ABSTRACT The methyltransferase-like 3 (Mettl3) is a key component of the large N6-adenosine-methyltransferase complex in mammalian responsible for RNA N6-methyladenosine (m6A) modification, which plays an important role in gene post-transcription modulation. Although RNA m6A is enriched in mammalian neurons, its regulatory function in nociceptive information processing remains elusive. Here, we reported that Complete Freund's Adjuvant (CFA)-induced inflammatory pain significantly decreased global m6A level and m6A writer Mettl3 in the spinal cord. Mimicking this decease by knocking down or conditionally deleting spinal Mettl3 elevated the levels of m6A in ten-eleven translocation methylcytosine dioxygenases 1 (Tet1) mRNA and TET1 protein in the spinal cord, leading to production of pain hypersensitivity. By contrast, overexpressing Mettl3 reversed a loss of m6A in Tet1 mRNA and blocked the CFA-induced increase of TET1 in the spinal cord, resulting in the attenuation of pain behavior. Furthermore, the decreased level of spinal YT521-B homology domain family protein 2 (YTHDF2), an RNA m6A reader, stabilized upregulation of spinal TET1 because of the reduction of Tet1 mRNA decay by the binding to m6A in Tet1 mRNA in the spinal cord after CFA. This study reveals a novel mechanism for downregulated spinal cord METTL3 coordinating with YTHDF2 contributes to the modulation of inflammatory pain through stabilizing upregulation of TET1 in spinal neurons.

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.

ing normal X-chromosomes and the alopecic area having mutated X-chromosomes, after random X-chromosome inactivation (lyonization). In a recent seminal work, Minic et al. documented different hair manifestations of IP. Whorled alopecia, being permanent similar to the dental anomalies, may be a useful clinical marker of incontinentia pigmenti in affected adults who may no longer have cutaneous manifestations. The novel dermoscopic finding of alopecia with greyish-blue dots in the absence of telangiectasia and follicular scaling/plugging can help in better characterization of such presentations.

Post-herpetic neuralgia (PHN) is a well-established clinical problem with potential severe personal and socioeconomic implications. GTP cyclohydrolase 1 (GCH1) gene, which encodes the rate-limiting enzyme in tetrahydrobiopterin synthesis, has been strongly implicated to be associated with neuropathic pain in previous animal and human studies. The rs3783641 (T > A) single-nucleotide polymorphism (SNP) in the GCH1 gene is functional. Here we examine the association between rs3783641 and PHN. A total of 292 subjects including 103 PHN patients, 87 herpes zoster (HZ) patients and 102 healthy controls were enrolled in this study. The rs3783641 polymorphisms were detected via the high-resolution melting curve (HRM) method. There were statistical differences between PHN group and the other two groups in genotype distribution (P = 0.029 and 0.017, respectively) and allele frequency (P = 0.032 and 0.005, respectively) of rs3783641. The proportion of subjects with AA genotype in the PHN group was significantly lower compared to HZ group and control group (P = 0.026 and 0.016, respectively). The frequency of A allele was lower in the PHN group than in control group (P = 0.005), and the frequency of T allele in the PHN group was higher than in HZ group and control group (P = 0.001 and 0.003, respectively). The results of this study suggest that the rs3783641 SNP in the GCH1 gene is associated with PHN, and the AA genotype showed a protective effect in PHN.

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.

Dysfunctions of gene transcription and translation in the nociceptive pathways play the critical role in development and maintenance of chronic pain. Circular RNAs (circRNAs) are emerging as new players in regulation of gene expression, but whether and how circRNAs are involved in chronic pain remain elusive. We showed here that complete Freund's adjuvant-induced chronic inflammation pain significantly increased circRNA-Filip1l (filamin A interacting protein 1-like) expression in spinal neurons of mice. Blockage of this increase attenuated complete Freund's adjuvant-induced nociceptive behaviors, and overexpression of spinal circRNA-Filip1l in naive mice mimicked the nociceptive behaviors as evidenced by decreased thermal and mechanical nociceptive threshold. Furthermore, we found that mature circRNA-Filip1l expression was negatively regulated by miRNA-1224 via binding and splicing of precursor of circRNA-Filip1l (pre-circRNA-Filip1l) in the Argonaute-2 (Ago2)-dependent manner. Increase of spinal circRNA-Filip1l expression resulted from the decrease of miRNA-1224 expression under chronic inflammation pain state. miRNA-1224 knockdown or Ago2 overexpression induced nociceptive behaviors in naive mice, which was prevented by the knockdown of spinal circRNA-Filip1l. Finally, we demonstrated that a ubiquitin protein ligase E3 component n-recognin 5 (Ubr5), validated as a target of circRNA-Filip1l, plays a pivotal role in regulation of nociception by spinal circRNA-Filip1l. These data suggest that miRNA-1224-mediated and Ago2-dependent modulation of spinal circRNA-Filip1l expression regulates nociception via targeting Ubr5, revealing a novel epigenetic mechanism of interaction between miRNA and circRNA in chronic inflammation pain.SIGNIFICANCE STATEMENT circRNAs are emerging as new players in regulation of gene expression. Here, we found that the increase of circRNA-Filip1l mediated by miRNA-1224 in an Ago2-dependent way in the spinal cord is involved in regulation of nociception via targeting Ubr5 Our study reveals a novel epigenetic mechanism of interaction between miRNA and circRNA in chronic inflammation pain.