| Objective:To investigate the effect and mechanism of CXC chemokine ligand 12-CXC chemokine receptor 4(CXCL12-CXCR4)axis on proneural-mesenchymal transition of glioblastomaMethods:Bioinformatic analyses were used to assess the clinical significance of CXCL12 and CXCR4 mRNA in GBM based on TCGA and CGGA gene databases The expressions of CXCL12 and CXCR4 in GBM samples were detected by immunohistochemistry.Expression of CXCL12 and CXCR4 in 5 kinds of human glioblastoma cell lines was detected by qRT-PCR and Western blot assay.Human glioma LN428 and U87MG cells were pretreated with different concentrations of CXCL12(0,20,40,60,80,100 ng/mL),different time(0,12,24,48,72 h)and 20 ?M plerixafor(selective CXCR4 antagonist),AICAR(0.5,1 mM),Rapamycin(25,50,100 nM)for 48 h,the expression levels of OLIG2,E-cadherin,YKL-40,N-cadherin,Vimentin SREBP1,SCD1,p-AMPK,AMPK,p-mTOR and mTOR were detected by Western blot,the proliferation ability was detected by CCK8 assay and colony formation assay.Oil Red O Staining and Transwell assay were used to test lipid synthesis,migration and invasion ability.The proliferation ability of tumor cells was detected by subcutaneous tumorigenesis experiment and the expression level of SREBP1 SCD1 protein in tissue sections of each group was detected by immunohistochemistry.Nude mice were injected with preconditioning glioma U87MG cells in the tail vein,and CT scans was used to detect tumor lung metastasisResults:TCGA-GBM database analysis showed that compared with healthy people,CXCR4 mRNA expression was increased in glioma patients.The expression level of CXCR4 mRNA was negatively correlated with the survival time of glioma patients and positively correlated with the WHO pathological grade of glioma patients.There was no significant difference in CXCL12 mRNA between the healthy and glioma patients.The results of qRT-PCR and Western blot showed that the expression of CXCR4 was different in glioma cells,with higher expression in LN428,U87MG,LN229 and lower expression in SW1783 and U251MG.The results of Western blot showed that,compared with control group,the expression of mesenchymal-associated proteins YKL-40,N-cadherin and Vimentin was significantly increased in CXCL12 group,while expression of the proneural-related proteins OLIG2,E-cadherin expression level significantly decreased,migration and invasion ability and proliferative ability of glioma LN428 and U87MG cells were significantly enhanced After treatment with plerixafor,migration and invasion ability and proliferative capacity of glioma LN428 and U87MG cells were significantly inhibited,expression of mesenchymal-related proteins was significantly decreased and expression of proneural-related proteins was increased.Western blot results showed that compared with the control group,the expression levels of SREBP1 and SCD1 in the CXCL12 group were significantly increased;compared with the CXCL12 group,the expression levels of SREBP1 and SCD1 protein were significantly reduced after treatment with plerixafor,AICAR,Rapamycin.Oil Red O staining and Transwell assay showed that compared with the control group,the ability of stably knock down SCD1 glioma LN428 and U87MG cells significantly reduced their lipid synthesis,migration and invasion capabilities.The results of animal experiments showed that the tumor growth rate and lung metastasis rate of CXCL12 group were significantly increased compared with the control group;compared with the CXCL12 group,the tumor growth rate and lung metastasis rate were significantly reduced after the plerixafor treatmentConclusions:CXCL12-CXCR4 axis promotes glioma cell proliferation,invasion and migration in vitro and in vivo.CXCL12-CXCR4 axis promotes proneural-mesenchymal transition of glioma.CXCL12-CXCR4 axis promotes lipid synthesis in glioma cells.Knockdown of SCD1 inhibits lipid synthesis,invasion and migration promoted by the CXCL12-CXCR4 axis in glioma cells.CXCL12-CXCR4 axis promotes glioma cell lipid synthesis depending on AMPK/mTOR signaling pathway. |
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