目的:明确HDAC6对人肝癌HepG2细胞迁移和侵袭的影响及其机制.方法:应用Western blot技术检测正常肝细胞系LO2和肝癌细胞系HepG2细胞中HDAC6的表达.应用HDAC6抑制剂Tubastatin A抑制HepG2细胞中HDAC6的表达,运用Western blot及荧光定量PCR技术检测HepG2细胞中转化生长因子β1(transforming growth factor-β1,TGF-β1)和上皮间充质转化(epithelial mesenchymal transition,EMT)相关分子标志物(N-cadherin,β-catenin,Vimentin)的表达;用TGF-β1抑制剂SB431542刺激HepG2细胞后,检测HepG2细胞中EMT标志蛋白的表达,应用TGF-β1刺激HepG2细胞后观察HepG2细胞的形态变化.应用过表达HDAC6的质粒P3-HDAC6、P3-HDAC6+ TGF-β1分别作用于HepG2细胞,通过Western blot检测HepG2细胞中EMT相关分子标志物的表达,应用划痕及Transwell技术检测HepG2细胞处理前后的迁移和侵袭能力变化.结果:肝癌细胞系HepG2细胞内HDAC6的蛋白表达量明显低于正常肝细胞系LO2(P ＜0.05).HepG2细胞Tubastatin A组中N-cadherin、β-catenin、Vimentin、TGF-β1的mRNA和蛋白表达水平相较于空白对照组明显增高(P均＜0.05).SB431542组中EMT标志蛋白表达水平相较于空白对照组明显降低(P均＜0.01).TGF-β1刺激HepG2细胞后细胞变得分散且狭长.同时发现P3-HDAC6+ TGF-β1组的细胞迁移和侵袭能力在48 h后明显强于P3-HDAC6组且弱于空白对照组(P均＜0.05).结论:HDAC6可通过下调TGF-β1的表达从而抑制HepG2细胞的EMT进而抑制HepG2细胞的迁移和侵袭.

Objective To analyze the correlation between herpes zoster neuralgia and the methylation status of the whole genome and GCH1 gene.Methods From June to October in 2017,patients with confirmed herpes zoster and obvious neuralgia were selected in Department of Dermatology,The Affiliated Hospital of Xuzhou Medical University,who achieved complete remission (no effect was observed on normal sleep) of neuralgia after antiviral and neurotrophic treatment.Finally,36 patients and 36 healthy controls were enrolled into this study.Peripheral blood samples were obtained from the healthy controls and patients before and after the treatment.Dot-blot hybridization assay was performed to determine the methylation status of the whole genome,methylated-DNA IP kit was used to enrich the methylation sites of the GCH1 gene,and real-time quantitative PCR was conducted to detect changes in methylation status of the GCH1 gene.Statistical analysis was carried out with GraphPad Prism v7.00 software by using paired t test for the comparison of methylation status before and after the treatment,and two-sample t test for the comparison between the patient group and control group.Results The relative methylation level of the whole genome was 135.94 ± 2.52 in the patients before treatment,significantly lower than that in the patients after treatment (144.76 ± 3.48,t =2.056,P ＜ 0.05) and healthy control group (146.84 ± 3.39,t =2.580,P ＜ 0.05).However,there was no significant difference in the methylation status of the whole genome between the patients after treatment and healthy controls (t =0.429,P ＞ 0.05).Compared with the patients after treatment (0.89 ± 0.13) and healthy control group (0.97 ± 0.07),the methylation status of the GCH1 gene significantly decreased in the patients before treatment (0.65 ± 0.17;t =3.977,4.648 respectively,P ＜ 0.05,＜ 0.01 respectively),while no significant difference between the patients after treatment and the healthy controls (t =0.506,P ＞ 0.05).Conclusion The methylation status of the whole genome and GCH 1 gene markedly decreased in the patients with herpes zoster neuralgia.

Guanosine triphosphate cyclohydrolase 1 (GTPCH1) is a protein encoded by the GCH1 gene,which catalyze GTP to tetrahydrofolinine (BH4) under physiological condition.BH4 is a coenzyme of aromatic amino acid hydroxylase and a cofactor of nitric oxide synthases.BH4 involves in the synthesis of various hormones and neurotransmitters and plays an important role in a series of pathophysiological processes in vivo.Recent studies showed that GTPCH1 is involved in the pathogenesis of neuropathic pain,doparesponsive dystonia,cancer and cardiovascular diseases.In this review,we will discuss the role of GTPCH1 in those diseases mentioned above.