ABSTRACT
<p><b>OBJECTIVE</b>To investigate the mechanism of high transcription of the glial cell-line derived neurotrophic factor (gdnf) gene induced by hyperacetylation of histone H3 lysine 9 (H3K9) at its promoter region II in rat C6 glioma cells.</p><p><b>METHODS</b>The acetylation level of H3K9 at Egr-1 binding site in gdnf gene promoter region II and the binding capacity of Egr-1 to its binding site in gdnf promoter were examined by ChIP-PCR in C6 astroglioma cells and normal rat astrocytes, and its changes were investigated in C6 astroglioma cells after treatment with histone acetyltransferase inhibitor curcumin or deacetylase inhibitor trichostatin A.</p><p><b>RESULTS</b>Compared normal astrocytes, C6 astroglioma cells showed significantly increased acetylation level of H3K9 at Egr-1 binding site in gdnf gene promoter region II and Egr-1 binding capacity (P<0.01). Curcumin treatment significantly reduced H3K9 acetylation level at Egr-1 binding site and decreased both the binding of Egr-1 to promoter region II and gdnf mRNA levels in C6 astroglioma cells (P<0.05). Conversely, increased H3K9 acetylation at the Egr-1 binding site induced by trichostatin A significantly increased the binding of Egr-1 to promoter region II and gdnf mRNA expression levels (P<0.05).</p><p><b>CONCLUSION</b>H3K9 hyperacetylation induces increased Egr-1 binding to gdnf gene promoter II, which might be the reason for the high transcription level of gdnf gene in rat C6 glioma cells.</p>
Subject(s)
Animals , Rats , Acetylation , Astrocytes , Metabolism , Binding Sites , Cell Line, Tumor , Early Growth Response Protein 1 , Metabolism , Glial Cell Line-Derived Neurotrophic Factor , Genetics , Glioma , Metabolism , Histones , Chemistry , Promoter Regions, Genetic , Protein Processing, Post-Translational , RNA, Messenger , Transcription, GeneticABSTRACT
Objective To observe the expression pattern of caspase-3 and HCLS1-associated protein X-1 (HAX-1) at different time after cerebral contusion in rat, and explore the new method for estimating the injury interval. Methods The cerebral contusion model was established using adult SD male rats. Then the rats were randomly allocated into 8 groups: 2 h, 6 h, 12 h, 1 d, 3 d, and 7 d after cerebral con-tusion, sham-operation and normal control. Expression of caspase-3 and HAX-1 protein after cerebral contusion in rat was detected by Western blotting. Laser scanning confocal microscope was used to ob-serve the number of HAX-1 positive cells and TUNEL-stained cells after cerebral contusion. Results The expression of caspase-3 increased parallelly with the time after cerebral contusion and reached the peak value on 3 d. The expression of caspase-3 decreased gradually and still maintained a high level expression on 7 d (P<0.05). The expression of HAX-1 positive cell went up after injury, and reached the peak value at 6 h (P<0.05), then turned down gradually after 12 h and went out of detection after 3 d. The number of TUNEL-stained cells increased obviously at 2 h and reached the peak value on 3 d. The number of TUNEL-stained apoptotic cells decreased gradually and still maintained a high level expres-sion on 7 d (P<0.05). Conclusion The expression of caspase-3 and HAX-1 after cerebral contusion has time sequential regularity, which may provide new evidence for forensic diagnosis of cerebral contusion interval.
ABSTRACT
Objective To study the mechanism that glial cell line-derived neurotrophic factor (GDNF)promotes human glio-ma cells proliferation.Methods We divided glioma samples into two groups,including low-grade glioma group and high-grade glio-ma group,while cerebral contusion patients were treated as the control group,12 cases in each group.C6 glioma cell lines were di-vided into three groups,such as GDNF group,BSA(bovine serum albumin)group and control group.CCK-8 (cell counting kit-8) was used to detect the cell proliferation,while Western blot was used to detect the expression of AKT,p-AKT,β-catenin and p-β-catenin in each group.Results Comparing with the control group,the expression levels of AKT,p-AKT,β-catenin and p-β-catenin in glioma group had a significantly increased (P <0.05).Meanwhile,the high-grade gliomas group also had a significant increase in those more than low-grade gliomas group (P <0.05).CCK-8 test showed that the cell proliferation in GDNF group was significant-ly higher than the control group (P <0.05),and the expression levels of p-AKT,β-catenin and p-β-catenin proteins all had a signifi-cant increase (P <0.05).However,the expression level of AKT had no obvious difference.Conclusion GDNF might promote the proliferation of glioma cells by up-regulating the expression of p-AKT,β-catenin and p-β-catenin.