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Objective To explore the expression,role and mechanism of long non-coding RNA (lncRNA) BANCR in astrocytoma.Methods (1) Twenty-four astrocytoma tissues and 6 normal brain tissues were collected from patients accepted surgical resection and conformed by pathology in our hospital from January 2016 to September 2017;the mRNA expressions of BANCR and signal transduction and transcriptional activator 3 (STA T3) were detected by real-time quantitative(qRT)-PCR;the glioma dataset GSE4290 including astrocytomas were downloaded and BANCR expressions in GSE4290 were analyzed by the web software R2.(2) Astrocytoma cell line LN18,cultured in vitro were divided into short hairpin RNA (shBANCR) group,full-length BANCR (BANCR) group,shControl group and empty vector group;cells in these groups were transfected with recombinant lentiviruses packing genomes encoding short hairpin RNA (shBANCR),full-length BANCR (BANCR),or their corresponding controls (shControl and empty vector);BANCR and STA T3 mRNA expressions in the 4 groups were detected by qRT-PCR;the cell proliferation,migration and invasion,and apoptosis were detected by CCK-8 assay,Transwell assay and flow cytometry,respectively;Western blotting was employed to detect the protein expressions of STAT3,matrix metalloproteinase (MMP)2,MMP9 and mitogen-activated protein kinase (MAPK),and Akt pathway protein expression.(3) Astrocytoma cell line LN18 were divided into si398 group,si1265 group,negative control group Ⅰ,and blank control group Ⅰ;cells in the blank control group Ⅰ were transfected with lipofectamine 2000,and cells in the other three groups were transfected with small interfering RNA si398,si1265 and negative control nonsense sequences targeting STAT3;48 h after transfection,BANCR and STAT3 mRNA expressions were detected by qRT-PCR;Western blotting was employed to detect the STAT3 protein expression.Results (1) In collected samples and glioma dataset GSE4290:the BANCR mRNA expression in astrocytoma tissues was significantly decreased as compared with that in the normal brain tissues (P＜0.05);a positive correlation was noted between BANCR and STA T3 mRNA expressions in astrocytomas (P＜0.05).(2) As compared with the negative control group,the shBANCR group had significantly decreased BANCR mRNA expression,and the BANCR mRNA expression in the BANCR group was significantly increased as compared with that in the empty vector group (P＜0.05);the number of migration and invasion cells in the shBANCR group was significantly larger as compared with that in the negative control group,and that in the BANCR group was significantly increased as compared with that in the empty vector group (P＜0.05);the protein levels of MMP2,MMP9,phosphorylated (p)-extracellular regulated protein kinase and p-mitogen-activated protein kinase in the shBANCR group were significantly increased as compared with those in the negative control group,and those in the BANCR group was significantly increased as compared with those in the empty vector group (P＜0.05).(3) As compared with negative control group Ⅰ and blank control group Ⅰ,si398 group and si1265 group had significantly decreased STA T3 and BANCR mRNA expressions and STAT3 protein expression (P＜0.05).Conclusion The BANCR expression is decreased in astrocytoma;STAT3-induced BANCR can inhibit cell migration and invasion by modulating MMP2,MMP9 and MAPK signaling pathway in astrocytoma.
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Objective To explore the effect of fibroblast growth factor receptor(FGFR)inhibitor BGJ398 on the vasculogenic mimicry(VM)formation of glioma cells. Methods The phosphor-FGFR(pFGFR)was de-tected by Western blot,the expressions of MMP2 and MMP14 were detected by Western blot and immunocytochem-istry;the VM formation of U87MG and U251MG was tested by tube formation assay;subcutaneously implanted tu-mor model in nude mouse was established and tumor sections were CD34/PAS double-stained to detect the forma-tion of VM in vivo. Results Western blot showed that pFGFR in the experimental groups decreased significantly (P < 0.05);western blot and immunohistochemical staining showed that the expression of MMP2 and MMP14 in the experimental groups decreased significantly compared to the control group. In the tube formation assay ,the tube formation of U87MG and U251MG cells were restrained. In the subcutaneously implanted tumor model ,the VM number of the experimental group(13.85 ± 3.96)was significantly lower than that in the control group(26.40 ± 5.06,P < 0.05). Conclusions In vivo and in vitro experiments confirmed that BGJ398 can inhibit the activa-tion of FGFR,and inhibit the VM formation of glioma cells. These indicate FGFR signaling pathway is involved in the formation of VM.
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Objective To explore the effect of fibroblast growth factor receptor (FGFR) receptor antagonist BGJ398 in growth, migration and invasiveness of gliomas. Methods (1) Glioma cells U87 and U251 were routinely cultured in vitro and divided into BGJ398 treatment group (10 μmol/L BGJ398 complete medium) and control group; the proliferation of U87 and U251 cells was detected by CCK-8 and colony formation; 2 d after cultivation, the migration and invasion of U87 and U251 cells were measured by wound-healing assay and Transwell assay. The phosphor-FGFR (pFGFR) level and vimentin expressions were detected by Western blotting. (2) Eight BALB/c nude mice were performed abdominal subcutaneous injection of 200 μL U87 cells (1×107 cells) and randomly divided into BGJ398 treatment group (giving physiological saline solution containing 20 mg/kg BGJ398) and control group (giving physiological saline solution); 15 d after cultivation, the quality of the subcutaneously implanted tumors was compared between the two groups, and the vimentin expression was detected by Western blotting. Results (1) Three, 4 and 5 d after cultivation, the optical density in the U87 cells of BGJ398 treatment group was significantly lower than that in the control group (3 d: t=4.059, P=0.015; 4 d: t=9.892, P=0.001; 5 d: t=10.259, P=0.001); 2, 3, 4 and 5 d after cultivation, the optical density in the U251 cells of BGJ398 treatment group was significantly lower than that in the control group (2 d: t=3.780, P=0.019; 3 d: t=4.515, P=0.011; 4 d: t=16.205, P=0.000; 5 d: t=17.613, P=0.000); 10 d after cultivation, the cloning number of U87 and U251 cells in the BGJ398 treatment group was significantly smaller than that in the control group (P<0.05); the results of wound-healing assay showed that the migration of U87MG cells in the BGJ398 treatment group was significantly slower than that in the control group (P<0.05); 24 h after cultivation, the number of U87 cells migration in the BGJ398 treatment group was significantly smaller as compared with that in the control group (P<0.05); 48 h after cultivation, the number of U87 and U251 cells passed the pore membrane in the BGJ398 treatment group was significantly smaller as compared with that in the control group (P<0.05). Western blotting showed that the content of pFGFR and vimentin in U87 and U251 cells of the BGJ398 treatment group decreased significantly as compared with that in the control group (P<0.05). (2) The subcutaneous tumor tissues in the BGJ398 treatment group[(0.186± 0.064) g] were significantly smaller than those in the control group[(0.450±0.106) g] (P<0.05); Vimentin expression in the BGJ398 treatment group (2.503±0.359) was significantly decreased than that in the control group (4.125±1.155, P<0.05). Conclusion Experiments in vivo and in vitro confirm that BGJ398 can inhibit the activation of FGFR and the growth, migration, and invasion of glioma cells, indicating that FGFR is one of effective targets for the treatment of gliomas.