PMC

CNTF/CNTFRα inhibited apoptosis by PI3K/AKT pathway in vitro. (A) U87 and (D) A172 cell apoptosis were analyzed with Annexin V and Propidium Iodide by flow cytometry assay after CNTF, siCNTFRα or LY294002 treatment. (B,C,E,F) Apoptosis- related protein BCL2 and BAX were detected by western blot after siRNA transfection, CNTF and LY294002 treatment in U87 and A172 cells. western blot was representative of three independent experiments.

CNTF/CNTFRα promoted glioma cell proliferation by PI3K/AKT pathway in vitro. (A) A172 and (B) U87 cells were seeded into 96 well plate after transfection with siRNA. Cell viability was assayed by CCK8 at indicated time points. (C,D and E) p-AKT was analyzed by western blot after transfecting siRNA or LY294002 treatment. (F) and (H) A172 and U87 cell viability was assayed with CNTF, LY294002 or siRNA treatment. (G) and (I) p-AKT was analyzed by Western blot after transfection with siRNA or LY294002 treatment.

CNTFRα hypomethylation could predict poor survival in LGG. (A) The methylation level of the cg20388256 probe region was quantitatively analyzed by MSP -PCR. LGG (n = 62) had significant lower methylation level than normal brain tissues (n = 6, p < 0.05). (B) CNTFRα ^hyper group was associated with longer patient survival compared with CNTFRα ^hypo group (p < 0.05). (C) CNTFRα ^hyper group was associated with longer survival compared with CNTFRα ^hypo in IDH ^wt patients. (D) CNTFRα ^hyper IDH ^mt group was associated with longer survival compared with CNTFRα ^hypo IDH ^wt (p < 0.05). hyper: hypermethylation; hypo: hypomethylation; mt: mutation; mt: wide type.

CNTFRα was overexpressed in LGG and CNTF can act as an autocrine ligand in glioma. (A) Bioinformatic analysis of CNTFRα expression in LGG (n = 530) and normal brain tissues (NB, n = 5, p < 0.05). (B) CNTFRα protein expression was analyzed in glioma samples (LGG n = 28) with normal brain tissue as control (n = 5). (C) Densitometric analysis of CNTFRα protein expression level of glioma samples. (D) and (E) Western blot results of CNTF and CNTFRα protein expression in three glioma primary cells and cell lines. (F) Immunofluorescence analysis of CNTF and CNTFRα expression showed membrane location (scale bar 10 um). (G) IHC analysis of CNTF and CNTFRα expression in glioma tissues. Western blots were representative of three independent experiments. Statistical results represent the mean ± SD of n = 3 determinations.

Knockdown of CNTFRα inhibited glioma xenograft growth in vivo. (A) A172 cells were injected subcutaneously into the addominal flanks of nude mice. When xenograft tumor volume was even on the nineteenth day, siRNA was injected into tumor xenografts every other day. Finally, xenograft tumors were harvested and photographed on the twenty-ninth day. (B) Xenograft tumor volume was decreased by 54% and (C) Xenograft tumor weight was decreased by 39% in the siCNTFRα group compared with control group. (D) and (E) In xenograft tumors, CNTFRα expression level was significantly decreased in the siCNTFRα group compared with the control group. Western blot results were representative of three independent experiments. (F) Immunohistochemistry analysis of the xenograft tumors showing decreased expression of p-AKT, BCL2, Ki67 and increased expression of cleaved caspase 3 and BAX in the siCNTFRα injection group.

Methylation of a CpG island shore in the CNTFRα gene regulated CNTFRα mRNA expression. (A) CNTFRα gene structure [based on RefSeq Feb. 2009 (GRCh37/hg19) assembly], and heat map showing DNA methylation level throughout the CNTFRα gene from the TCGA LGG cohort (n = 530). Blue boxes represent the first and second exons. Green, purple and aqua represent the CpG context (CpG island, CpG shore and CpG shelf), respectively. DNA methylation level throughout CNTFRα gene in LGG. (B) Scatter plot of the correlation analysis between CNTFRα mRNA and methylation level of probe cg20388256 (r = −0.45, p < 0.0001). (C) Correlation analysis between DNA methylation and gene expression levels. Each point represented one CpG site and the dashed line indicateed the variation of correlation throughout CNTFRα gene. Orange lines represented the statistically significant threshold for the correlation analysis (p = 0.05). (D,E,F and G) U138 and SH66 cells were treated with 5-Aza-2-dC or control, and CNTFRα expression was quantified by real time PCR and Western blot. Error bars represented means ± SD from replicates (n = 3).