression levels of Ki67 and Active-?-catenin showed no differences between the shLGR5 and Wnt-C59 groups.Furthermore,Spearman correlation tests verified that the expression of LGR5 was positively correlated with Active-?-catenin,as well as Ki67,between the shLGR5 and shCtrl groups.6.Significant differences in LGR5 levels were observed between high-gradeand low-grade gliomas.In addition,both Ki67 and N-cadherin levels increased with grade.Positive correlations were observed between the levels of LGR5 and Ki67and between the levels of LGR5 and N-cadherin.In addition,many glioma cells with high expression of LGR5 and N-cadherin were observed in the invasive front of human glioma tissues,while glioma cells that rarely expressed LGR5 and N-cadherin were mostly observed inside tumor.7.Log-rank tests revealed that in the HGG group,LGR5high patients had significantly lower PFS and OS compared with that of patients with(LGR5low patients.The median PFS and OS were 7.0 and 17.5 months for LGR5high patients and 10.5 and 23.0 months for LGR5low patients,respectively.In the LGG group,LGR5high patients had significantly lower RFS than LGR5low patients,while the OS was not statistically significant between LGR5high patients and LGR5low patients.The median PFS of LGR5high patients was 23.5 months.8.In the HGG group,univariate analysis revealed that LGR5 expression,peritumoral edema and chemoradiotherapy were correlated with worse PFS.Multivariate Cox regression analysis demonstrated that LGR5 expression and chemoradiotherapy were independent indicators of postoperative recurrence.Moreover,LGR5 expression,the Karnofsky Performance Scale(KPS),IDH1 mutation and chemoradiotherapy were associated with worse OS according to the log-rank test.Multivariate analysis showed that LGR5 expression,IDH1 mutation and chemoradiotherapy were independent indicators of OS.In the LGG group,IDH1 mutation and chemoradiotherapy significantly decreased both RFS and OS,whereas LGR5 expression was only related with shorter PFS but not OS.In multivariate analysis,LGR5 expression and chemoradiotherapy were key factors resulting in shorter PFS.Conclusion1.LGR5 can be used as a new functional GSCs,it significantly influenced the expression of some CSC markers and stem genes in GSCs.2.LGR5 upregulate the Wnt/?-catenin signaling pathway by promoting the EMT related cell invasion and migration ability.3.LGR5 is the independent prognostic factors for glioma patients,and would become an important therapeutic targets for GSCs.Innovation points1.LGR5 is confirmed as a new functional glioma stem cell markers for the first time,and the relationship between LGR5 and CSC markers were determined.2.The function that LGR5 promote the EMT and its regulatory mechanism are demonstrated in GSCs.3.LGR5 is proved to be an effective target for GSCs treatment by orthotopic transplantation model in nude mice. |
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