Background/Aims: Glial cell line-derived neurotrophic factor (GDNF) is an important factor promoting invasive glioma growth. This study was performed to reveal a unique mechanism of glioma cell proliferation and migration. Methods: Human U251 glioma cells were used to screen the optimal GDNF concentration and treatment time to stimulate proliferation and migration. MicroRNA (MiRNA) expression profiles were detected by microarray and confirmed by real-time polymerase chain reaction (PCR). The target genes of differentially expressed miRNAs were predicted by miRWalk, and those targeted by multiple miRNAs were screened with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. A regulatory miRNA network was constructed using ingenuity pathway analysis (IPA). Target gene expression of differentially expressed miRNAs was examined by real-time PCR or mRNA microarray. Results: The results show that 50 ng/mL GDNF for 24 h significantly promotes U251 glioma cell proliferation and migration (P < 0.05). Seven miRNAs (hsa-miR-194-5p, hsa-miR-152-3p, hsa-miR-205-5p, hsa-miR-629-5p, hsa-miR-3609, hsa-miR-183-5p, and hsa-miR-487b-3p) were significantly up-regulated after GDNF treatment (P < 0.05). These miRNAs are primarily involved in signal transduction, cell adhesion and cell cycle through mitogen-activated protein kinase (MAPK) signaling, focal adhesion and glioma signal pathways. Five of these miRNAs (hsa-miR-194-5p, hsa-miR-152-3p, hsa-miR-205-5p, hsa-miR-183-5p, and hsa-miR-487b-3p) co-regulate TP53 and Akt. mRNA expression levels of four genes co-targeted by two or more up-regulated miRNAs were significantly decreased after GDNF treatment (P < 0.05). Conclusion: GDNF treatment of U251 glioma cells significantly increased the expression of seven miRNAs involved in cell adhesion and the cell cycle.

Background: Glial cell line-derived neurotrophic factor (GDNF) is highly expressed in glioblastoma (GBM) and blocking its expression can inhibit the initiation and development of GBM. GDNF is a dual promoter gene, and the promoter II with two enhancers and two silencers plays a major role in transcription initiation. We had previously reported that histone hyperacetylation and DNA hypermethylation in GDNF promoter II region result in high transcription of GDNF in GBM cells, but the mechanism remains unclear. In this study, we investigated whether these modifications synergistically regulate high GDNF transcription in GBM. Results: Cyclic AMP response element binding protein (CREB) expression and phosphorylation at S133 were significantly increased in human GBM tissues and GBM cell lines (U251 and U343). In U251 GBM cells, high expressed CREB significantly enhanced GDNF transcription and promoter II activity. CREB regulated GDNF transcription via the cyclic AMP response elements (CREs) in enhancer II and silencer II of GDNF promoter II. However, the two CREs played opposite regulatory roles. Interestingly, hypermethylation of CRE in silencer II occurred in GBM tissues and cells which led to decreased and increased phosphorylated CREB (pCREB) binding to silencer II and enhancer II, respectively. Moreover, pCREB recruited CREB binding protein (CBP) with histone acetylase activity to the CRE of GDNF enhancer II, thereby increasing histone H3 acetylation and RNA polymerase II recruitment there and at the transcription start site (TSS), and promoted GDNF high transcription in U251 cells. The results indicated that high GDNF transcription was attributable to DNA hypermethylation in CRE of GDNF silencer II increasing pCREB binding to CRE in enhancer II, which enhanced CBP recruitment, histone H3 acetylation, and RNA polymerase II recruitment there and at the TSS. Conclusions: Our results demonstrate that pCREB-induced crosstalk between DNA methylation and histone acetylation at the GDNF promoter II enhanced GDNF high transcription, providing a new perspective for GBM treatment.

Research Paper

Background: Studies have shown that astrocytes play an important role in a variety of biological processes, so damage to astrocytes can cause a series of related diseases. Glial cell line-derived neurotrophic factor (GDNF) has always been considered a protective factor for dopamine neurons. However, it remains unclear whether GDNF has a protective effect on glial cells, especially astrocytes. In this study, we put forward the hypothesis that a high concentration of GDNF in the microenvironment of astrocytes exerts an inhibitory effect on the apoptosis of astrocytes by DNA-damaging reagents. Methods: We isolated, purified, and identified primary astrocytes from neonate rats. Astrocytes were exposed to mitoxantrone (MTN, a DNA-damaging compound) for 24 h. The effects of MTN on astrocytes were tested by Hoechst 33342 staining, CCK-8 assay, and flow cytometry assay. One of the concentrations of MTN was applied to construct an apoptotic model of astrocytes. The astrocytes were then treated with GDNF together with a selected concentration of MTN for 24 h. The cell viability, cell nucleus morphology, and apoptosis ratio of the cells was assessed by Hoechst 33342 staining, CCK-8 assay, and flow cytometry assay, respectively. RNA sequencing (RNA-Seq), quantitative PCR analysis, and KEGG pathway mapping were performed to examine the genes involved in the procedure. Finally, Western blot analysis was applied to confirm the expression levels of the proteins of interest. Results: Hoechst 33342 staining revealed a one-tenth change in the percentage of Hoechst-positive cells after the addition of 500 ng/mL GDNF combined with 1,000 nM MTN for 24 h. The viability of the cells treated the same as described above was 1.4-fold that of the control group. Flow cytometry assays indicated that the apoptotic rates were 17.67, 8.67, and 4.34% for 0, 200, and 500 ng/mL GDNF, respectively. Birc2, Birc3, and Gadd45b were linked to the antiapoptotic process induced by GDNF in astrocytes. Western blot analysis confirmed the elevated expression of Birc2 and Gadd45b. Conclusions: Our studies revealed that GDNF has a noticeable antiapoptotic effect on gene-injured astrocytes. This may provide critical clues for the treatment of a series of diseases in which damaged astrocytes are involved.